WO2024156726A1 - MAINTAINING REGULATORY T CELL (Treg) FUNCTIONALITY UNDER CONDITIONS TRIGGERING Treg DYSFUNCTIONALITY - Google Patents

MAINTAINING REGULATORY T CELL (Treg) FUNCTIONALITY UNDER CONDITIONS TRIGGERING Treg DYSFUNCTIONALITY Download PDF

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WO2024156726A1
WO2024156726A1 PCT/EP2024/051599 EP2024051599W WO2024156726A1 WO 2024156726 A1 WO2024156726 A1 WO 2024156726A1 EP 2024051599 W EP2024051599 W EP 2024051599W WO 2024156726 A1 WO2024156726 A1 WO 2024156726A1
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tregs
treg
cells
cell
isolated
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Markus Kleinewietfeld
Ibrahim HAMAD
Beatriz CÔRTE-REAL
Dominik N. Mueller
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Vib Vzw
Universiteit Hasselt
Max-Delbrueck-Centrum Fuer Molekulare Medizin In Der Helmholtz-Gemeinschaft
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to protecting regulatory T cells (Tregs) against dysfunctionality as caused in e.g. auto-immune disease settings.
  • Tregs regulatory T cells
  • Such protection is warranted in case of adoptive transfer or Tregs as a means of treatment of auto-immune and other diseases, and as a means of controlling transplant or graft rejection.
  • the protection is enabled by inhibiting the Treg mitochondrial sodium/lithium/calcium exchanger.
  • Regulatory F0XP3+ T cells play an essential role for the maintenance of peripheral tolerance and immune cell homeostasis (Kleinewietfeld & Hafler 2014, Immunol Rev 259:231-244; Sakaguchi et al. 2010, Nat Rev Immunol 10:490-500).
  • they Depending on environment and tissue, they have the ability to suppress and neutralize responses of the innate and adaptive immune system by various mechanisms like secretion of anti-inflammatory cytokines as interleukin (IL)-10 or by cell-cell contact dependent mechanism involving co-stimulatory receptors like cytotoxic T-lymphocyte-associated protein 4 (CTLA- 4).
  • IL interleukin
  • CTLA- 4 cytotoxic T-lymphocyte-associated protein 4
  • Treg dysfunction is frequently associated with the development of a pro- inflammatory, cytokine producing phenotype, termed Treg plasticity or fragility (Hatzioannou et al. 2021, Front Immunol 12:731947; Overacre-Delgoffe & Vignali 2018, Cancer Immunol Res 6:882-887; Kleinewietfeld & Hafler 2013, Semin Immunol 25:305-312).
  • High salt (NaCI) content in food has been established to have detrimental effects on several pathologies and autoimmune disorders by shifting the immune cell balance towards a pro-inflammatory state (Muller et al. 2019, Nat Rev Immunol 19:243- 254; Manzel et al. 2014, Curr Allergy Asthma Rep 14:404).
  • T effector cells like T helper (Th)17 responses (Wu et al. 2013, Nature 496:513-517; Kleinewietfeld et al. 2013, Nature 496:518-522), we and others have shown that HS could also severely affect the function of Tregs (Hernandez et al.
  • HS has been shown to dynamically induce a pro-inflammatory Th 1-like Treg phenotype with high expression levels of interferon (IFN)-y and lower levels of IL-10 (Hernandez et al. 2015, J Clin Invest 125:4212-4222; Sumida et al. 2018, Nat Immunol 19:1391-1402).
  • IFN interferon
  • T cell subsets highly depend on various metabolic needs depending on environment and activation status (Geltink et al. 2018, Annu Rev Immunol 36:461-488).
  • Tregs are believed to closely mimic memory T cells in their metabolic needs, preferentially depending on oxidative phosphorylation (OXPHOS) and fatty acid p-oxidation (FAO) while T effector cells rather use aerobic glycolysis (Geltink et al. 2018, Annu Rev Immunol 36:461-488; Newton et al. 2016, Nat Immunol 17:618-625; Chapman et al.
  • WO2011/048589 links both inhibition and activation of NCLX with treatment of amongst other inflammatory diseases, and activation of NCLX with treatment of amongst other autoimmune diseases.
  • the invention relates to isolated regulatory T cells (Treg cells) or populations of isolated Treg cells characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited in the Treg cells. More in particular, the function of NCLX can be inhibited by a pharmacological compound, or the expression of NCLX can be inhibited by a DNA nuclease specifically knocking out or disrupting NCLX, by an RNase specifically targeting NCLX, or by an inhibitory oligonucleotide specifically targeting NCLX.
  • the Treg cells can be polyclonal Treg cells, in vitro amplified or expanded Tregs, antigenspecific Tregs, engineered T cell receptor (TCR)-Tregs, engineered chimeric antigen receptor (CAR)-Tregs, monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs, B-cell-targeting antibody receptor (BAR) Tregs, design Tregs, or chimeric cytokine receptor (CCR) Tregs.
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the invention also relates to compositions comprising isolated Treg cells or populations of isolated Treg cells as defined above.
  • compositions can be pharmacological compositions.
  • the isolated Treg cells or population of isolated Treg cells or the (pharmaceutical) compositions as defined above can be for use as a medicament; for use in treating an auto-immune disease; for use in treating an inflammatory disease; for use in suppressing transplant, graft or allograft rejection; or for use in treating a cardiovascular disease.
  • the isolated Treg cells or population of isolated Treg cells or the (pharmaceutical) compositions as defined above can be adoptively transferred in a subject.
  • the Treg cells are autologous Treg cells, allogeneic Treg cells, or induced Treg cells.
  • the invention further relates to methods of producing isolated Treg-cells as defined above, such methods comprising a step of isolating Treg-cells from peripheral blood, umbilical cord blood, thymus or leukapheresis product obtained from a subject, or comprising a step of reprogramming conventional CD4+ T-cells obtained from a subject into Tregs.
  • An optional further step in such methods is a step of ex-vivo expanding the isolated or reprogrammed Tregs.
  • Another step in such methods can be a step of ex-vivo manipulation to inhibit the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in the Tregs by means of pharmacological inhibition or by means of a DNA nuclease specifically knocking out or disrupting NCLX, an RNase specifically targeting NCLX, or an inhibitory oligonucleotide specifically targeting NCLX.
  • the invention also relates to pharmaceutical kits comprising at least one vial comprising isolated Treg cells or populations of Treg cells as defined above, or comprising a composition comprising such Treg cells or populations of Treg cells.
  • the invention also relates to isolated TCR-engineered Treg-cells, CAR-engineered Treg-cell, or BAR- engineered Treg cells characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited in the engineered Treg cells.
  • FIGURE 1 Salt induced changes in Treg phenotype and function closely mimic human autoimmune Tregs.
  • FIGURE 2 High salt induces discrete changes in gene expression in human Tregs indicative for metabolic reprogramming.
  • FIGURE 3 High salt inhibits mitochondrial respiration in human Tregs.
  • OCR Oxygen consumption rate
  • TMRE tetramethylrhodamine ethyl ester
  • MFI fluorescence intensity
  • FIGURE 4 Salt mediated disruption of mitochondrial respiration perturbs human Treg function.
  • A-B FOXP3 expression after HS exposure on Tregs activated under control (“CTRL”) or HS conditions ("+40mM NaCI”).
  • CTRL Tregs activated under control
  • FIGURE 5 Impact of salt on Treg fitness and function in vivo.
  • A-C Salt intake and its impact on Tregs in healthy human subjects.
  • E-F Impact of salt on Treg fitness and function to contain experimental autoimmune encephalomyelitis (EAE) by adoptive transfer.
  • EAE was induced in recipient mice and CD4 + CD25 + Tregs of naive donor mice were pre-incubated in the presence of HS (+40mM NaCI) or control (CTRL) conditions for a period of 24 hours prior to being adoptively transferred to contain EAE into recipient mice 6 days after EAE induction.
  • CTR control
  • FIG. 1 Impact of salt on human Treg fitness and function to contain xGvHD.
  • FIG. 1 Schematic experimental set-up for xGvHD.
  • Human CD25 + Tregs were activated in control media, media supplemented with lOpM of antimycin A (AA) or media supplemented with +40mM NaCI for 6 hours.
  • FIGURE 6 Salt induced blockade of mitochondrial function depends on NCLX.
  • Tregs were pre-incubated under HS conditions (+40mM NaCI) in the presence or absence of lOpM NCLX inhibitor CGP-37157 (CGP) for 24 hours and later co-cultured with CFSE labelled PBMCs at a 1 to 1 ratio.
  • A-E Data is depicted as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01. Normal distribution was calculated by Shapiro- Wilk normality test with a significance level of 0.05. Significance was calculated by paired two-tailed (B, D) t-test for normal distributed data, by paired one-tailed Wilcoxon test (E) for non-normal distributed data or by unpaired two-tailed (A) or one-tailed (C) for not normally distributed data. In Na + quantifications, outliers were identified with ROUT method and not included in the analysis (A).
  • FIGURE 7 Indel signature of representative SLC8B1-KO in human Tregs.
  • Treg high salt
  • Tregs regulatory T cells
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • MS multiple sclerosis
  • T regs regulatory T cells
  • Dysfunctional autoimmune Tregs display pro-inflammatory features and altered mitochondrial metabolism but factors contributing to this phenotype remain elusive.
  • High salt (HS) intake has been identified to alter immune function and to promote autoimmunity.
  • HS high salt
  • extracellular HS raises intracellular Na + that perturbs mitochondrial respiration by directly interfering with complex ll/lll of the electron transport chain (ETC).
  • Increased sodium concentrations can moreover be present in inflamed tissues independent of high salt dietary intake (e.g., Jantsch et al. 2015, Cell Metab 21:493-501; Jobin et al. 2021, Trends Immunol 42:469-479; Huhn et al. 2021, Proc Natl Acad Sci USA 118:e2102549118).
  • the invention therefore in a first aspect relates to (an) isolated regulatory T cell (s) (Treg cell (s), or simply Treg(s)) or to a population of isolated Treg cells characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited, in the Treg cell or cells.
  • Treg cell s
  • Treg cell simply Treg(s)
  • the advantage of Tregs with such modification resides in their increased functionality under unfavorable conditions as can occur when transferred into subjects having or suffering from an autoimmune disease, or by extension an (uncontrolled, such as persistent or chronic) inflammatory disease, or from a cardiovascular disease, independent of being or having been on a high salt diet prior to (therapeutic, adoptive) transfer of the Tregs.
  • the function of NCLX can be inhibited, or substantially inhibited by means of incubating Tregs with a pharmacological compound, such as a compound capable of selectively or specifically inhibiting, or substantially inhibiting, NCLX activity.
  • a pharmacological compound such as a compound capable of selectively or specifically inhibiting, or substantially inhibiting, NCLX activity.
  • the pharmacological compound is 7-Chloro-5-(2-chlorophenyl)-l,5-dihydro-4,l-benzothiazepin-2(3H)-one (CGP-37157).
  • the expression of NCLX in Tregs can be inhibited, or substantially inhibited by means of a specific or selective inhibitor of NCLX expression.
  • a specific or selective inhibitor may be a DNA nuclease specifically knocking out or disrupting NCLX, an RNase specifically targeting NCLX, an inhibitory oligonucleotide specifically targeting NCLX, or may be encoded on a transposon specifically targeting NCLX.
  • Such inhibitory oligonucleotides may be selected from (the group consisting of) an antisense oligomer, a siRNA, a shRNA, a gapmer, and the likes; such DNA nucleases may be selected from (the group consisting of) a ZFN, a TALEN, a CRISPR-Cas, and a meganuclease; and such RNases may be selected from (the group consisting of) a ribozyme and a CRISPR-C2c2. More general details of these modalities are provided hereinafter.
  • the step of inhibiting, or substantially inhibiting function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in Tregs is performed in vitro or ex vivo, i.e. in Tregs such as obtained from a subject or in induced Tregs (more details further hereinafter).
  • the inhibitor or function or expression of NCLX can e.g. be targeted to the mitochondria of the Tregs by means of e.g. a mitochondriotropic nanocarrier (see further).
  • NCLX is also known as SLC8B1 (solute carrier family 8 member Bl) or (mitochondrial) sodium/lithium/calcium exchanger.
  • SLC8B1 synthetic carrier family 8 member Bl
  • mitochondrial sodium/lithium/calcium exchanger.
  • human chrl2:113, 298, 759-113, 359, 493 GRCh38/hg38; minus strand
  • human chrl2:113, 736, 576-113, 772, 914 GRCh37/hgl9 by Entrez Gene; minus strand
  • Protein symbol Q6J4K2-NCLX human; protein accession Q6J4K2.
  • CGP37157 or CGP-37157 is a small molecule compound inhibiting NCLX. It is also known under CAS No. : 75450-34-9, has the chemical formula CisHnCLNOS, and as chemical name e.g. 7-Chloro-5-(2- chlorophenyl)-l,5-dihydro-4,l-benzothiazepin-2(3H)-one.
  • the InChi Key for CGP37157 is KQEPIRKXSUIUTH-UHFFFAOYSA-N.
  • the chemical structure of CGP37157 is:
  • Tregs as cell-based immunotherapy
  • GvHD Graft-versus-Host Disease
  • T1D type 1 diabetes
  • Treg cells have been developed using gene editing (e.g. Boardman et al. 2022, J Allergy Clin Immunol 149:1-11).
  • One way of increasing the efficacy of Treg cell therapy resides in using Treg cells that are specific for a disease-related antigen (e.g. Boardman et al. 2016, Biochem Soc Trans 44:342-348; Fujio et al. 2006, J Immunol 177: 8140-8147).
  • Autoimmune antigens, or autoantigens are known for e.g. MS (e.g.
  • MBP myelin basic protein
  • PBP proteolipid protein
  • MOG myelin oligodendrocyte glycoprotein
  • GAD65 glutamic acid decarboxylase 65
  • IA-2 insulinoma antigen-2
  • HSP heat shock protein
  • IGRP islet-specific glucose-6-phosphatase catalytic subunit related protein
  • ZnT8 zinc transporter-8
  • PDX1 pancreatic duodenal homeobox factor 1
  • CHGA chromogranin A
  • IAPP islet amyloid polypeptide
  • autoantigens include thyroglobulin, thyroid peroxidase, TSH receptor in thyroid diseases; insulin (proinsulin), glutamic acid decarboxylase (GAD), tyrosine phosphatase IA-2, heat-shock protein HSP65, islet-specific glucose6-phosphatase catalytic subunit related protein (IGRP) in type 1 diabetes; 21-OH hydroxylase in autoimmune adrenalitis; 17-alpha hydroxylase, histidine decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, in autoimmune polyendocrine syndromes; H+/K+ ATPase intrinsic factor in autoimmune gastritis and pernicious anemia; myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), proteolipid protein (PLP) in multiple sclerosis; acetyl-choline receptor in myasthenia gravis; retinol-binding protein (RBP) in autoimmune
  • Tregs constitute about 5% of circulating CD4+ T cells and can be identified by the lineage marker forkhead box protein P3 (FOXP3).
  • Tregs can be isolated from peripheral blood or umbilical cord blood (Brunstein et al. 2011, Blood 117:1061), from discarded paediatric thymuses (Dijke et al. 2016, Am J Transplant 16: 58), or via a GMP procedure from a standard leukapheresis product (Hoffmann et al. 2006, Biol Blood Marrow Transplant 12: 267). The latter procedure entails in a first step depletion of CD19+ cells followed by a second step of enrichment of cells expressing CD25 molecules.
  • CD25 High expression of CD25 is only one of the Treg markers; other surface markers that could be employed in a Treg isolation and/or enrichment procedure include CCR8, CTLA4, CD38, TIGIT, ICOS, OX-40, 4-1BB, and GITR, as well as CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA, CD31, Foxp3, CD45RA, or combinations thereof such as e.g. CD4+CD25+CD127low selection (Piekarska et al. 2021, Folia Histochem Cytobiol 59:75-85; Santegoets et al. 2015, Cancer Immunol Immunother 64: 1271-1286).
  • Non-Treg contaminants can be removed by using e.g. CD49b antibodies optionally in combination with CD127 antibodies; as the Tregs do not expose CD49b or CD127, they remain untouched (Kleinewietfeld et al. 2009, Blood 113:827-836; Haase et al. 2015, J Immunother 38:250-258).
  • a further source of Tregs consists of conventional CD4+ T-cells that are reprogrammed into Tregs such as including inducing strong FOXP3 expression (also termed induced Tregs; e.g. Kanamori et al. 2016, Trends Immunol 37: 803-11; Honaker et al. 2020, Sci Transl Med 12:eaay6422).
  • Tregs can by polyclonal Tregs (such as expanded ex vivo using anti-CD3/CD28-coated beads in the presence of high dose of IL-2), in vitro amplified/expanded Tregs (such as in protocols using drugs like rapamycin or all-trans retinoic acid (ATRA); Golovina et al. 2011, PLoS One 6: el5868; Scotta et al. 2013, Haematologica 98: 1291), antigen-specific Tregs (amplified in the presence of antigen-presenting cells; or activated (by CD40 ligand) allogeneic B cells (in the presence of IL-2), e.g. Putnam et al.
  • polyclonal Tregs such as expanded ex vivo using anti-CD3/CD28-coated beads in the presence of high dose of IL-2
  • in vitro amplified/expanded Tregs such as in protocols using drugs like rapamycin or all-trans retinoic
  • CAR chimeric antigen receptor
  • BAR B-cell-targeting antibody receptor
  • CCR chimeric cytokine receptor
  • Modular CARs incorporate interchangeable antigen-targeting moieties (Koristka et al. 2018, J Autoimmun 90:116-131).
  • Tregs Gene transfer into Tregs is, similar as into conventional T cells, possible via lentiviruses or retroviruses or by using transposase-based systems (Zhou et al.2015, J Immunol 195:2493-2501; Ivies et al. 1997, Cell 91:501-510).
  • Genome editing with Zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) of Tregs has become possible (see e.g. Boardman et al. 2022, J Allergy Clin Immunol 149:1-11); rapid and efficient Crispr-Cas based editing procedures have been added to the repertoire (e.g. Van Zeebroeck et al.
  • transgenes e.g. CAR-encoding genes
  • transposon/transposase systems e.g. the sleeping beauty system
  • Tregs Delivery of the gene transfer or genome editing components into Tregs is possible via electroporation, delivery in nanoparticles, or via viruses (including adeno-associated viruses, AAVs). Genome editing may also be applied to open the Treg adoptive transfer field to allogeneic Treg transfer: indeed, removal of the endogenous TCR limits stimulation of TCR-muted Tregs by allo-recognition (Boardman et al. 2022, J Allergy Clin Immunol 149:1-11; Boardman et al. 2016, Curr Transplant Rep 3:275-283).
  • Treg allogeneic HLA molecules can be replaced with nonclassical HLA molecules or Tregs can be modified to overexpress molecules such as Siglec ligands or CD47 to reduce NK cell-mediated lysis (Boardman et al. 2022, J Allergy Clin Immunol 149:1-11).
  • a series of Treg modifications is included in Table 1 of Amini et al. 2021 (Front Immunol 11 :611638).
  • Knockdown of gene expression in Tregs by means of shRNA e.g. Li et al. 2021, Front Cell Dev Biol 9:708562
  • siRNA e.g. Zhang et al.
  • Tregs In vitro or ex vivo expansion of isolated and/or enriched Tregs is feasible, and often required to get hold of an amount of Tregs sufficient for enabling adoptive transfer. These hurdles can at present already be overcome as witnessed by the expanding number of clinical trials relying on adoptive transfer of Tregs. Kits for isolation and expansion of human Tregs are commercially available (e.g. Miltenyi Biotec: "CD4 + CD25 + CD45RA + Regulatory T Cell Isolation Kit, human", “Treg Expansion Kit, human”). Furthermore, the Treg field in general and the field of Treg expansion in particular is in full development and reported advances include for instance ex vivo procedures assisted by co-culture with multipotent adult progenitor cells (Reading et al.
  • Treg expansion protocols may need to take into account looking for proper CD39/CD73 coexpression in the expanded Tregs (Jarvis et al. 2021, Communications Biology 4:1186). All above features of Treg isolation and/or expansion, Treg engineering, adoptive Treg cell transfer, etc., are frequently subject of review papers, e.g. Ferreira et al. 2019, Nat Rev Drug Discov 18:749-769.
  • Tregs can be further be coated with e.g. a nanoparticle "backpack” loaded with interleukin-2 (IL-2) to improve initial engraftment after adoptive transfer (Marshall et al. 2023, J Biomed Mater Res 111:185- 197; these authors designed loadable nanoparticles of acceptable sizes and electrostatic properties allowing for conjugation to the surface of viable Tregs).
  • IL-2 interleukin-2
  • the isolated regulatory T cell(s) (Treg cell(s), or simply Treg(s)) or population of isolated Treg cells characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited, in the Treg cell or cells, are more in particular any of polyclonal Treg cells, in vitro amplified or expanded Tregs, antigen-specific Tregs, engineered T cell receptor (TCR)-Tregs, engineered chimeric antigen receptor (CAR)-Tregs, monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs, B-cell- targeting antibody receptor (BAR) Tregs, design Tregs, or chimeric cytokine receptor (CCR) Tregs.
  • Treg cell(s) or simply Treg(s)
  • the invention relates to isolated TCR-engineered Treg-cells, CAR-engineered Treg- cells (monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs), BAR-Treg cells, design Tregs, or chimeric cytokine receptor (CCR) Tregs characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited in this/these Treg cell or cells.
  • CCR chimeric cytokine receptor
  • compositions comprising any of the above described isolated Treg cells/Tregs or populations of isolated Treg cells/Tregs in which the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited.
  • such compositions are pharmacological compositions or pharmacologically acceptable compositions.
  • the invention relates to Treg cell therapy, one goal of it being to induce or re-establish immune tolerance.
  • the invention therefore relates to any of the above described isolated Tregs in which the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited, or to any composition comprising such Tregs, for use as a medicament, or for use in the manufacture of a medicament.
  • the medicament is a medicament for/for use in/or for use in a method of treating or suppressing (progression of) an auto-immune disease; for/for use in/or for use in a method of treating or suppressing (progression of) an inflammatory disease (such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease); is for/for use in/or for use in a method of treating or suppressing (progression of) an uncontrolled infection (at least those type of infections in which normalization of Tregs can be of help in treating or suppressing it); for/for use in/or for use in a method of treating or suppressing (progression of) transplant, graft or allograft rejection; or is for/for use in/or for use in a method of treating or inhibiting (progression of) cardiovascular disease (CVD).
  • an inflammatory disease such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease
  • an uncontrolled infection at least those type of infections in which normalization of Tregs can be of help in treating
  • Such medicament may be administered (in a therapeutically effective amount) to a subject being or having been on a diet containing high salt (although not a prerequisite, as high salt conditions in inflamed tissue can occur independent of dietary salt, see above).
  • isolated Treg(s) or population of isolated Tregs or composition comprising such Tregs are for use in Treg cell therapy, in particular for use in adoptive (Treg) cell transfer (ACT) therapy.
  • Tregs are autologous Treg cells (i.e. Tregs obtained from the subject to be treated, and then optionally further expanded), allogeneic Treg cells (i.e.
  • induced Treg cells such as induced starting from autologous or allogeneic CD4+ T-cells.
  • methods for (or for use in) treating or suppressing (progression of) an auto-immune disease in a subject methods for (or for use in) treating or suppressing (progression of) an inflammatory disease in a subject (such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease); methods for (or for use in) treating or suppressing (progression of) an uncontrolled infection (at least those type of infections in which normalization of Tregs can be of help in treating or suppressing it) in a subject; methods for (or for use in) for treating or suppressing (progression of) transplant, graft or allograft rejection in a subject; and methods for (or for use in) for treating or suppress
  • the auto-immune disease, inflammatory disease, or infection is treated or (its progression) suppressed; or the transplant, graft or allograft rejection is treated or (its progression) suppressed; or the CVD is treated or (its progression) inhibited.
  • Tregs may be any of polyclonal Treg cells, in vitro amplified or expanded Tregs, antigen-specific Tregs, engineered T cell receptor (TCR)-Tregs, engineered chimeric antigen receptor (CAR)-Tregs, monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs, B-cell-targeting antibody receptor (BAR) Tregs, design Tregs, or chimeric cytokine receptor (CCR) Tregs.
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the step of inhibiting or substantially inhibiting function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in Tregs is performed by administering an inhibitor of function or expression of NCLX selectively or specifically to Tregs of a subject in need thereof.
  • Treg selectivity or specificity can be approximated or obtained by targeting the NCLX inhibitor to Tregs e.g. by targeting the NCLX inhibitor to a surface marker of Tregs.
  • Such surface marker can be any of the markers relied on in procedures for isolation and/or enrichment of Tregs (see further).
  • selectivity or specificity for mitochondria can optionally be added to the Treg selectivity or specificity.
  • Dual cell/mitochondria targeting (specificity or selectivity) has been established (reviewed by e.g. Liew et al . 2021, Angew Chem Int Ed Engl 60:2232-2256).
  • the invention therefore relates to Treg-selective or -specific inhibitors or function or expression of NCLX, optionally to Treg mitochondria-selective or -specific inhibitors of function or expression of NCLX.
  • Such inhibitors are in particular for use as a medicament, or for use in the manufacture of a medicament.
  • Treg-selective inhibitors or function or expression of NCLX is for/for use in/or for use in a method of treating or suppressing (progression of) an auto-immune disease; for/for use in/or for use in a method of treating or suppressing (progression of) an inflammatory disease (such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease); for/for use in/or for use in a method of treating or suppressing (progression of) an uncontrolled infection (at least those type of infections in which normalization of Tregs can be of help in treating or suppressing it); for/for use in/or for use in a method of, treating or suppressing (progression of) transplant, graft or allograft rejection; or for/for use in/or for use in a method of treating or inhibiting (progression of) cardiovascular disease.
  • an inflammatory disease such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease
  • an uncontrolled infection at least those type of infections in which normalization of Tre
  • compositions comprising a Treg-selective or -specific inhibitor or function or expression of NCLX, optionally to a Treg mitochondria-selective or -specific inhibitor of function or expression of NCLX.
  • Such compositions are in particular for use as medicament or for use in the manufacture of a medicament.
  • composition or medicament is for/for use in/or for use in a method of treating or suppressing (progression of) an auto-immune disease; for/for use in/or for use in a method of treating or suppressing (progression of) an inflammatory disease (such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease); for/for use in/or for use in a method of treating or suppressing (progression of) an uncontrolled infection (at least those type of infections in which normalization of Tregs can be of help in treating or suppressing it); for/for use in/or for use in a method of treating or suppressing (progression of) transplant, graft or allograft rejection; or for/for use in/or for use in a method of treating or inhibiting (progression of) cardiovascular disease.
  • an inflammatory disease such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease
  • an uncontrolled infection at least those type of infections in which normalization of Tregs can be of help in treating or suppressing it
  • any such methods of treating, suppressing or inhibiting include a step of administering to the subject a therapeutically effective amount of the composition or medicament comprising a Treg-selective inhibitor of function or expression of NCLX.
  • the auto-immune or inflammatory disease, or infection is treated or (its progression) suppressed; the transplant, graft or allograft rejection is treated or (its progression) suppressed; or the cardiovascular disease is treated or (its progression) inhibited.
  • a further aspect of the invention refers to methods of inhibiting function or expression of NCLX in Tregs, such methods comprising selectively or specifically targeting Tregs (optionally selectively or specifically targeting Treg mitochondria) with an inhibitor of function or expression of NCLX.
  • the inhibitor of function of NCLX is a pharmacological inhibitor (small molecule)
  • such methods can comprise selectively targeting mitochondria in Tregs with the inhibitor of function of NCLX.
  • autoimmune diseases include multiple sclerosis, type 1 diabetes, autoimmune thyroid diseases, autoimmune adrenalitis, autoimmune polyendocrine syndromes, autoimmune gastritis, pernicious anemia, myasthenia gravis, autoimmune ocular syndromes, autoimmune inner ear diseases, celiac disease, inflammatory bowel disease, and Parkinson's disease, autoimmune encephalitis, lupus, autoimmune hepatitis, etc.
  • Inflammatory diseases include ankylosing spondylitis, gout, arthritis/psoriatic arthritis/rheumatoid arthritis, scleroderma, vasculitis, Kawasaki disease, mixed connective tissue disease, myositis, Sjogren's syndrome, spondyloarthritis, spondyloarthropathy, idiopathic arthritis, uveitis. Inflammation is moreover an important factor in the development of cardiovascular diseases (CVDs) and Treg therapy has been suggested as modality for targeting CVDs (e.g. Meng et al. 2016, Nat Rev Cardiol 13:167-179).
  • CVDs cardiovascular diseases
  • Treg therapy has been suggested as modality for targeting CVDs (e.g. Meng et al. 2016, Nat Rev Cardiol 13:167-179).
  • transplant as referred to herein is embracing organ transplants or allografts (e.g. liver, kidney, heart, lung) as well as cellular transplants (e.g. bone marrow cells, (hematopoietic or embryonic or adult) stem cells, Langerhans cells or pancreatic islets, etc.).
  • organ transplants or allografts e.g. liver, kidney, heart, lung
  • cellular transplants e.g. bone marrow cells, (hematopoietic or embryonic or adult) stem cells, Langerhans cells or pancreatic islets, etc.
  • Improper immunosuppression in a subject receiving the transplant is a cause underlying hyperacute, acute or chronic transplant rejection, or underlying graft vs host disease (GvHD).
  • Skin, cornea, vascular and bone grafts are usually of autologous origin and normally do not cause rejection reactions - if, however, of allogeneic origin, the current invention could be applied in suppressing such allograf
  • antagonist or "inhibitor” of a target as used interchangeable herein refers to inhibitors of function or to inhibitors of expression of a target of interest.
  • Interchangeable alternatives for “antagonist” include inhibitor, repressor, suppressor, inactivator, and blocker.
  • An “antagonist” thus refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with target expression, activation, function, or activity.
  • Biopharmaceutical and genetic antagonists include such entities as antisense oligonucleotides, gapmers, siRNA, shRNA, zinc- finger nucleases, meganucleases, TAL effector nucleases, CRISPR-Cas effectors, etc. (general description of these compounds included hereinafter).
  • Inactivation or inhibition of a process as envisaged in the current invention refers to different possible levels of inactivation or inhibition, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even 100% or more if inactivation or inhibition (compared to a normal situation or compared to the situation prior to starting the inactivation or inhibition).
  • the nature of the inactivating/inhibitory compound is not vital/essential to the invention as long as the process envisaged is inactivated/inhibited such as to treat or inhibit (progression of) auto-immune diseases or inflammatory diseases etc. as described herein.
  • agents include entities such as antisense oligonucleotides, gapmers, siRNA, shRNA, zinc-finger nucleases, meganucleases, Argonaute, TAL effector nucleases, CRISPR-Cas effectors, and nucleic acid aptamers.
  • any of these agents is specifically, selectively, or exclusively acting on or antagonizing the target of interest; or any of these agents is designed for specifically, selectively, or exclusively acting on or antagonizing the target of interest.
  • the target of interest in particular is NCLX.
  • ASO antisense oligonucleotides
  • An antisense oligonucleotide (ASO) is a short strand of nucleotides and/or nucleotide analogues that hybridizes with the complementary mRNA in a sequence-specific or -selective manner. Formation of the ASO-mRNA complex ultimately results in downregulation of target protein expression (Chan et al. 2006, Clin Exp Pharmacol Physiol 33:533-540; this reference also describes some of the software available for assisting in design of ASOs).
  • Modifications to ASOs can be introduced at one or more levels: phosphate linkage modification (e.g. introduction of one or more of phosphodiester, phosphoramidate or phosphorothioate bonds), sugar modification (e.g. introduction of one or more of LNA (locked nucleic acids), 2'-O-methyl, 2'-O-methoxy-ethyl, 2' -fluoro, S-constrained ethyl or tricyclo-DNA and/or non-ribose modifications (e.g. introduction of one or more of phosphorodiamidate morpholinos or peptide nucleic acids).
  • phosphate linkage modification e.g. introduction of one or more of phosphodiester, phosphoramidate or phosphorothioate bonds
  • sugar modification e.g. introduction of one or more of LNA (locked nucleic acids)
  • 2'-O-methyl, 2'-O-methoxy-ethyl, 2' -fluoro S-
  • a gapmer antisense oligonucleotide consists of a central DNA region (usually a minimum of 7 or 8 nucleotides) with (usually 2 or 3) 2'-modified nucleosides flanking both ends of the central DNA region.
  • RNAseH RNAseH
  • Antidote strategies are available as demonstrated by administration of an oligonucleotide fully complementary to the antisense oligonucleotide (Crosby et al. 2015, Nucleic Acid Ther 25:297-305). Uptake of oligonucleotides by cells can be spontaneous or be assisted by e.g. transfection etc..
  • RNA interference Another process to modulate expression of a gene/target gene of interest is based on the natural process of RNA interference. It relies on double-stranded RNA (dsRNA) that is cut by an enzyme called Dicer, resulting in double stranded small interfering RNA (siRNA) molecules which are 20-25 nucleotides long. siRNA then binds to the cellular RNA-lnduced Silencing Complex (RISC) separating the two strands into the passenger and guide strand. While the passenger strand is degraded, RISC is cleaving mRNA specifically or selectively at a site instructed by the guide strand. Destruction of the mRNA prevents production of the protein of interest and the gene is 'silenced'.
  • dsRNA double-stranded RNA
  • siRNA double stranded small interfering RNA
  • siRNAs are dsRNAs with 2 nt 3' end overhangs whereas shRNAs are dsRNAs that contains a loop structure that is processed to siRNA.
  • shRNAs are introduced into the nuclei of target cells using a vector (e.g. bacterial or viral) that optionally can stably integrate into the genome.
  • a vector e.g. bacterial or viral
  • manufacturers of RNAi products provide guidelines for designing siRNA/shRNA.
  • siRNA sequences between 19-29 nt are generally the most effective. Sequences longer than 30 nt can result in nonspecific silencing. Ideal sites to target include AA dinucleotides and the 19 nt 3' of them in the target mRNA sequence.
  • siRNAs with 3' dUdU or dTdT dinucleotide overhangs are more effective. Other dinucleotide overhangs could maintain activity but GG overhangs should be avoided. Also to be avoided are siRNA designs with a 4-6 poly(T) tract (acting as a termination signal for RNA pol III), and the G/C content is advised to be between 35-55%.
  • shRNAs should comprise sense and antisense sequences (advised to each be 19-21 nt in length) separated by loop structure, and a 3' AAAA overhang. Effective loop structures are suggested to be 3-9 nt in length.
  • shRNAs are usually transcribed from vectors, e.g. driven by the Pol III U6 promoter or Hl promoter.
  • Vectors allow for inducible shRNA expression, e.g. relying on the Tet-on and Tet-off inducible systems commercially available, or on a modified U6 promoter that is induced by the insect hormone ecdysone.
  • a Cre-Lox recombination system has been used to achieve controlled expression in mice.
  • Synthetic shRNAs can be chemically modified to affect their activity and stability.
  • Plasmid DNA or dsRNA can be delivered to a cell by means of transfection (lipid transfection, cationic polymer-based nanoparticles, lipid or cellpenetrating peptide conjugation) or electroporation.
  • Vectors include viral vectors such as lentiviral, retroviral, adenoviral and adeno-associated viral vectors.
  • Ribozymes are another type of molecules that can be used to modulate expression of a gene/target gene of interest. They are RNA molecules capable of catalyzing specific biochemical reactions, in the current context capable of targeted cleavage of nucleotide sequences, in particular targeted cleavage of a RNA/RNA target of interest. Examples of ribozymes include the hammerhead ribozyme, the Varkud Satellite ribozyme, Leadzyme and the hairpin ribozyme.
  • RNA knock-out can be a gene knockdown or the gene can be knocked out, knocked down, disrupted or modified by a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques such as described hereafter, including, but not limited to, retroviral gene transfer.
  • a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques such as described hereafter, including, but not limited to, retroviral gene transfer.
  • One way in which genes can be knocked out, knocked down, disrupted or modified is by the use of zinc finger nucleases.
  • Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain.
  • Zinc finger domains can be engineered to target a desired DNA sequence/DNA sequence of interest, which enable zinc-finger nucleases to target unique sequence within a complex genome.
  • these reagents can be used to precisely alter the genomes of higher organisms.
  • a TALEN® is composed of a TALE DNA binding domain for sequence-specific or sequence-selective recognition fused to the catalytic domain of an endonuclease that introduces double strand breaks (DSB).
  • DSB double strand breaks
  • the DNA binding domain of a TALEN® is capable of targeting with high precision a large recognition site (for instance 17bp).
  • Meganucleases are sequence-specific or sequence-selective endonucleases, naturally occurring "DNA scissors", originating from a variety of single-celled organisms such as bacteria, yeast, algae and some plant organelles. Meganucleases have long recognition sites of between 12 and 30 base pairs. The recognition site of natural meganucleases can be modified in order to target native genomic DNA sequences (such as endogenous genes) or DNA sequences of interest. Another recent genome editing technology is the CRISPR/Cas system, which can be used to achieve RNA- guided genome engineering (including knock-out, knock-down or disruption of a gene of interest).
  • CRISPR interference is a genetic technique which allows for sequence-specific or sequence-selective control of expression of a gene of interest in prokaryotic and eukaryotic cells. It is based on the bacterial immune system-derived CRISPR (clustered regularly interspaced palindromic repeats) pathway. Recently, it was demonstrated that the CRISPR-Cas editing system can also be used to target RNA. It has been shown that the Class 2 type Vl-A CRISPR-Cas effector C2c2 (Casl3a; CRISPR-Casl3a or CRISPR-C2c2) can be programmed to cleave single stranded RNA targets carrying complementary protospacers (Abudayyeh et al. 2016 Science353/science.aaf5573). C2c2 is a single-effector endoRNase mediating ssRNA cleavage once it has been guided by a single crRNA guide toward a target RNA/RNA of interest.
  • Methods for administering nucleic acid-based therapeutic modalities/agents include methods applying non-viral (DNA or RNA) or viral nucleic acids (DNA or RNA viral vectors).
  • Methods for non-viral nucleic acid administration include the injection of naked DNA (circular or linear), electroporation, the gene gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes (e.g. complexes of nucleic acid with DOTAP or DOPE or combinations thereof, complexes with other cationic lipids), dendrimers, viral- like particles, inorganic nanoparticles, hydrodynamic delivery, photochemical internalization (Berg et al. 2010, Methods Mol Biol 635:133-145) or combinations thereof.
  • adenovirus or adeno-associated virus vectors retrovirus vectors , naked or plasmid DNA, and lentivirus vectors. Combinations are also possible, e.g. naked or plasmid DNA combined with adenovirus, or RNA combined with naked or plasmid DNA to list just a few.
  • Other viruses e.g. alphaviruses, vaccinia viruses such as vaccinia virus Ankara
  • alphaviruses, vaccinia viruses such as vaccinia virus Ankara
  • nucleic acid e.g. in liposomes (lipoplexes) or polymersomes (synthetic variants of liposomes), as polyplexes (nucleic acid complexed with polymers), carried on dendrimers, in inorganic (nano)particles (e.g. containing iron oxide in case of magnetofection), or combined with a cell penetrating peptide (CPP) to increase cellular uptake.
  • Organ- or cellular-targeting strategies may also be applied to the nucleic acid (nucleic acid combined with organ- or cell-targeting moiety); these include passive targeting (mostly achieved by adapted formulation) or active targeting (e.g.
  • nucleic acid-comprising nanoparticle by coupling a nucleic acid-comprising nanoparticle with any compound (e.g. an aptamer or antibody or antigen binding molecule) binding to a target organ- or cell-specific antigen) (e.g. Steichen et al. 2013, Eur J Pharm Sci 48:416-427).
  • any compound e.g. an aptamer or antibody or antigen binding molecule binding to a target organ- or cell-specific antigen
  • CPPs enable translocation of their payload of interest across the plasma membrane.
  • CPPs are alternatively termed Protein Transduction Domains (TPDs), usually comprise 30 or less (e.g. 5 to 30, or 5 to 20) amino acids, and usually are rich in basic residues, and are derived from naturally occurring CPPs (usually longer than 20 amino acids), or are the result of modelling or design.
  • TPDs Protein Transduction Domains
  • CPPs include the TAT peptide (derived from HIV-1 Tat protein), penetratin (derived from Drosophila Antennapedia - Antp), pVEC (derived from murine vascular endothelial cadherin), signal-sequence based peptides or membrane translocating sequences, model amphipathic peptide (MAP), transportan, MPG, polyarginines; more information on these peptides can be found in Torchilin 2008 (Adv Drug Deliv Rev 60:548-558) and references cited therein.
  • CPPs can be coupled to carriers such as nanoparticles, liposomes, micelles, or generally any hydrophobic particle.
  • Coupling can be by absorption or chemical bonding, such as via a spacer between the CPP and the carrier.
  • an antibody binding to a target-specific antigen can further be coupled to the carrier (Torchilin 2008, Adv Drug Deliv Rev 60:548-558).
  • CPPs have already been used to deliver payloads as diverse as plasmid DNA, oligonucleotides, siRNA, peptide nucleic acids (PNA), proteins and peptides, small molecules and nanoparticles inside the cell (Stalmans et al. 2013, PloS One 8:e71752).
  • any other modification of the DNA or RNA to enhance efficacy of nucleic acid therapy is likewise envisaged to be useful in the context of the applications as outlined herein.
  • the enhanced efficacy can reside in enhanced expression, enhanced delivery properties, enhanced stability and the like.
  • a specific or selective inhibitor of a target of interest may exert the desired level of inhibition of the target of interest with an IC50 of 1000 nM or less, with an IC50 of 500 nM or less, with an IC50 of 100 nM or less, with an IC50 of 50 nM or less, with an IC50 of 10 nM or less, with an IC50 of 1 nM or less, with an IC50 between 1 pM and InM, or with an IC50 between 0.1 pM and 10 nM.
  • Cross-inhibition of more than one target is possible; for clinical development it can e.g. be desired to be able to test an inhibitor in a suitable in vitro model or in vivo animal model before starting clinical testing with the same inhibitor in a human population, which may require the inhibitor to cross-inhibit the animal (or other non-human) target and the orthologous human target.
  • Specificity or selectivity of inhibition refers to the situation in which an inhibitor is, at a certain concentration (sufficient to inhibit the target of interest) inhibiting the target protein with higher efficacy (e.g. with an at least 2-fold, 5-fold, or 10-fold lower IC50, e.g. at least 20-, 50- or 100-fold or more lower IC50) than the efficacy with which it is possibly (if at all) inhibiting other targets (targets not of interest).
  • Such specificity or selectivity of inhibition is in particular determined within the setting of the target subject (e.g. human patient, or animal model) and thus can encompass/does not exclude inhibition of (at least one) orthologous target.
  • Exclusivity of inhibition refers to the situation in which an inhibitor is inhibiting only the target of interest.
  • Specificity or selectivity of cell targeting refers to the situation in which a composition, at a certain concentration, is interacting with the intended target cell (such as binding to, or such as causing inhibition of function or expression of NCLX in the intended target cell) with higher efficacy (e.g. with an at least 2-fold, 5-fold, or 10-fold higher efficacy, or e.g. with at least 20-, 50- or 100- fold higher efficacy) than the efficacy with which the composition is interacting with other cells (not intended as target cell).
  • Exclusivity of cell targeting refers to the situation in which a composition is interacting only with the intended target cell.
  • therapeutic modality therapeutic agent, agent, and drug are used interchangeably herein, and likewise relate to the Tregs as described herein (with inhibited function or expression of NCLX). All refer to a therapeutically active compound, or to a therapeutically active composition (comprising one or more therapeutically active compounds).
  • Treatment refers to any rate of reduction, delaying or retardation of the progress of the disease or disorder, or a single symptom thereof, compared to the progress or expected progress of the disease or disorder, or single symptom thereof, when left untreated. This implies that a therapeutic modality on its own may not result in a complete or partial response (or may even not result in any response), but may, in particular when combined with other therapeutic modalities (such as other immunosuppressants or therapeutic modalities other than the Tregs of the invention for treating or suppressing autoimmune or inflammatory diseases or transplant, graft or allograft rejection), contribute to a complete or partial.
  • other therapeutic modalities such as other immunosuppressants or therapeutic modalities other than the Tregs of the invention for treating or suppressing autoimmune or inflammatory diseases or transplant, graft or allograft rejection
  • the treatment results in no/zero progress of the disease or disorder, or single symptom thereof (i.e. "inhibition” or “inhibition of progression”), or even in any rate of regression of the already developed disease or disorder, or single symptom thereof.
  • “Suppression/suppressing” can in this context be used as alternative for “treatment/treating”.
  • Treatment/treating also refers to achieving a significant amelioration of one or more clinical symptoms associated with a disease or disorder, or of any single symptom thereof. Depending on the situation, the significant amelioration may be scored quantitatively or qualitatively. Qualitative criteria may e.g. by patient well-being.
  • the significant amelioration is typically a 10% or more, a 20% or more, a 25% or more, a 30% or more, a 40% or more, a 50% or more, a 60% or more, a 70% or more, a 75% or more, a 80% or more, a 95% or more, or a 100% improvement over the situation prior to treatment.
  • the time-frame over which the improvement is evaluated will depend on the type of criteria/disease observed and can be determined by the person skilled in the art.
  • a “therapeutically effective amount” refers to an amount of a therapeutic agent to treat, inhibit or prevent a disease or disorder in a subject (such as a mammal). Efficacy in vivo can, e.g., be measured by assessing the duration of survival (e.g. overall survival), time to disease progression (TTP), response rates (e.g., complete response and partial response, stable disease), length of progression-free survival (PFS), duration of response, and/or quality of life.
  • duration of survival e.g. overall survival
  • TTP time to disease progression
  • response rates e.g., complete response and partial response, stable disease
  • PFS length of progression-free survival
  • the term "effective amount” or “therapeutically effective amount” may depend on the dosing regimen of the agent/therapeutic agent or composition comprising the agent/therapeutic agent (e.g. medicament or pharmaceutical composition).
  • the effective amount will generally depend on and/or will need adjustment to the mode of contacting or administration.
  • the effective amount of the agent or composition comprising the agent is the amount required to obtain the desired clinical outcome or therapeutic effect without causing significant or unnecessary toxic effects (often expressed as maximum tolerable dose, MTD).
  • MTD maximum tolerable dose
  • the agent or composition comprising the agent may be administered as a single dose or in multiple doses.
  • the effective amount may further vary depending on the severity of the condition that needs to be treated; this may depend on the overall health and physical condition of the subject or patient and usually the treating doctor's or physician's assessment will be required to establish what is the effective amount.
  • the effective amount may further be obtained by a combination of different types of contacting or administration.
  • the aspects and embodiments described above in general may comprise the administration of one or more therapeutic compounds to a subject (such as a mammal) in need thereof or in need of treatment.
  • a (therapeutically) effective amount of (a) therapeutic compound(s) is administered to the mammal in need thereof in order to obtain the described clinical response(s).
  • administering means any mode of contacting that results in interaction between an agent or composition comprising the agent (such as a medicament or pharmaceutical composition) and an object (e.g. cell, tissue, organ, body lumen) with which said agent or composition is contacted.
  • agent or composition comprising the agent
  • object e.g. cell, tissue, organ, body lumen
  • the interaction between the agent or composition and the object can occur starting immediately or nearly immediately with the administration of the agent or composition, can occur over an extended time period (starting immediately or nearly immediately with the administration of the agent or composition), or can be delayed relative to the time of administration of the agent or composition. More specifically the "contacting" results in delivering an effective amount of the agent or composition comprising the agent to the object.
  • the invention relates to methods of producing the isolated Tregs in which the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited, such methods comprising a step of isolating Treg-cells from peripheral blood, umbilical cord blood, thymus or leukapheresis product obtained from a subject, or a step of reprogramming conventional CD4+ T-cells obtained from a subject into Tregs (or, in other words, a step of producing induced Tregs) (cfr. supra). Such methods may further comprise a step of ex-vivo expanding the isolated or reprogrammed Tregs (cfr. supra).
  • such methods may further comprise a step of ex-vivo manipulation to inhibit, or to substantially inhibit, the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in the Tregs by means of pharmacological inhibition or by means of a DNA nuclease specifically knocking out or disrupting NCLX, an RNase specifically targeting NCLX, or an inhibitory oligonucleotide specifically targeting NCLX (cfr. supra).
  • kits comprising at least one vial comprising at least one of the isolated Treg cell or population of Treg cells, or comprising a composition comprising such Tregs, wherein the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in said Tregs is inhibited, or substantially inhibited.
  • said Tregs are of allogeneic origin (independent of having been isolated, enriched and/or expanded from natural Tregs or from induced Tregs).
  • kits comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising a Treg cell according to the invention or comprising a composition comprising a Treg cell according to the invention.
  • Other optional components of such kit include use instructions; one or more containers with sterile pharmaceutically acceptable carriers, excipients or diluents [such as for producing or formulating a (pharmaceutically acceptable) composition of the invention]; one or more syringes; one or more needles; etc.
  • kits may be pharmaceutical kits.
  • a "subject" in general is a mammalian species.
  • the mammalian species in general is a higher species including primates, cattle (e.g. cows, sheep, goats, pigs), horses, and pets (e.g. dogs, cats).
  • the subject is a human subject.
  • CD4 + T cells were isolated by incubating whole blood with RosetteSepTM Human CD4+ T Cell Enrichment Cocktail (15062, Stemcell) followed by Ficoll-Paque PLUS (GE17-1440-02, Sigma) gradient centrifugation or by using EasySepTM Human CD4 + T Cell Isolation Kit (17952, StemCell).
  • CD25- enriched and -depleted CD4 + cells were isolated with CD25 microbeads II (130-097-044, Miltenyi Biotec).
  • Tregs Regulatory T cells
  • FACS Aria II BD Biosciences
  • CD4 APC-Cy7 clone RPA-T4
  • CD25 PeCy7 clone M-A251
  • CD127 PerCP-Cy5.5 clone A019D5
  • T effector (Teff) cells were isolated by untouched CD4 + T cell isolation (17952, Stemcell) or were FACS sorted for CD25-CD127 + cells.
  • Tregs were isolated according to the following parameters: CD4 + CD25 hl CD127 low to a FOXP3 purity >96%.
  • T effector cells were labelled with CellTrace CFSE (C34554, ThermoFisher Scientific) at lpM and cultured with fresh or pre-treated Tregs (as indicated in Figure legends) in 96-well round-bottom plates in XVIVO15 medium (LONZA) supplemented with 5% Fetal Bovine Serum.
  • Tregs were stained with cell trace violet (CTV) (C34557, ThermoFisher Scientific) at a final concentration of 2.5pM.
  • CTV cell trace violet
  • Cells were stimulated with Treg Inspector beads (aCD2/aCD3/aCD28-coated beads) at 1 bead/cell ratio (130-092-909, Miltenyi Biotec) for 4 to 5 days before analyzed by flow cytometry.
  • Treg Inspector beads aCD2/aCD3/aCD28-coated beads
  • 1 bead/cell ratio 130-092-909, Miltenyi Biotec
  • Tregs (5xl0 4 cell/well) were stimulated in XVIVO15 medium supplemented with 5% FBS for a period of 6-, 12-, 24-, 72 hours or 4 days in the presence of lpg/mL of plate-bound aCD3 (clone UCHT1), 1 or 5pg/mL of soluble aCD28 (clone 28.2) and 25U/mL of IL-2 (11147528001, Sigma) in the presence or absence of +40mM NaCI. Where indicated, Tregs were also incubated in the presence of lOpM antimycin A (AA) (A8674, Sigma Aldrich) or lOpM of CGP-37157 (220005, EMD Millipore).
  • AA lOpM antimycin A
  • CGP-37157 220005, EMD Millipore
  • CD14 + monocytes were magnetically bead-isolated from PBMCs (17858, StemCell Technologies) and cultured with 50U/ml IL-4 (11340045, Immunotools) and 50ng/ml GM-CSF (300-03, Peprotech) in X-VIVO15 supplemented with 10% FBS. After 5 days of incubation, DCs were harvested and stored in liquid nitrogen for later use.
  • Treg suppression assays in the presence of DCs were performed as before (Arroyo-Hornero et al. 2022, Allergy 77:2818-2821) . Briefly, CD4 + CD25‘ Tconvs (referred as Teff) were stained with lpM Cell Trace CFSE Cell Proliferation Kit (C34554, ThermoFisher Scientific) and cultured with autologous Tregs in 96- well U-bottom plates in X-VIVO15 supplemented with 5% FBS. Cells were stimulated for 4 days using allogenic moDCs in the presence of 0.5ug/mL of soluble aCD3 (555329, BD Bioscience).
  • MLR Mixed lymphocyte reaction
  • MLR was performed as described before (Kleinewietfeld et al. 2009, Blood 113:827-836). Briefly, human Treg cells were preincubated under CTRL or HS (+40mM NaCI) conditions with plate bound aCD3, soluble aCD28 and IL-2 for a period of 24 hours. Autologous PBMCs (100000 cells/well) were stained with CFSE (C34554, ThermoFisher Scientific) and later stimulated with irradiated (3000 rad) allogeneic PBMCs (100000 cells/well) in RPMI + 10% FCS for 5 days. For suppression of proliferation, pre-incubated autologous Treg cells were added at the indicated ratio.
  • Treg cell suppression assays For murine Treg cell suppression assays, spleens were harvested from 15-week-old C57BL/6J mice.
  • CD4 + CD25 + Treg and CD4 + CD25‘ T effector cells (Teff) were isolated using the mouse CD4 + CD25 + Regulatory T Cell Isolation Kit according to the manufacturer's instructions (Miltenyi Biotec #130-091- 041, Bergisch Gladbach, Germany).
  • CD4 + CD25‘ Teff cells were stained with eF450 Proliferation Dye (Thermo Fisher, Schrete, Germany).
  • Cells were then cultured at a 1:2, 1:4, 1:8 and 1:16 ratio with 100.000 CD4 + CD25‘ Teffs in 96-well plates (Sarstedt, Numbrecht, Germany) together with 50.000 Treg expansion beads (130-092-909, Miltenyi Biotec) in RPMI1640 medium (Thermo Fisher) in the presence or absence of +40mM NaCI for 4 days.
  • Cells were analyzed by flow cytometry (FACS) as described before 14 if not specified elsewhere. Duplicates were performed for the different experiments and analysis. Cells were first stained with LIVE/DEAD cell kit (L34972, Invitrogen) or ef780 viability dye (Thermo Fisher) to exclude dead cells. For surface staining, cells were labelled with respective antibodies for 15 minutes in MACS buffer (0.5% BSA, 2mM EDTA) at 4°C.
  • LIVE/DEAD cell kit L34972, Invitrogen
  • ef780 viability dye Thermo Fisher
  • eBioscienceTM Foxp3 / Transcription Factor Staining Buffer Set (00-5523-00, Invitrogen) according to manufacturer's instructions and later labelled with respective intracellular antibodies in Perm buffer for 30 minutes at 4°C, washed and assayed in MACS buffer.
  • cytokine detection cells were stimulated with 50 ng/ml phorboll2-myristatel3-acetate (PMA) (P1585, Sigma) and 250ng/ml lonomycin (10634, Sigma) in the presence of GolgiPlug (555029, BD) for 5 hours. Data was acquired on a BD LSR Fortessa II, BD FACS Calibur or BD FACS Canto II and analyzed with FlowJo software (TreeStar).
  • PMA phorboll2-myristatel3-acetate
  • GolgiPlug 555029, BD
  • Tregs were purified by FACS-sorting and expanded for a period of 6 days on 24 wellplates in the presence of lOpg/mL of plate-bound aCD3, lpg/mL of soluble aCD28 and 300U/mL of IL-2 in XVIVO15 medium supplemented with 5% FBS.
  • XVIVO15 medium supplemented with 5% FBS
  • lOOU/mL of IL-2 were resuspended in XVIVO15 medium supplemented with 5% FBS and lOOU/mL of IL-2 and further incubated in 6 well-plates for a period of 24 hours.
  • cells and RNP mixture was pipetted to a well of a 16-well strip nucleovette (AmaxaTM P3 Primary cell 4D-NucleofectorTM X Kit S V4XP-3032, LONZA) and placed on a 4D- NucleofectorTM (LONZA) using EO115 program. Afterwards, cells were plated in 24-well plates with XVIVO15 medium supplemented with 5% FBS and lOOU/mL IL-2. Western blotting was done as described before (Kleinewietfeld et al.
  • Treg Inspector beads aCD2/aCD3/aCD28-coated beads
  • BD LSR Fortessa II
  • the metabolic profile was evaluated in 24- and 72 hours cultured Tregs stimulated with aCD3, aCD28 and IL-2, in the presence or absence of +40mM NaCI.
  • Real-time measurements of oxygen consumption rate (OCR) were made using an XF-96 Extracellular Flux Analyzer (Agilent). After incubation times, cells were collected, counted and plated in Cell-tak (354240, Corning) coated XF-96 plates (102416-100, Agilent) at the concentration of 2 x 10 5 cells/well. Cells were let to incubate for 60 min at 37°C, without CO2.
  • a mitochondrial stress test designed to repeat 4 cycles (of 3 minutes mixing followed by 3 minutes measuring) per phase was used to measure OCR under basal conditions and in response to 5pM oligomycin (75351, Sigma Aldrich), 1.5pM of carbonylcyanide-4- (trifluoromethoxy) -phenylhydrazone (FCCP) (C2920, Sigma Aldrich) and l .M of antimycin A (A8674, Sigma Aldrich) and Rotenone (R8875, Sigma Aldrich).
  • Tregs were stimulated as described above for a period of 4 hours in control medium or media supplemented with +40mM NaCI, lOpM antimycin A (AA), 5pM or 200pM of Etomoxir. Controls were incubated in the presence of lOOU/mL IL-2 for 30 minutes of stimulation. Cells were fixed with BD CytofixTM Fixation Buffer (554655, BD Biosciences) for 10 minutes at 37°C and permeabilized with Perm Buffer III (BD Biosciences, 58050) for 30 minutes on ice. Cells were stained with anti-pSTAT5 antibody (560311, BD Biosciences) for 30 minutes at 4°C and were acquired on a BD LSR Fortessa II.
  • BD CytofixTM Fixation Buffer 554655, BD Biosciences
  • Perm Buffer III BD Biosciences, 58050
  • Tregs were cultured as mentioned before for a period of 6 hours in the presence or absence of +40mM NaCI. Oligomycin A (5pM) was used as a positive control for ATP decrease.
  • ATPIite Luminescence Assay System 6016941, Perkin Elmer was used for ATP measurements according to the manufacturer's instructions. Briefly, after incubation time, cells were lysed using a cell lysis solution for 5 minutes in constant shaking followed by the addition of a substrate solution. Cells were further incubated for 15 minutes in dark-adapted conditions and luminescence was measured on a FLUOstar OPTIMA reader (BMG Labtech).
  • TMRE-Mitochondrial membrane potential assay kit (abll3852, Abeam) was used according to the manufacturer's instructions. Briefly, Tregs were cultured and activated as described above for a period of 6 hours in the presence or absence of +40mM NaCI and AA (lOpM). FCCP (lOpM) was used as a positive control and added to the cells 10 minutes before the end of incubation period. After incubation, TMRE was added to the cells and incubated for 20 minutes at 37°C. After, cells were washed and acquired by flow cytometry on a BD Calibur (BD Bioscience).
  • MitoTracker Green M7514, ThermoFisher Scientific was used at a final concentration of lOOnM directly into cell culture medium and let to incubate for 60 minutes at 37°C. After, cells were washed in ice-cold PBS (17-516F, LONZA) and further acquired by flow cytometry using a BD LSR Fortessa II.
  • a Complex II Enzyme activity microplate assay kit (abl09908, Abeam) was performed according to the manufacturer's instructions. Briefly, Tregs were isolated by FACS sorting. Protein extraction was carried using a detergent solution and final concentration was adjusted to recommended dilution for plate loading. Samples were loaded to a 96-well microplate coated with antiComplex II monoclonal antibody and let to incubate for 2 hours at RT. After, plate was washed, and an activity solution control buffer or buffer containing +4mM NaCI was added to the corresponded wells.
  • Optical density was measured at OD600nm in an iMark Microplate Reader (BIORAD) under kinetic mode for 60 minutes allowing interval measures of 20 seconds.
  • Complex ll/lll activity was assessed using the MitoTox Complex ll+lll OXPHOS Activity Assay Kit (abl09905, Abeam) according to the manufacturer's instructions.
  • Activity solution was mixed with increasing concentrations of NaCI (serial dilutions from +64mM to +0.0625mM NaCI) or with AA as positive control (serial dilutions from 352nM to 0.3438nM).
  • Bovine heart mitochondria were added and absorbance at 550nm was measured in kinetic mode on a Spectramax 190 plate reader.
  • Complex ll/lll activity was calculated relative to the solvent control (water). In some experiments lOpM of CGP-37157 (220005, EMD Millipore) was added.
  • ROS production was assessed on the mitochondria of Tregs using MitoSOXTM Red Mitochondrial Superoxide Indicator kit (M36008, ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, after Tregs incubation for 6 hours in the presence or absence of +40mM NaCI, cells were washed of medium and MitoSOXTM reagent working solution was added and incubated for 10 minutes at 37°C. After, cells were washed and fluorescence was measured by flow cytometry on a BD LSR Fortessa II.
  • Tregs were cultured as described above for a period of 12 hours in the presence or absence of +40mM NaCI. Metabolites were extracted on both cells and medium fractions and identified using Liquid chromatography-mass spectrometry (LC-MS). For metabolite extraction on cells, medium was removed and cells washed with ice cold 0.9% NaCI solution. After, a cellular extraction buffer (80% methanol, containing 2pM d27 myristic acid) was added to the cells and let to incubate for 2-3 minutes on ice. Cells were later centrifuged and supernatant was used for further metabolite identification. Protein pellets were kept in order to determine protein concentration by BCA assay.
  • LC-MS Liquid chromatography-mass spectrometry
  • 990pL of medium extraction buffer was added to lOpL of medium and stored overnight at -80°C. After incubation, medium samples were centrifuged and supernatants were used for metabolites analysis. Following extraction, the complex mixture of metabolites was separated prior to MS measurement using a Dionex UltiMate 3000 LC System (ThermoFisher Scientific) coupled to a Q Exactive Orbitrap mass spectrometer (ThermoFisher Scientific) operating in negative ion mode. Practically, lOpI of the sample was injected on a C18 column (Aquility UPLC®HSS T3 1.8pm 2.1x100mm).
  • a gradient using solvent A (H2O, lOmM Tributyl-Amine, 15mM acetic acid) and solvent B (100% Methanol) was applied as follows: 0 minutes, 0%B; 2 minutes, 0%B; 7 minutes, 37%B; 14 minutes, 41%B; 26 minutes, 100%B; 30 minutes, 100%B; 31 minutes, 0%B; 40 minutes, 0%B.
  • the flowrate was kept constant at 0.250ml/min and the column was kept at 40°C throughout the analysis.
  • the MS operated in full scan mode (range 70-1050 Th in negative normalized mode) using a spray voltage of 3.2kV, a capillary temperature of 320°C, sheath gas at 50.0, auxiliary gas at 10.0.
  • the AGC target was set at 3e6 using a resolution of 140.000, with a maximum IT of 512ms. Data collection was performed using the Xcalibur software, version 4.2.47 (ThermoFisher Scientific).
  • CD4 + T cells were enriched using CD4 + T cell Isolation Kit (Miltenyi 130-104-454) and incubated for 30 minutes with HS (+10mM or +40mM NaCI) and washed with iso-osmolal sucrose solution. The pellet was lysed with 0.1% Triton and total Na + was quantified using an iCE 3000 Series atomic absorption spectrometer (Thermo Scientific) as described before (Neubert et al. 2020, PLoS Biol 18:e3000722). Animal care and use followed the regulations of the German Animal Welfare Act. The procedures followed were approved by the shoot der Stadt Regensburg and performed in accordance with institutional guidelines.
  • Tregs were cultured as described above in the presence or absence of +40mM NaCI for a period of 6 hours. After incubation, cells were fixed with specific fixative (8% formaldehyde (FA) (EM-grade), 5% glutaraldehyde (GA) (EM-grade), 0.1M Cacodylate buffer) on an equal volume as cells. Cells were fixed for 30 minutes RT in constant rotation. After incubation, fixative was removed and cells were washed 3 times for 30 minutes at 4°C in 0.1M Cacodylate buffer in constant rotation. Once washes were complete, equal volume of 1% Osmium tetroxide (OsO4) was added to the cells and samples were shaken for 1 hour at 4°C.
  • OsO4 Osmium tetroxide
  • samples were washed by constant shaking for 20 minutes in ddH2O. This step was repeated 4 times. After last washing step, samples were left for 1 hour in 1% Uranyl Acetate (UrAc) under cold and dark conditions for bulk staining. After staining incubation, samples were once again washed by constant shaking in ddl-120 for 20 minutes. Washing step was done at 4°C and repeated 4 times. After, samples were dehydrated at 4°C in constant shaking, infiltrated at 4°C and embedding was performed using Spurr's resin. Final step of polymerization was done at 70°C.
  • Uranyl Acetate UrAc
  • RNA isolation and quantitative polymerase chain reaction with reverse transcription qRT-PCR
  • Rneasy plus Micro Kit (74034, Qiagen) was used according to the manufacturer's instructions and further converted to cDNA using qScriptTM cDNA SuperMix kit (95048, QuantaBio) according to manufacturer's instructions.
  • Real Time PCR was performed on a Step ONE Plus RT-PCR machine (Applied Biosciences) using the TaqMan Fast Universal PCR Master Mix (4367846, ThermoFisher Scientific).
  • RNA sequencing (RNAseq)
  • RNA sequencing analysis RNA was isolated as described above. Sequencing libraries were prepared with the NEB Next Ultra DNA Library Prep Kit for Illumina (version 6.0-2/18), according to the manufacturer's protocol including a size selection to 250bp insert size. Sequence-libraries of each sample were equimolarly pooled and sequenced on 4 NextSeq500 v2 flow-cell at 1 x 75 bp (76-8-8-0). Quality of raw sequence reads was checked using FastQC version 0.11.8, and nucleotide calls with a quality score of 28 or higher were considered high quality. Due to low read quality, one sample from the 6 hours control group was removed from analysis. Adapters were removed using cutadapt v.2.4.
  • EGAS00001004470 (Alissafi et al. 2020, Cell Metab 32:591-604. e7) were downloaded from the European Genome-phenome Archive (EGA). The quality of raw sequence reads was checked using FastQC version 0.11.8, and the data was analyzed as described above. Droplet-based single cell (sc)RNAseq preparation
  • Tregs were incubated for a period of 6 hours in the presence and absence of +40mM NaCI. After incubation, cells were collected and converted to barcoded scRNAseq libraries by using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (lOx Genomics), aiming for an estimated 5,000 cells per library and following the manufacturer's instructions. Samples were processed using a kit associated to V2 barcoding chemistry of lOx Genomics. Single samples were processed in a single well of a PCR plate, allowing all cells from a sample to be treated with the same master mix and in the same reaction vessel. scRNAseq profile
  • RNA-Seq profiling of single Treg cells was performed with an average sequencing saturation metric of >80%, as calculated by Cell Ranger. Aggregation of sample conditions was done using the Cell Ranger Aggr software from lOx Genomics. Digital gene expression matrices were pre-processed and filtered using the SCRAN and ScaterR packages. Outlier cells were first identified based on three metrics (library size, number of expressed genes and mitochondrial proportion); cells were tagged as outliers when they were four median absolute deviations distant from the median value of each metric across all cells.
  • a principal component analysis plot was generated based on the following metrics: 'pct_counts_in_top_100_features', 'total_features_by_ counts', 'pct_counts_feature_control', 'total_features_by_counts_feature_control', 'loglO_total_counts_endogenous' and
  • Unsupervised clustering of the cells was performed using graph-based clustering based on SNN-Cliq and PhenoGraph as implemented in the Seurat v.2.3Rpackage (default parameters). Clustering was visualized in two-dimensional scatter plots (via tSNE) using the Seurat v.2.3Rpackage.
  • the metabolic landscape of Tregs was modeled using the Compass method (version 0.9.5) (Wagner et al. 2021, Cell 184:4168-4185) by leaving the standard settings unaltered.
  • the gene expression matrix of Tregs single cell data was used as input.
  • the Compass output data was concatenated and transformed as described (Wagner et al. 2021, Cell 184:4168-4185). To determine which reactions and metabolites were significantly different between groups (CTRL and +40mM NaCI), a Wilcoxon rank sum tests on Compass scores was performed.
  • Ingenuity Pathway Analysis (IPA; Ingenuity Systems/Qiagen) was used to map lists of significant genes (FDR ⁇ 0.05) to gene ontology groups and biological pathways. The functional and canonical pathway analysis was used to identify the significant biological functions and pathways. Functions and pathways with p-values less than 0.05 (Fischer's exact test) were considered to be statistically significant.
  • GSEA Gene set enrichment analysis
  • MsigDB Molecular Signatures Database
  • HS and NS intake was defined by a cutoff of >7g/day or ⁇ 7g/day, respectively.
  • the cohort was comprised of 8 male and 10 female participants with an age range of 35.8 ⁇ 10.4yrs.
  • PBMCs of participants were isolated from peripheral blood and analyzed by FACS as described above.
  • EAE Experimental autoimmune encephalomyelitis
  • CD4 + CD25 + Treg cells were isolated using the mouse CD4 + CD25 + Regulatory T Cell Isolation Kit according to the manufacturer's instructions (Miltenyi Biotec #130-091-041, Bergisch Gladbach, Germany) from C57BL/6J mice. Cells were plated for 24hrs in 48-well plates (Sarstedt, Numbrecht, Germany) at 750 thousand cells per well in RPMI 1640 with 10% FCS (both Thermo Fisher) and 2.000 lU/ml recombinant murine IL-2 (Miltenyi Biotec) in the presence or absence of 40mM NaCI or in the presence of 40mM NaCI plus lOpM of CGP-37157 (220005, EMD Millipore).
  • mice Six- to ten-week-old male NSG mice were purchased from Charles River and housed randomly on different IVC cages at the in-house animal care facility. Mice received autoclaved chow and tap water ad libitum for 9 days before induction.
  • each animal received 1.3xl0 7 CD25-depleted PBMCs alone (PBMC group) or together with 0.2x107 CD25-enriched Tregs pre-activated with aCD3 (lpg/mL), aCD28 (lpg/mL) and IL-2 (25U/mL) for 6 hours in control media or media supplemented with +40mM NaCI (HS group) or in media containing lOpM of antimycin A (AA group) or in media supplemented with +40mM NaCI containing lOpM of CGP-37157 (CGP group). After thorough washing, cells were resuspended in PBS and injected through the tail vein.
  • aCD3 lpg/mL
  • aCD28 lpg/mL
  • IL-2 25U/mL
  • mice The weight and clinical symptoms of the mice were monitored during the entire course of the experiment. Clinical symptoms were scored according to general appearance and mobility as described before (Hernandez et al. 2015, J Clin Invest 125:4212-4222). At sacrifice, organs were collected, processed and cells were used for FACS. Engraftment of human cells was monitored by FACS analysis of peripheral blood.
  • Statistical analyses were performed with GraphPad Prism Version 8. All data were presented as mean ⁇ standard error of the mean (SEM), unless stated otherwise. Value of n is always displayed in the Figure as individual data point, referring to an independent biological replicate, more information about absolute n numbers can be found in the Figure legends. All statistical test used were also indicated in the respective Figure legends. Normality of the data was tested by Shapiro-Wilk normality test. Significance between two groups was analyzed by t-test (when normal distributed) or Wilcoxon matched-pairs signed rank test (for non-normal distributed). For more than two groups with one variable only, one-way ANOVA with Tukey's post-hoc test (for normal distributed data) was used.
  • Tregs Perturbed function of Tregs is associated to autoimmunity, however, factors contributing to this phenotype still remain elusive.
  • RNAseq bulk-RNA sequencing
  • GSEA gene set enrichment analysis
  • the RNA sequencing data from healthy individual- and patient derived Tregs were downloaded from the European Genome-phenome Archive (EGAS00001004470) and reanalyzed accordingly.
  • Such enrichment plots comprise an enrichment score (ES) curve and a ranked list metric curve.
  • the ES curve represents the running sum of the weighted enrichment score obtained from the GSEA algorithm.
  • the ranked list metric curve represents the degree of correlation of genes with the depicted pathway (red for positive and blue for negative correlation). Furthermore the normalized enrichment score (NES) and the corresponding FDR are reported within the plot.
  • RNA- seq single cell RNA- seq
  • scRNA-Seq single cell RNA- seq
  • the tSNE projections were performed on 4785 cells and 10 clusters were identified based on the expression of key signature genes.
  • HS-treated Tregs were highly differentially represented within the clusters. While in clusters 0 and 3 there was an overrepresentation of HS-treated Tregs; clusters 1, 2 and 4 depicted an underrepresentation of these cells (Figure 2D).
  • SDH succinate dehydrogenase
  • malate dehydrogenase isocitrate dehydrogenase
  • isocitrate dehydrogenase isocitrate dehydrogenase
  • RNA-seq and scRNA-seq data pointed towards a disturbed mitochondrial metabolism of HS- treated Tregs
  • OCR oxygen consumption rate
  • Tregs cultured under HS conditions showed a dramatic reduction of ATP production, even to a similar extend as compared to oligomycin exposure, a potent inhibitor of mitochondrial complex V of the ETC at 6 hours post activation (Figure 3C).
  • Figure 3C To examine whether changes in the mitochondrial mass could account for the observed effects on Tregs metabolism, we analyzed mitochondria of human Tregs by FACS using Mitotracker. No changes were observed between HS and control conditions, indicating that the previously observed effects were not due to changes in mitochondrial mass. In line with these findings, analysis of HS and control activated human Tregs by transmission electron microscopy (tEM) did not reveal any major morphological changes.
  • tEM transmission electron microscopy
  • HS tetramethylrhodamine ethyl ester
  • FCCP carbonyl cyanide p- (trifluoromethoxy)phenylhydrazone
  • AA m and antimycin A
  • HS led to a collapse of the mitochondrial membrane potential and significantly reduced TMRE signal in Tregs even to a comparable extend to AA at 6 hours post activation ( Figure 3D).
  • mtROS mitochondrial reactive oxygen species
  • FOXP3 is essential for Treg stability and function and mutations in the gene can lead to the development of severe autoimmunity (Kleinewietfeld & Hafler 2014, Immunol Rev 259:231-244; Sakaguchi et al. 2010, Nat Rev Immunol 10:490-500; Arroyo Hornero et al. 2020, Front Immunol 11:253; Kleinewietfeld et al. 2013, Nature 496:518-522), its intriguing to speculate that the rapid downregulation of FOXP3 expression after HS exposure could account for the observed loss of function in Tregs. In the light of these findings, it is of interest that recent data revealed a direct link between FOXP3 expression and OXPHOS (Howie et al.
  • EXAMPLE 6 Salt induced inhibition of mitochondrial function disrupts T reg fitness and function in vivo.
  • Daily salt consumption of participants was calculated based on a specifically designed comprehensive dietary questionnaire covering individual food habits as described before (Mahler et al. 2022, Nutrients 14:253) and volunteers were grouped into 'low salt' (LS) and 'high salt' (HS) groups based on a cutoff of 7g/day NaCI intake (Figure 5A).
  • Tregs peripheral blood mononuclear cells
  • PBMCs isolated from individual participants were then used to assess the cytokine expression of Tregs by FACS. Strikingly, the analysis of Tregs revealed a significant reduction of IL-10 expression in FOXP3 + Tregs from the HS group in comparison to the LS group ( Figure 5B) and the percentage of IL-10 expression in Tregs negatively correlated with salt intake in a significant manner (Figure 5C). However, we only observed tendencies for decreases in FOXP3 and increases in IFN-0 expression in the HS group (data not shown).
  • EAE was induced in 12-week-old C57BL/6 recipient mice and Tregs were isolated from naive donor mice and pre-incubated in either control or HS conditions for a period of 24 hours and subsequently injected into recipient mice after 6 days of EAE induction and mice were scored daily to monitor EAE development (Figure 5E).
  • mice that received control Tregs (CTRL) animals receiving salt pre-treated Tregs (+40mM NaCI) failed to contain the development of EAE, resulting in significantly higher disease scores (Figure 5F).
  • mice receiving CD25-depleted PBMC together with untreated control Tregs were significantly able to prevent severe xGvHD.
  • HS or AA pre-treated Tregs were unable to restrain disease in CD25" PBMC receiving animals ( Figure 5J).
  • the analysis of splenocytes on sacrifice showed that only animals receiving functional Tregs displayed a lower percentage of human CD8 + T cells, known as major drivers of xGvHD (Guichelaar et al. 2013, Clin Cancer Res 19:1467-1475; Mutis et al. 2006, Clin Cancer Res 12:5520-5525) (data not shown).
  • our data demonstrates that even the short-term perturbation of mitochondrial respiration in Tregs by treatment of either HS or AA, similarly lead to long-term disruption of human and murine Treg function in vivo.
  • gRNA highly efficient guide (g)RNA for the human SLC8B1 gene was designed and selected as described before (Van Zeebroeck et al. 2021, Front Immunol 12:655122).
  • a schematic representation is given in Figure 7.
  • KO score is generated by ICE analysis tool (Synthego) that indicates the percentage of KO on the genomic level.
  • the sequence of the gRNA is included in the Table 2.
  • HS severely affects cellular metabolism and suppressive function of Tregs.
  • Th 1-1 ike profile which closely overlaps with the transcriptomic features of Tregs isolated from patients with autoimmune disorders such as MS, SLE and RA.
  • Our data demonstrates that raised physiological HS conditions, similar to Na + concentrations that can be reached inside inflamed tissues in vivo, perturbs the function of Tregs by metabolic reprogramming through the interference with mitochondrial respiration.
  • Hallmarks of the phenotype induced by the interference with mitochondrial respiration in human Tregs are a lack of IL-2 responsiveness by hampered STAT5 phosphorylation and decreased F0XP3, IL-10 and CTLA-4 levels as well as increases in mtROS and IFN-y production and activation of the WNT/beta-catenin pathway, consequently leading to a loss of Treg suppressive function.
  • Tregs not only play a role in autoimmunity, but have also been shown to be important mediators to contain chronic inflammation in cardiovascular diseases (CVD) such as atherosclerosis (Ait-Oufella et al. 2006, Nat Med 12:178-180), hypertensive target organ damage (Kvakan et al. 2009, Ciruculation 119:2904-2912) and myocardial infarction (Weirather et al. 2014, Circ Res 115:55-67).
  • CVD cardiovascular diseases
  • atherosclerosis Ait-Oufella et al. 2006, Nat Med 12:178-180
  • hypertensive target organ damage Kvakan et al. 2009, Ciruculation 119:2904-2912
  • myocardial infarction Weirather et al. 2014, Circ Res 115:55-67.
  • Treg numbers and a dysfunctional Treg phenotype have also been reported in CVD and were associated with progression of disease (Meng e
  • Tregs in CVD also displayed a pro-inflammatory Thl-like phenotype (Butcher et al. 2016, Circ Res 119:1190-1203; Bansal et al. 2019, Circulation 139:206-221), it would be of interest to investigate if excess sodium may further contribute to chronic inflammation and CVD by disabling Tregs.
  • HS-perturbed Tregs seem to rather resemble 'fragile' Tregs than 'ex- Tregs'. While exhibiting decreases in F0XP3, HS-perturbed Tregs still largely maintained demethylation of the FOXP3 locus and showed a Th-like phenotype and further expression of characteristic markers for Treg fragility (data not shown). Although this becomes a detrimental scenario in autoimmunity or CVD, it can be beneficial in cancer, promoting more efficient anti-tumor immunity (Hatzioannou et al.
  • HS perturbs mitochondrial function of human Tregs by inhibition of mitochondrial respiration on the level of ETC complex ll/lll.
  • the critical disturbance of energy production induces metabolic reprogramming in Tregs and goes in line with severe changes in gene expression and functional disturbances including swift alterations in F0XP3 expression.
  • the HS effect could be mimicked by pharmacological inhibition or genetic disruption of mitochondrial complex III, leading to a similar phenotype observed in dysfunctional Tregs of patients with autoimmunity and our data indicates that salt intake could impact functional features of Tregs in humans.

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Abstract

The invention relates to protecting regulatory T cells (Tregs) against dysfunctionality as caused in e.g. the auto-immune disease setting. In particular, such protection is warranted in case of adoptive transfer or Tregs as a means of treatment of auto-immune and other diseases, and as a means of controlling transplant or graft rejection. Furthermore in particular, the protection is enabled by inhibiting the Treg mitochondrial sodium/lithium/calcium exchanger.

Description

MAINTAINING REGULATORY T CELL (Treg) FUNCTIONALITY UNDER CONDITIONS TRIGGERING Treg DYSFUNCTIONALITY
FIELD OF THE INVENTION
The invention relates to protecting regulatory T cells (Tregs) against dysfunctionality as caused in e.g. auto-immune disease settings. In particular, such protection is warranted in case of adoptive transfer or Tregs as a means of treatment of auto-immune and other diseases, and as a means of controlling transplant or graft rejection. Furthermore in particular, the protection is enabled by inhibiting the Treg mitochondrial sodium/lithium/calcium exchanger.
BACKGROUND OF THE INVENTION
Regulatory F0XP3+ T cells (Tregs) play an essential role for the maintenance of peripheral tolerance and immune cell homeostasis (Kleinewietfeld & Hafler 2014, Immunol Rev 259:231-244; Sakaguchi et al. 2010, Nat Rev Immunol 10:490-500). Depending on environment and tissue, they have the ability to suppress and neutralize responses of the innate and adaptive immune system by various mechanisms like secretion of anti-inflammatory cytokines as interleukin (IL)-10 or by cell-cell contact dependent mechanism involving co-stimulatory receptors like cytotoxic T-lymphocyte-associated protein 4 (CTLA- 4). Mutations in the FOXP3 gene lead to fatal autoimmunity and dysfunctional Tregs have been linked to autoimmune disorders such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) or type-1 diabetes (T1D) and chronic infections (Kleinewietfeld & Hafler 2014, Immunol Rev 259:231-244; Sakaguchi et al. 2010, Nat Rev Immunol 10:490-500; Arroyo Hornero et al. 2020, Front Immunol 11:253). Interestingly, Treg dysfunction is frequently associated with the development of a pro- inflammatory, cytokine producing phenotype, termed Treg plasticity or fragility (Hatzioannou et al. 2021, Front Immunol 12:731947; Overacre-Delgoffe & Vignali 2018, Cancer Immunol Res 6:882-887; Kleinewietfeld & Hafler 2013, Semin Immunol 25:305-312). In contrast to so-called "ex-Tregs" that lose their Foxp3 expression in line with epigenetic alterations, fragility in Tregs is associated with the acquisition of a pro-inflammatory Th-like phenotype and loss of function, while, at least to a certain extent, maintaining Foxp3 demethylation and expression (Hatzioannou et al. 2021, Front Immunol 12:731947; Overacre-Delgoffe & Vignali 2018, Cancer Immunol Res 6:882-887; Overacre-Delgoffe et al. 2017, Cell 169:1130-1141; Dominguez-Villar et al. 2018, Nat Immunol 19:665-673; Junius et al. 2021, Sci Immunol 6:eabe4723; Saxena et al. 2021, Eur J Immunol 51:1956-1967).
High salt (NaCI) content in food, particularly associated to the so-called "western-diet", has been established to have detrimental effects on several pathologies and autoimmune disorders by shifting the immune cell balance towards a pro-inflammatory state (Muller et al. 2019, Nat Rev Immunol 19:243- 254; Manzel et al. 2014, Curr Allergy Asthma Rep 14:404). Besides impacting T effector cells, like T helper (Th)17 responses (Wu et al. 2013, Nature 496:513-517; Kleinewietfeld et al. 2013, Nature 496:518-522), we and others have shown that HS could also severely affect the function of Tregs (Hernandez et al. 2015, J Clin Invest 125:4212-4222; Sumida et al. 2018, Nat Immunol 19:1391-1402; Safa et al. 2015, J Am Soc Nephrol 26:2341-2347, Yang et al. 2020, Cell Rep 30:1515-1529; Luo et al. 2019, Cell Rep 26:1869- 1879). HS has been shown to dynamically induce a pro-inflammatory Th 1-like Treg phenotype with high expression levels of interferon (IFN)-y and lower levels of IL-10 (Hernandez et al. 2015, J Clin Invest 125:4212-4222; Sumida et al. 2018, Nat Immunol 19:1391-1402). Interestingly, the HS induced Treg phenotype closely resembles dysfunctional Tregs frequently noted in patients with autoimmunity like MS (Kleinewietfeld & Hafler 2014, Immunol Rev 259:231-244; Dominguez-Villar et al. 2018, Nat Immunol 19:665-673). These findings indicate that excess salt intake could contribute to an overall immune imbalance and therefore potentially represents an environmental (risk) factor contributing to disease. Despite the above findings, the exact mode of action on how salt impairs Treg function is still unknown. Immunometabolism has gained increased attention over recent years and it is now clear that many immune reactions are governed through changes in cellular metabolism (Geltink et al. 2018, Annu Rev Immunol 36:461-488; Norata et al. 2015, Immunity 43:421-434). Especially T cell subsets highly depend on various metabolic needs depending on environment and activation status (Geltink et al. 2018, Annu Rev Immunol 36:461-488). Tregs are believed to closely mimic memory T cells in their metabolic needs, preferentially depending on oxidative phosphorylation (OXPHOS) and fatty acid p-oxidation (FAO) while T effector cells rather use aerobic glycolysis (Geltink et al. 2018, Annu Rev Immunol 36:461-488; Newton et al. 2016, Nat Immunol 17:618-625; Chapman et al. 2020, Nat Rev Immunol 20:55-70; Binger et al. 2017, Front Immunol 8:311; Kurniawan et al. 2020, Cell Metab 31:920-936; Pompura et al. 2021, J Clin Invest 131(2):el38519). Several studies have highlighted the importance of mitochondrial metabolism for optimal Treg stability and function (Geltink et al. 2018, Annu Rev Immunol 36:461-488; Binger et al. 2017, Front Immunol 8:311) and a recent report has demonstrated the importance of mitochondrial respiration for Treg function (Weinberg et al. 2019, Nature 565:495-499). The ablation of mitochondrial electron transport chain (ETC) complex III in murine Tregs, by knocking-out Uqcrfsl, encoding for Rieske iron-sulfur polypeptide 1 (RISP), resulted in Treg loss of function and development of lethal inflammation. Interestingly, recent studies observed that Tregs isolated from patients with MS showed dampening in OXPHOS, with diminished maximal respiration levels compared to healthy controls (Duscha et al. 2020, Cell 180:1067-1080; La Rocca et al. 2017, Metabolism 77:39-46) and expression analysis of Tregs from autoimmune patients displayed deregulation of mitochondrial genes (Alissafi et al. 2020, Cell Metab 32:591-604.e7), indicating defects in mitochondrial function of Tregs in autoimmune patients. However, factors contributing to this phenotype are unknown and if and how metabolic alterations impact the pro-inflammatory signature and function of autoimmune Tregs are poorly understood.
Very recently, two studies described the role of Na+ in the regulation of mitochondrial function in endothelial cells, fibroblasts, monocytes and macrophages (Hernansanz-Agustin et al. 2020, Nature 586:287-291; Geisberger et al. 2021, Circulation 144:144-158). In endothelial cells and fibroblasts under hypoxic conditions, Na+ acts as a second messenger that controls OXPHOS function and redox signaling by modulating the fluidity of the inner mitochondrial membrane. Mechanistically, Na+ effects were mediated through the mitochondrial Na+/Ca2+ exchanger (NCLX) (Hernansanz-Agustin et al. 2020, Nature 586:287-291). In monocytes and Ml and M2 macrophages, Na+ entered in the intracellular compartment followed by inhibition of the ETC and blunted ATP production (Geisberger et al. 2021, Circulation 144:144-158).
A role for NCLX in asthmatic airway remodeling and hyperresponsiveness was reported (Johnson et al. 2022, J Biol Chem 298:102259). In a non-enabling disclosure, WO2011/048589 links both inhibition and activation of NCLX with treatment of amongst other inflammatory diseases, and activation of NCLX with treatment of amongst other autoimmune diseases.
SUMMARY OF THE INVENTION
In a first aspect, the invention relates to isolated regulatory T cells (Treg cells) or populations of isolated Treg cells characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited in the Treg cells. More in particular, the function of NCLX can be inhibited by a pharmacological compound, or the expression of NCLX can be inhibited by a DNA nuclease specifically knocking out or disrupting NCLX, by an RNase specifically targeting NCLX, or by an inhibitory oligonucleotide specifically targeting NCLX.
In the above, the Treg cells can be polyclonal Treg cells, in vitro amplified or expanded Tregs, antigenspecific Tregs, engineered T cell receptor (TCR)-Tregs, engineered chimeric antigen receptor (CAR)-Tregs, monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs, B-cell-targeting antibody receptor (BAR) Tregs, design Tregs, or chimeric cytokine receptor (CCR) Tregs.
The invention also relates to compositions comprising isolated Treg cells or populations of isolated Treg cells as defined above. In particular, such compositions can be pharmacological compositions.
Furthermore, the isolated Treg cells or population of isolated Treg cells or the (pharmaceutical) compositions as defined above can be for use as a medicament; for use in treating an auto-immune disease; for use in treating an inflammatory disease; for use in suppressing transplant, graft or allograft rejection; or for use in treating a cardiovascular disease. In particular, the isolated Treg cells or population of isolated Treg cells or the (pharmaceutical) compositions as defined above can be adoptively transferred in a subject. In particular, the Treg cells are autologous Treg cells, allogeneic Treg cells, or induced Treg cells.
The invention further relates to methods of producing isolated Treg-cells as defined above, such methods comprising a step of isolating Treg-cells from peripheral blood, umbilical cord blood, thymus or leukapheresis product obtained from a subject, or comprising a step of reprogramming conventional CD4+ T-cells obtained from a subject into Tregs. An optional further step in such methods is a step of ex-vivo expanding the isolated or reprogrammed Tregs. Another step in such methods can be a step of ex-vivo manipulation to inhibit the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in the Tregs by means of pharmacological inhibition or by means of a DNA nuclease specifically knocking out or disrupting NCLX, an RNase specifically targeting NCLX, or an inhibitory oligonucleotide specifically targeting NCLX.
The invention also relates to pharmaceutical kits comprising at least one vial comprising isolated Treg cells or populations of Treg cells as defined above, or comprising a composition comprising such Treg cells or populations of Treg cells.
The invention also relates to isolated TCR-engineered Treg-cells, CAR-engineered Treg-cell, or BAR- engineered Treg cells characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited in the engineered Treg cells.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1. Salt induced changes in Treg phenotype and function closely mimic human autoimmune Tregs.
(A) CD4+ T effector cells (Teff) were labelled with CFSE and stimulated with aCD2/aCD3/aCD28-coated beads and co-cultured with Tregs (1:1 ratio). Cells were cultured in control (CTRL) or HS media (+40mM NaCI). Teff proliferation was measured by flow cytometry after 4 to 5 days of incubation. The percentage of proliferation of HS group ("+40 mM NaCI") was calculated by normalizing to the control ("CTRL") group (n=12 from 6 independent experiments).
(B) IFN-y expression was assessed by FACS in activated Tregs in the presence of control ("CTRL") or HS media ("+40mM NaCI") (n=3 from 3 independent experiments). Data is depicted as fold-change over CTRL group.
(C) Treg proliferation was assessed by CTV dye dilution after 4 to 5 days of incubation in suppression assays alongside CFSE labelled CD4+ T effector cells under control ("CTRL") or HS ("+40mM NaCI") conditions. Representative quantification of Treg proliferation is shown; gating was on viable cells. Data is depicted as fold-change over CTRL group (n=12 from 5 independent experiments). Data is depicted as mean ± SEM. **p<0.01, ***p<0.001, *p<0.05. Normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by paired two- tailed t-test for normal distributed data.
FIGURE 2. High salt induces discrete changes in gene expression in human Tregs indicative for metabolic reprogramming.
(A) Hierarchical clustering showing changes in gene expression of unstimulated Treg cells (0 hours) and activated Tregs after 6- and 72 hours activation under control ("CTRL") or HS conditions ("+40mM NaCI"). Each column contains the average measurements for differential gene expression by RNAseq for 2 to 5 biological replicates. Relative gene expression is indicated by upregulation (>0) and downregulation (<0).
(B) Volcano plot of differentially expressed genes (DEGs) in HS vs control activated Tregs for 6 hours. Points represent genes: the x-axis showcases the Iog2 fold change for the ratio of HS vs untreated Tregs, whereas the y-axis showcases the statistical significance (FDR-adjusted p-value); up- and downregulated genes are depicted at Iog2 FC>1 and Iog2 FC<-1, respectively.
(C) Ratio between IFNG and IL10 gene expression at 96 hours. Data is depicted as fold-change under HS conditions over control conditions (n=7 from 4 independent experiments). Data is depicted as mean ± SEM. *p<0.05, normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by paired one-tailed Wilcoxon t-test for non-normal distributed data.
(D) Gene expression and metabolic analysis of human Tregs. Histogram visualization showing the absolute number of cells belonging to each cluster identified in scRNA-seq dataset within Tregs under control (CTRL) or HS (+40mM NaCI) conditions. Clusters are numbered 0 to 9 and represented in both histogram bars with cluster 9 at the bottom up to cluster 0 at the top of each bar.
FIGURE 3. High salt inhibits mitochondrial respiration in human Tregs.
(A) Oxygen consumption rate (OCR) of Tregs activated under control ("CTRL") or HS conditions ("+40mM NaCI") for a period of 72 hours. Overlay of maximal OCR of control and +40mM NaCI cultured Tregs after 72 hours of activation (n=10 from 7 independent experiments).
(B) Cell viability was assessed after oxygen consumption rate was performed on Tregs (n=3). Each dot represents a biological replicate. Data is depicted as mean ± SEM. *p<0.05. Normal distribution was calculated by Shapiro-Wilk normality test with a significance lever of 0.05. Significance was calculated by paired two-tailed Wilcoxon t-test for non-normal distributed data.
(C) Relative ATP content of 6 hour activated Tregs under control ("CTRL") or HS conditions ("+40mM NaCI") (n=7 from 3 independent experiments). Oligomycin A (5pM) treatment was used as positive control (n=5 from 2 independent experiments).
(D) Relative TMRE (tetramethylrhodamine ethyl ester) mean fluorescence intensity (MFI) in Tregs activated for a period of 6 hours under control ("CTRL") or HS ("+40mM NaCI") conditions and in the presence of antimycin A ("AA") or (trifluoromethoxy)phenylhydrazone ("FCCP") (n=8 from 4 independent experiments).
Data is depicted as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by Friedman test with Benjamini-Hochberg FDR-correction.
(E) Mitochondrial ROS was measured using MitoSOX staining on Tregs incubated under control ("CTRL") or HS ("+40mM NaCI") conditions for a period of 6 hours (n=6 from 3 independent experiments). A dot plot is depicted. Each dot represents a biological replicate. Data is depicted as mean ± SEM. *p<0.05. Normal distribution was calculated by Shapiro-Wilk normality test with a significance lever of 0.05. Significance was calculated by paired two-tailed t-test for normal distributed data.
FIGURE 4. Salt mediated disruption of mitochondrial respiration perturbs human Treg function.
(A-B) FOXP3 expression after HS exposure on Tregs activated under control ("CTRL") or HS conditions ("+40mM NaCI"). (A) Relative gene expression of FOXP3 in Tregs activated for 6 hours under control or HS conditions assessed by qRT-PCR. Data is depicted as fold-change over control (n=5 from 3 independent experiments). (B) FACS analysis of FOXP3 expression in isolated Tregs, activated for 24 hours under control or HS conditions. A dot plot showing MFI levels normalized to the control group is depicted (n=5 independent donors). Data is depicted as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by paired two-tailed t-test for normal distributed data.
(C) Quantification of FOXP3 MFI calculated in total CD4+ T cells incubated under control conditions ("CTRL") or in the presence of antimycin A ("AA") for 24 hours. A dot plot showing MFI levels normalized to the control group is depicted (n=5 independent donors).
(D) Relative gene expression of IL10 and CTLA4 in Tregs activated for 6 hours under control (CTRL), HS (+40mM NaCI) conditions or in the presence of AA assessed by qRT-PCR. Data is depicted as fold-change over control (n=7-9 from 4-5 independent experiments). Data is depicted as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Normal distribution was calculated by Shapiro-Wilk normalitytest with a significance level of 0.05. Significance was calculated by one-tailed.
(E) Phosphorylation of STAT5 (pSTAT5) assessed in Tregs activated for a period of 4 hours under control ("CTRL"), HS ("+40mM NaCI") conditions or in the presence of AA ("AA") by FACS. Dot plot depicts MFI levels (n=5-8 from 2-3 independent experiments).
(F) Tregs were pre-incubated under control ("CTRL"), HS ("+40mM NaCI") conditions or in the presence of AA ("AA") for 72 hours and then co-cultured with CFSE labelled Teffs at a 1 to 1 ratio. Teff proliferation was assessed by FACS after 4 days. Percentage of proliferation of HS and AA group was calculated by normalizing to controls (n=6 from 4 independent experiments). Data is depicted as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Normal distribution was calculated by Shapiro-Wilk normalitytest with a significance level of 0.05. Significance was calculated by one-way ANOVA with Tukey's post-hoc test.
(G) Human RISP KO and control (Mock CTRL) Tregs were co-cultured with CFSE-labelled total PBMCs at a ratio of 1 to 3. Percentage of proliferation of CD3+ T cells co-cultured with RISP KO Tregs was calculated by normalizing to control Tregs (Mock CTRL) (n=3 from 2 independent experiments). Data is depicted as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by paired two- tailed t-test for normal distributed data.
(H) Ratio between IFNG and IL10 gene expression after RISP KO (n=5 from 4 independent experiments).
FIGURE 5. Impact of salt on Treg fitness and function in vivo.
(A-C) Salt intake and its impact on Tregs in healthy human subjects. (A) Healthy individuals were separated into 2 groups based on their daily salt intake ("LS"= low salt group (<7g NaCI/day); "HS"= high salt group (>7g NaCI/day)). (B) Analysis of IL-10 expression in FOXP3+ Tregs of PBMCs isolated from participants. Quantification of I L-10+ cells in FOXP3+ Tregs is depicted (n=8/HS and n=10/LS independent donors). (C) The Spearman Correlation scatter plot showing the association between percentage of IL- 10+FOXP3+ Tregs and daily salt intake (g) in healthy volunteers (n=18). The black line corresponds to the regression line (correlation R = -0.67; p = 0.0023). Data is depicted as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by unpaired two-tailed t-test for normal distributed data.
(D) Murine Treg suppressive capacity was assessed in suppression assays in co-cultures with T effector cells (Teff). Teff proliferation was measured under HS conditions (+40mM NaCI) or control conditions (CTRL) at different Treg:Teff ratios (n=2 from 2 independent experiments).
(E-F) Impact of salt on Treg fitness and function to contain experimental autoimmune encephalomyelitis (EAE) by adoptive transfer. (E) EAE was induced in recipient mice and CD4+CD25+ Tregs of naive donor mice were pre-incubated in the presence of HS (+40mM NaCI) or control (CTRL) conditions for a period of 24 hours prior to being adoptively transferred to contain EAE into recipient mice 6 days after EAE induction. (F) Mice were kept for a period of 15 days post induction and monitored daily for the development of EAE symptoms (n=4-5 per group from 2 independent experiments). Data is depicted as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by Man-Whitney test for separate days during EAE development.
(G) Impact of salt on human Treg fitness and function to contain xGvHD. Schematic experimental set-up for xGvHD. Human CD25+ Tregs were activated in control media, media supplemented with lOpM of antimycin A (AA) or media supplemented with +40mM NaCI for 6 hours. Pre-incubated Tregs were mixed with autologous CD25" PBMCs and injected into NSG mice (PBMC alone; PBMC & Treg CTRL; PBMC & Treg NaCI, PBMC & Treg AA; n=5-6/group). Mice were monitored for a period of 60 days for development of xGvHD.
(H) Functional characterization of Tregs in vivo. Percentage of engrafted human CD3+ T cells (mCD45_ hCD3+) in xGvHD model of (G) at day 15 post-injection.
(I) Functional characterization of Tregs in vivo. Percentage of weight of mice during the course of xGvHD over time.
(J) Survival rates of xGvHD experiment (see (G)) over time. Significance was calculated by curve comparison analysis using Mantel-Cox and Gehan-Breslow-Wilcoxon tests.
FIGURE 6. Salt induced blockade of mitochondrial function depends on NCLX.
(A) Intracellular sodium content was calculated in murine CD4+T cells after exposure to HS environments for a period of 30 minutes (n=16 from 3 independent experiments).
(B) Relative ETC complex II ("Complex II") activity after purification of complex II from Tregs under control conditions (CTRL) or in the presence of additional NaCI (+4mM) (n=3). Data is normalized to controls.
(C) Relative complex ll+lll activity was a measured in bovine heart mitochondria under HS conditions in the presence or absence of lOpM NCLX inhibitor CGP-37157 (CGP).
(D) Tregs were pre-incubated under HS conditions (+40mM NaCI) in the presence or absence of lOpM NCLX inhibitor CGP-37157 (CGP) for 24 hours and later co-cultured with CFSE labelled PBMCs at a 1 to 1 ratio. CD4+ Teff proliferation was assessed by FACS after 4 days. The percentage proliferation normalized to the HS group is depicted (n=5 from 3 independent experiments).
(E) Ratio between IFNG and I LIO gene expression in Tregs incubated under HS conditions (+40mM NaCI) in the presence or absence of lOpM NCLX inhibitor CGP-37157 (CGP) for 72 hours (n=5 from 3 independent experiments).
(F) Inhibition of NCLX restores perturbed Treg function in vivo. FOXP3 gene expression was assessed by qRT-PCR on Tregs pre-incubated in the presence of HS (+40mM NaCI) or HS plus CGP (+40mM NaCI + CGP) for a period of 6 hours (n=5 from 3 independent experiments).
(G) Murine Tregs were pre-incubated with +40mM NaCI and +40mM NaCI + CGP for 24 hours, before adoptively transferred to contain EAE development (n=4-5) as described in Figure 5G.
(H) Human Tregs were preincubated under +40mM NaCI and +40mM NaCI + CGP before being coinjected with CD25' PBMC into NSG mice (n=4) as described in Figure 5F-G. Mice were monitored for survival (left plot) and weight loss (day 40, right plot).
(A-E) Data is depicted as mean ± SEM. *p<0.05, **p<0.01. Normal distribution was calculated by Shapiro- Wilk normality test with a significance level of 0.05. Significance was calculated by paired two-tailed (B, D) t-test for normal distributed data, by paired one-tailed Wilcoxon test (E) for non-normal distributed data or by unpaired two-tailed (A) or one-tailed (C) for not normally distributed data. In Na+ quantifications, outliers were identified with ROUT method and not included in the analysis (A).
(F-H) Data is depicted as mean ± SEM. *p<0.05. Normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by unpaired two-tailed (H) and one-tailed (G) Man-Whitney t-test and by paired one-tailed Wilcoxon t-test for non-normal distributed data (F).
FIGURE 7. Indel signature of representative SLC8B1-KO in human Tregs.
Schematic representation of the SLC8B1 gene. Grey boxes indicate exons. The gRNA sequence (20 bp) is indicated by the left open black box and the PAM sequence (3 bp) is indicated by the right open black box. The dotted line and scissors indicate the Cas9 cutting site. The different KO sequences are listed, percentages indicate the relative contribution of each sequence for final KO score. For KO calculation, isolated DNA from the different conditions alongside specific primers targeting the gene of interest were investigated by Sanger sequencing (LCG Genomics). KO score is generated by ICE analysis tool (Synthego) that indicates the percentage of KO on the genomic level. SEQ ID NO:2 corresponds to nucleotides 562- 619 or GenBank accession No NM_024959.4 (human SLC8B1, transcript variant 1, mRNA).
DETAILED DESCRIPTION
One hypothesis to explain the rising incidence of autoimmune diseases in the western world refers to a component/components of the western diet being responsible. One characteristic of western diet and processed foods is an elevated salt content. In work leading to the present invention, the effects of high salt (HS) on immunometabolism of regulatory T cells (Tregs) and the functional consequences of HS on human Tregs were investigated in relation to autoimmunity.
It was first established that high salt (HS) conditions are leading to dysfunctionality in regulatory T cells (Tregs), even after short-term exposure to high salt conditions physiologically mimicking NaCI concentrations reached in vivo after a high salt diet. Furthermore, it was established the HS Treg (function and fitness) dysfunctionality resembles Treg dysfunctionality as occurring in Tregs of subjects suffering from an autoimmune disease (resemblance established with Tregs from rheumatoid arthritis (RA) patients, systemic lupus erythematosus (SLE) patients and multiple sclerosis (MS) patients). The observed Treg dysfunctionalities include changes in gene expression and immunometabolism in particular affecting Treg mitochondrial function. These findings were further corroborated in autoimmune disease (AID) models showing that only adoptive transfer of functional Tregs, and not of HS-treated Tregs, can contain AID. Under in vivo autoimmune context (and alike contexts) conditions which are similar to HS conditions as established herein, functional Tregs, such as adoptively transferred functional Tregs, will encounter conditions unfavorable for maintaining their functionality, and will become dysfunctional. In order to protect these functional Tregs from becoming dysfunctional, it was further established that inhibition of the mitochondrial Na+/Ca2+ exchanger NCLX in Tregs provides such protection. Adoptive transfer of Tregs in which NCLX has been blocked or eliminated (as illustrated using the pharmacological inhibitor CGP37157) prior to transfer were able to treat AID and ameliorate AID progression.
FOXP3+ regulatory T cells (T regs) are central for peripheral tolerance and their deregulation is associated with autoimmunity. Dysfunctional autoimmune Tregs display pro-inflammatory features and altered mitochondrial metabolism but factors contributing to this phenotype remain elusive. High salt (HS) intake has been identified to alter immune function and to promote autoimmunity. By investigating transcriptional changes of human Tregs over time, we identified that HS induces severe metabolic reprogramming, recapitulating key features of autoimmune Tregs. Mechanistically, extracellular HS raises intracellular Na+ that perturbs mitochondrial respiration by directly interfering with complex ll/lll of the electron transport chain (ETC). Metabolic disturbance of Tregs by temporary HS encounter or blockade of complex III rapidly induces a pro-inflammatory signature and FOXP3 downregulation, leading to long-term dysfunction in vitro and in different disease models in vivo. In line with this, Tregs of human subjects with HS-intake show distorted functional features. Importantly, the detrimental salt-induced effect could be reversed by inhibition of mitochondrial Na+/Ca2+ exchanger (NCLX), as illustrated using the pharmacological inhibitor CGP37157 . These results indicate that salt contributes to metabolic reprogramming as observed in autoimmune Tregs and that short-term HS encounter perturb metabolic fitness and long-term function of human Tregs with important implications for autoimmunity. Increased sodium concentrations can moreover be present in inflamed tissues independent of high salt dietary intake (e.g., Jantsch et al. 2015, Cell Metab 21:493-501; Jobin et al. 2021, Trends Immunol 42:469-479; Huhn et al. 2021, Proc Natl Acad Sci USA 118:e2102549118).
The invention therefore in a first aspect relates to (an) isolated regulatory T cell (s) (Treg cell (s), or simply Treg(s)) or to a population of isolated Treg cells characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited, in the Treg cell or cells. As indicated hereinabove, the advantage of Tregs with such modification resides in their increased functionality under unfavorable conditions as can occur when transferred into subjects having or suffering from an autoimmune disease, or by extension an (uncontrolled, such as persistent or chronic) inflammatory disease, or from a cardiovascular disease, independent of being or having been on a high salt diet prior to (therapeutic, adoptive) transfer of the Tregs. In one embodiment thereto, the function of NCLX can be inhibited, or substantially inhibited by means of incubating Tregs with a pharmacological compound, such as a compound capable of selectively or specifically inhibiting, or substantially inhibiting, NCLX activity. In a specific embodiment, the pharmacological compound is 7-Chloro-5-(2-chlorophenyl)-l,5-dihydro-4,l-benzothiazepin-2(3H)-one (CGP-37157).
In another embodiment, the expression of NCLX in Tregs can be inhibited, or substantially inhibited by means of a specific or selective inhibitor of NCLX expression. In particular, such specific or selective inhibitor may be a DNA nuclease specifically knocking out or disrupting NCLX, an RNase specifically targeting NCLX, an inhibitory oligonucleotide specifically targeting NCLX, or may be encoded on a transposon specifically targeting NCLX. Such inhibitory oligonucleotides may be selected from (the group consisting of) an antisense oligomer, a siRNA, a shRNA, a gapmer, and the likes; such DNA nucleases may be selected from (the group consisting of) a ZFN, a TALEN, a CRISPR-Cas, and a meganuclease; and such RNases may be selected from (the group consisting of) a ribozyme and a CRISPR-C2c2. More general details of these modalities are provided hereinafter.
In a particular embodiment to any of the above, the step of inhibiting, or substantially inhibiting function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in Tregs is performed in vitro or ex vivo, i.e. in Tregs such as obtained from a subject or in induced Tregs (more details further hereinafter). Herein, the inhibitor or function or expression of NCLX can e.g. be targeted to the mitochondria of the Tregs by means of e.g. a mitochondriotropic nanocarrier (see further).
NCLX
NCLX is also known as SLC8B1 (solute carrier family 8 member Bl) or (mitochondrial) sodium/lithium/calcium exchanger. Located on human chrl2:113, 298, 759-113, 359, 493 (GRCh38/hg38; minus strand), alternatively on human chrl2:113, 736, 576-113, 772, 914 (GRCh37/hgl9 by Entrez Gene; minus strand), or alternatively on human chrl2:113, 736, 564-113, 797, 298 (GRCh37/hgl9 by Ensembl; minus strand). Protein symbol: Q6J4K2-NCLX human; protein accession Q6J4K2. Reference sequence (REFSEQ) mRNAs: GenBank accession Nos. NM_001330466.2, NM_001358345.2, and NM_024959.4.
CGP37157
CGP37157 or CGP-37157 is a small molecule compound inhibiting NCLX. It is also known under CAS No. : 75450-34-9, has the chemical formula CisHnCLNOS, and as chemical name e.g. 7-Chloro-5-(2- chlorophenyl)-l,5-dihydro-4,l-benzothiazepin-2(3H)-one. The InChi Key for CGP37157 is KQEPIRKXSUIUTH-UHFFFAOYSA-N. Finally, the chemical structure of CGP37157 is:
Figure imgf000013_0001
Treg isolation and expansion, Treg engineering
Therapeutic application of Tregs as cell-based immunotherapy is a growing field. Several clinical trials are ongoing using Treg cell therapy in Graft-versus-Host Disease (GvHD; acute as well as chronic), and in the solid organ transplant setting (see, e.g., Table 1 in Romano et al. 2017, Transplant International 30: 745-753). Application of Tregs in managing autoimmune diseases (such as RA, SLE, MS, inflammatory bowel disease (IBD), type 1 diabetes (T1D)) and in managing the immunogenicity of gene therapeutics and biologies can also be envisaged. Applying Tregs in these context brings specificity to the immunosuppression, which is currently lacking when using standard immunosuppressants. Many efforts are ongoing to further increase the efficacy, function, and survival of human Treg cells in the adoptive transfer setting and these efforts include gene editing (e.g. Boardman et al. 2022, J Allergy Clin Immunol 149:1-11). One way of increasing the efficacy of Treg cell therapy resides in using Treg cells that are specific for a disease-related antigen (e.g. Boardman et al. 2016, Biochem Soc Trans 44:342-348; Fujio et al. 2006, J Immunol 177: 8140-8147). Autoimmune antigens, or autoantigens, are known for e.g. MS (e.g. myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG), FABP7, PROK2, RTN3, and SNAP91; Bronge et al. 2022, Sci Adv 8:eabnl823), T1D (e.g. insulin, glutamic acid decarboxylase 65 (GAD65), insulinoma antigen-2 (IA-2), heat shock protein (HSP), islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP), imogen-3, zinc transporter-8 (ZnT8), pancreatic duodenal homeobox factor 1 (PDX1), chromogranin A (CHGA), and islet amyloid polypeptide (IAPP); e.g. Han et al. 2013, Am J Transl Res 5:379-392). Other autoantigens include thyroglobulin, thyroid peroxidase, TSH receptor in thyroid diseases; insulin (proinsulin), glutamic acid decarboxylase (GAD), tyrosine phosphatase IA-2, heat-shock protein HSP65, islet-specific glucose6-phosphatase catalytic subunit related protein (IGRP) in type 1 diabetes; 21-OH hydroxylase in autoimmune adrenalitis; 17-alpha hydroxylase, histidine decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, in autoimmune polyendocrine syndromes; H+/K+ ATPase intrinsic factor in autoimmune gastritis and pernicious anemia; myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), proteolipid protein (PLP) in multiple sclerosis; acetyl-choline receptor in myasthenia gravis; retinol-binding protein (RBP) in autoimmune ocular syndromes; type II and type IX collagen in autoimmune inner ear diseases; tissue transglutaminase in celiac disease; pANCA histone HI protein in inflammatory bowel diseases; heat-shock protein HSP60 and oxy-light density lipoproteins in atherosclerosis, and; synuclein in Parkinson disease.
Tregs constitute about 5% of circulating CD4+ T cells and can be identified by the lineage marker forkhead box protein P3 (FOXP3). Tregs can be isolated from peripheral blood or umbilical cord blood (Brunstein et al. 2011, Blood 117:1061), from discarded paediatric thymuses (Dijke et al. 2016, Am J Transplant 16: 58), or via a GMP procedure from a standard leukapheresis product (Hoffmann et al. 2006, Biol Blood Marrow Transplant 12: 267). The latter procedure entails in a first step depletion of CD19+ cells followed by a second step of enrichment of cells expressing CD25 molecules. High expression of CD25 is only one of the Treg markers; other surface markers that could be employed in a Treg isolation and/or enrichment procedure include CCR8, CTLA4, CD38, TIGIT, ICOS, OX-40, 4-1BB, and GITR, as well as CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA, CD31, Foxp3, CD45RA, or combinations thereof such as e.g. CD4+CD25+CD127low selection (Piekarska et al. 2021, Folia Histochem Cytobiol 59:75-85; Santegoets et al. 2015, Cancer Immunol Immunother 64: 1271-1286). It is further possible to obtain Tregs by negative isolation procedures involving removal of non-Treg contaminants. Non-Treg contaminants can be removed by using e.g. CD49b antibodies optionally in combination with CD127 antibodies; as the Tregs do not expose CD49b or CD127, they remain untouched (Kleinewietfeld et al. 2009, Blood 113:827-836; Haase et al. 2015, J Immunother 38:250-258).
A further source of Tregs consists of conventional CD4+ T-cells that are reprogrammed into Tregs such as including inducing strong FOXP3 expression (also termed induced Tregs; e.g. Kanamori et al. 2016, Trends Immunol 37: 803-11; Honaker et al. 2020, Sci Transl Med 12:eaay6422).
Tregs can by polyclonal Tregs (such as expanded ex vivo using anti-CD3/CD28-coated beads in the presence of high dose of IL-2), in vitro amplified/expanded Tregs (such as in protocols using drugs like rapamycin or all-trans retinoic acid (ATRA); Golovina et al. 2011, PLoS One 6: el5868; Scotta et al. 2013, Haematologica 98: 1291), antigen-specific Tregs (amplified in the presence of antigen-presenting cells; or activated (by CD40 ligand) allogeneic B cells (in the presence of IL-2), e.g. Putnam et al. 2013, Am J Transplant 13: 3010; or via engineering with a T cell receptor/TCR - dependent on subject's HLA type), or engineered chimeric antigen receptor (CAR)-Tregs (monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs; independent on subject's HLA-type), B-cell-targeting antibody receptor (BAR) Tregs, design Tregs, or chimeric cytokine receptor (CCR) Tregs. Modular CARs incorporate interchangeable antigen-targeting moieties (Koristka et al. 2018, J Autoimmun 90:116-131).
Gene transfer into Tregs is, similar as into conventional T cells, possible via lentiviruses or retroviruses or by using transposase-based systems (Zhou et al.2015, J Immunol 195:2493-2501; Ivies et al. 1997, Cell 91:501-510). Genome editing with Zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) of Tregs has become possible (see e.g. Boardman et al. 2022, J Allergy Clin Immunol 149:1-11); rapid and efficient Crispr-Cas based editing procedures have been added to the repertoire (e.g. Van Zeebroeck et al. 2021, Front Immunol. 12:655122). Plasmid-based delivery of transgenes (e.g. CAR-encoding genes) via transposon/transposase systems, e.g. the sleeping beauty system, has also been reported (Fritsche et al. 2019, Trends Biotechnol 38:1099-1112).
Delivery of the gene transfer or genome editing components into Tregs is possible via electroporation, delivery in nanoparticles, or via viruses (including adeno-associated viruses, AAVs). Genome editing may also be applied to open the Treg adoptive transfer field to allogeneic Treg transfer: indeed, removal of the endogenous TCR limits stimulation of TCR-muted Tregs by allo-recognition (Boardman et al. 2022, J Allergy Clin Immunol 149:1-11; Boardman et al. 2016, Curr Transplant Rep 3:275-283). Alternatively, Treg allogeneic HLA molecules can be replaced with nonclassical HLA molecules or Tregs can be modified to overexpress molecules such as Siglec ligands or CD47 to reduce NK cell-mediated lysis (Boardman et al. 2022, J Allergy Clin Immunol 149:1-11). A series of Treg modifications is included in Table 1 of Amini et al. 2021 (Front Immunol 11 :611638). Knockdown of gene expression in Tregs by means of shRNA (e.g. Li et al. 2021, Front Cell Dev Biol 9:708562), siRNA (e.g. Zhang et al. 2009, Beijing Da Xue Xue Bao Yi Xue Ban 41:313-318), antisense RNA (e.g. Revenko et al. 2022, J Immunother Cancer 10:e003892), and antisense RNA targeted to Tregs via an aptamer (e.g. Manrique-Rincon et al. 2021, Mol Ther Nucleic Acids 25:143-151) all have been described. A procedure to obtain Treg-specific aptamers has been described (Veeramani et al. 2015, Cancer Res 75 (15 Supplement):5022). All this indicates that the common tools of genetic modification of a cell are readily applicable to Tregs.
In vitro or ex vivo expansion of isolated and/or enriched Tregs is feasible, and often required to get hold of an amount of Tregs sufficient for enabling adoptive transfer. These hurdles can at present already be overcome as witnessed by the expanding number of clinical trials relying on adoptive transfer of Tregs. Kits for isolation and expansion of human Tregs are commercially available (e.g. Miltenyi Biotec: "CD4+ CD25+ CD45RA+ Regulatory T Cell Isolation Kit, human", "Treg Expansion Kit, human"). Furthermore, the Treg field in general and the field of Treg expansion in particular is in full development and reported advances include for instance ex vivo procedures assisted by co-culture with multipotent adult progenitor cells (Reading et al. 2021, Front Immunol 12:716606). Furthermore, it was reported that therapeutic Treg expansion protocols may need to take into account looking for proper CD39/CD73 coexpression in the expanded Tregs (Jarvis et al. 2021, Communications Biology 4:1186). All above features of Treg isolation and/or expansion, Treg engineering, adoptive Treg cell transfer, etc., are frequently subject of review papers, e.g. Ferreira et al. 2019, Nat Rev Drug Discov 18:749-769.
Tregs can be further be coated with e.g. a nanoparticle "backpack" loaded with interleukin-2 (IL-2) to improve initial engraftment after adoptive transfer (Marshall et al. 2023, J Biomed Mater Res 111:185- 197; these authors designed loadable nanoparticles of acceptable sizes and electrostatic properties allowing for conjugation to the surface of viable Tregs).
Mitochondrial targeting
The field of mitochondriotopic/mitochondria-targeted nanocarriers is expanding with some even having entered clinical phase studies. Such nanocarriers were demonstrated to deliver therapeutic payloads such as small molecules into mitochondria as well as to be capable of gene or nucleic acid delivery into mitochondria. The use of receptor-ligand pairs has been applied to mitochondria-targeted nanocarriers designed for therapeutic payload delivery specifically in cancer cells, therewith indicating that "hierarchical targeting" and passing different physiological barriers are feasible (reviewed by e.g. Liew et al . 2021, Angew Chem Int Ed Engl 60:2232-2256).
Thus, in a further embodiment to the first aspect of this invention, the isolated regulatory T cell(s) (Treg cell(s), or simply Treg(s)) or population of isolated Treg cells characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited, in the Treg cell or cells, are more in particular any of polyclonal Treg cells, in vitro amplified or expanded Tregs, antigen-specific Tregs, engineered T cell receptor (TCR)-Tregs, engineered chimeric antigen receptor (CAR)-Tregs, monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs, B-cell- targeting antibody receptor (BAR) Tregs, design Tregs, or chimeric cytokine receptor (CCR) Tregs.
In a further aspect, the invention relates to isolated TCR-engineered Treg-cells, CAR-engineered Treg- cells (monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs), BAR-Treg cells, design Tregs, or chimeric cytokine receptor (CCR) Tregs characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited in this/these Treg cell or cells.
In a further aspect, the invention relates to compositions comprising any of the above described isolated Treg cells/Tregs or populations of isolated Treg cells/Tregs in which the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited. In one embodiment thereto, such compositions are pharmacological compositions or pharmacologically acceptable compositions.
In a further aspect, the invention relates to Treg cell therapy, one goal of it being to induce or re-establish immune tolerance. The invention therefore relates to any of the above described isolated Tregs in which the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited, or to any composition comprising such Tregs, for use as a medicament, or for use in the manufacture of a medicament. In one embodiment, the medicament is a medicament for/for use in/or for use in a method of treating or suppressing (progression of) an auto-immune disease; for/for use in/or for use in a method of treating or suppressing (progression of) an inflammatory disease (such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease); is for/for use in/or for use in a method of treating or suppressing (progression of) an uncontrolled infection (at least those type of infections in which normalization of Tregs can be of help in treating or suppressing it); for/for use in/or for use in a method of treating or suppressing (progression of) transplant, graft or allograft rejection; or is for/for use in/or for use in a method of treating or inhibiting (progression of) cardiovascular disease (CVD). Furthermore, such medicament may be administered (in a therapeutically effective amount) to a subject being or having been on a diet containing high salt (although not a prerequisite, as high salt conditions in inflamed tissue can occur independent of dietary salt, see above). Furthermore in particular, such isolated Treg(s) or population of isolated Tregs or composition comprising such Tregs, are for use in Treg cell therapy, in particular for use in adoptive (Treg) cell transfer (ACT) therapy. In a further embodiment, such Tregs are autologous Treg cells (i.e. Tregs obtained from the subject to be treated, and then optionally further expanded), allogeneic Treg cells (i.e. Tregs obtained from a subject different from the subject to be treated, and then optionally further expanded), or induced Treg cells (such as induced starting from autologous or allogeneic CD4+ T-cells). Further included in the invention are methods for (or for use in) treating or suppressing (progression of) an auto-immune disease in a subject; methods for (or for use in) treating or suppressing (progression of) an inflammatory disease in a subject (such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease); methods for (or for use in) treating or suppressing (progression of) an uncontrolled infection (at least those type of infections in which normalization of Tregs can be of help in treating or suppressing it) in a subject; methods for (or for use in) for treating or suppressing (progression of) transplant, graft or allograft rejection in a subject; and methods for (or for use in) for treating or suppressing or inhibiting (progression of) cardiovascular disease (CVD) in a subject; such methods including a step of administering to the subject (a therapeutically effective amount of) any isolated Tregs in which the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited, or including a step of administering to the subject (a therapeutically effective amount of) any composition comprising such Tregs. By means of such administering, the auto-immune disease, inflammatory disease, or infection, is treated or (its progression) suppressed; or the transplant, graft or allograft rejection is treated or (its progression) suppressed; or the CVD is treated or (its progression) inhibited. Besides being characterized by having function or expression of their mitochondrial Na+/Ca2+ exchanger NCLX being inhibited, such Tregs may be any of polyclonal Treg cells, in vitro amplified or expanded Tregs, antigen-specific Tregs, engineered T cell receptor (TCR)-Tregs, engineered chimeric antigen receptor (CAR)-Tregs, monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs, B-cell-targeting antibody receptor (BAR) Tregs, design Tregs, or chimeric cytokine receptor (CCR) Tregs.
In another aspect of the invention, the step of inhibiting or substantially inhibiting function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in Tregs is performed by administering an inhibitor of function or expression of NCLX selectively or specifically to Tregs of a subject in need thereof. Treg selectivity or specificity can be approximated or obtained by targeting the NCLX inhibitor to Tregs e.g. by targeting the NCLX inhibitor to a surface marker of Tregs. Such surface marker can be any of the markers relied on in procedures for isolation and/or enrichment of Tregs (see further). In case of a pharmacological NCLX inhibitor (small molecule), selectivity or specificity for mitochondria can optionally be added to the Treg selectivity or specificity. Dual cell/mitochondria targeting (specificity or selectivity) has been established (reviewed by e.g. Liew et al . 2021, Angew Chem Int Ed Engl 60:2232-2256). The invention therefore relates to Treg-selective or -specific inhibitors or function or expression of NCLX, optionally to Treg mitochondria-selective or -specific inhibitors of function or expression of NCLX. Such inhibitors are in particular for use as a medicament, or for use in the manufacture of a medicament. In particular, such Treg-selective inhibitors or function or expression of NCLX is for/for use in/or for use in a method of treating or suppressing (progression of) an auto-immune disease; for/for use in/or for use in a method of treating or suppressing (progression of) an inflammatory disease (such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease); for/for use in/or for use in a method of treating or suppressing (progression of) an uncontrolled infection (at least those type of infections in which normalization of Tregs can be of help in treating or suppressing it); for/for use in/or for use in a method of, treating or suppressing (progression of) transplant, graft or allograft rejection; or for/for use in/or for use in a method of treating or inhibiting (progression of) cardiovascular disease. Alternatively, the invention relates to compositions comprising a Treg-selective or -specific inhibitor or function or expression of NCLX, optionally to a Treg mitochondria-selective or -specific inhibitor of function or expression of NCLX. Such compositions are in particular for use as medicament or for use in the manufacture of a medicament. In particular, such composition or medicament is for/for use in/or for use in a method of treating or suppressing (progression of) an auto-immune disease; for/for use in/or for use in a method of treating or suppressing (progression of) an inflammatory disease (such as an uncontrolled inflammatory disease, or such as persistent or chronic inflammatory disease); for/for use in/or for use in a method of treating or suppressing (progression of) an uncontrolled infection (at least those type of infections in which normalization of Tregs can be of help in treating or suppressing it); for/for use in/or for use in a method of treating or suppressing (progression of) transplant, graft or allograft rejection; or for/for use in/or for use in a method of treating or inhibiting (progression of) cardiovascular disease. Any such methods of treating, suppressing or inhibiting, include a step of administering to the subject a therapeutically effective amount of the composition or medicament comprising a Treg-selective inhibitor of function or expression of NCLX. By means of such administering, the auto-immune or inflammatory disease, or infection, is treated or (its progression) suppressed; the transplant, graft or allograft rejection is treated or (its progression) suppressed; or the cardiovascular disease is treated or (its progression) inhibited.
A further aspect of the invention refers to methods of inhibiting function or expression of NCLX in Tregs, such methods comprising selectively or specifically targeting Tregs (optionally selectively or specifically targeting Treg mitochondria) with an inhibitor of function or expression of NCLX. In particular, when the inhibitor of function of NCLX is a pharmacological inhibitor (small molecule), then such methods can comprise selectively targeting mitochondria in Tregs with the inhibitor of function of NCLX.
Autoimmune diseases
Over 50 million subjects in the United States alone and more than 4% of the world population are thought to be affected by an autoimmune disorder. As derivable from the previous sections, autoimmune diseases include multiple sclerosis, type 1 diabetes, autoimmune thyroid diseases, autoimmune adrenalitis, autoimmune polyendocrine syndromes, autoimmune gastritis, pernicious anemia, myasthenia gravis, autoimmune ocular syndromes, autoimmune inner ear diseases, celiac disease, inflammatory bowel disease, and Parkinson's disease, autoimmune encephalitis, lupus, autoimmune hepatitis, etc.
Inflammatory diseases
Inflammatory diseases include ankylosing spondylitis, gout, arthritis/psoriatic arthritis/rheumatoid arthritis, scleroderma, vasculitis, Kawasaki disease, mixed connective tissue disease, myositis, Sjogren's syndrome, spondyloarthritis, spondyloarthropathy, idiopathic arthritis, uveitis. Inflammation is moreover an important factor in the development of cardiovascular diseases (CVDs) and Treg therapy has been suggested as modality for targeting CVDs (e.g. Meng et al. 2016, Nat Rev Cardiol 13:167-179).
Transplant
The term transplant as referred to herein is embracing organ transplants or allografts (e.g. liver, kidney, heart, lung) as well as cellular transplants (e.g. bone marrow cells, (hematopoietic or embryonic or adult) stem cells, Langerhans cells or pancreatic islets, etc.). Improper immunosuppression in a subject receiving the transplant is a cause underlying hyperacute, acute or chronic transplant rejection, or underlying graft vs host disease (GvHD). Skin, cornea, vascular and bone grafts are usually of autologous origin and normally do not cause rejection reactions - if, however, of allogeneic origin, the current invention could be applied in suppressing such allograft rejection.
Inhibition of a target of interest
The term "antagonist" or "inhibitor" of a target as used interchangeable herein refers to inhibitors of function or to inhibitors of expression of a target of interest. Interchangeable alternatives for "antagonist" include inhibitor, repressor, suppressor, inactivator, and blocker. An "antagonist" thus refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with target expression, activation, function, or activity.
Downregulating of expression of a gene encoding a target is feasible through "genetic" means (e.g., by administering siRNA, shRNA or antisense oligonucleotides to the target gene). Biopharmaceutical and genetic antagonists include such entities as antisense oligonucleotides, gapmers, siRNA, shRNA, zinc- finger nucleases, meganucleases, TAL effector nucleases, CRISPR-Cas effectors, etc. (general description of these compounds included hereinafter).
Inactivation or inhibition of a process as envisaged in the current invention refers to different possible levels of inactivation or inhibition, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even 100% or more if inactivation or inhibition (compared to a normal situation or compared to the situation prior to starting the inactivation or inhibition). The nature of the inactivating/inhibitory compound is not vital/essential to the invention as long as the process envisaged is inactivated/inhibited such as to treat or inhibit (progression of) auto-immune diseases or inflammatory diseases etc. as described herein.
Downregulating expression of a gene encoding a target is feasible through agents include entities such as antisense oligonucleotides, gapmers, siRNA, shRNA, zinc-finger nucleases, meganucleases, Argonaute, TAL effector nucleases, CRISPR-Cas effectors, and nucleic acid aptamers. In particular, any of these agents is specifically, selectively, or exclusively acting on or antagonizing the target of interest; or any of these agents is designed for specifically, selectively, or exclusively acting on or antagonizing the target of interest. In the context of the present invention, the target of interest in particular is NCLX.
One process of modulating/downregulating expression of a gene/target gene of interest relies on antisense oligonucleotides (ASOs), or variants thereof such as gapmers. An antisense oligonucleotide (ASO) is a short strand of nucleotides and/or nucleotide analogues that hybridizes with the complementary mRNA in a sequence-specific or -selective manner. Formation of the ASO-mRNA complex ultimately results in downregulation of target protein expression (Chan et al. 2006, Clin Exp Pharmacol Physiol 33:533-540; this reference also describes some of the software available for assisting in design of ASOs). Modifications to ASOs can be introduced at one or more levels: phosphate linkage modification (e.g. introduction of one or more of phosphodiester, phosphoramidate or phosphorothioate bonds), sugar modification (e.g. introduction of one or more of LNA (locked nucleic acids), 2'-O-methyl, 2'-O-methoxy-ethyl, 2' -fluoro, S-constrained ethyl or tricyclo-DNA and/or non-ribose modifications (e.g. introduction of one or more of phosphorodiamidate morpholinos or peptide nucleic acids). The introduction of 2'-modifications has been shown to enhance safety and pharmacologic properties of antisense oligonucleotides. Antisense strategies relying on degradation of mRNA by RNase H requires the presence of nucleotides with a free 2' -oxygen, i.e. not all nucleotides in the antisense molecule should be 2'-modified. The gapmer strategy has been developed to this end. A gapmer antisense oligonucleotide consists of a central DNA region (usually a minimum of 7 or 8 nucleotides) with (usually 2 or 3) 2'-modified nucleosides flanking both ends of the central DNA region. This is sufficient for the protection against exonucleases while allowing RNAseH to act on the (2'-modification free) gap region. Antidote strategies are available as demonstrated by administration of an oligonucleotide fully complementary to the antisense oligonucleotide (Crosby et al. 2015, Nucleic Acid Ther 25:297-305). Uptake of oligonucleotides by cells can be spontaneous or be assisted by e.g. transfection etc..
Another process to modulate expression of a gene/target gene of interest is based on the natural process of RNA interference. It relies on double-stranded RNA (dsRNA) that is cut by an enzyme called Dicer, resulting in double stranded small interfering RNA (siRNA) molecules which are 20-25 nucleotides long. siRNA then binds to the cellular RNA-lnduced Silencing Complex (RISC) separating the two strands into the passenger and guide strand. While the passenger strand is degraded, RISC is cleaving mRNA specifically or selectively at a site instructed by the guide strand. Destruction of the mRNA prevents production of the protein of interest and the gene is 'silenced'. siRNAs are dsRNAs with 2 nt 3' end overhangs whereas shRNAs are dsRNAs that contains a loop structure that is processed to siRNA. shRNAs are introduced into the nuclei of target cells using a vector (e.g. bacterial or viral) that optionally can stably integrate into the genome. Apart from checking for lack of cross-reactivity with non-target genes, manufacturers of RNAi products provide guidelines for designing siRNA/shRNA. siRNA sequences between 19-29 nt are generally the most effective. Sequences longer than 30 nt can result in nonspecific silencing. Ideal sites to target include AA dinucleotides and the 19 nt 3' of them in the target mRNA sequence. Typically, siRNAs with 3' dUdU or dTdT dinucleotide overhangs are more effective. Other dinucleotide overhangs could maintain activity but GG overhangs should be avoided. Also to be avoided are siRNA designs with a 4-6 poly(T) tract (acting as a termination signal for RNA pol III), and the G/C content is advised to be between 35-55%. shRNAs should comprise sense and antisense sequences (advised to each be 19-21 nt in length) separated by loop structure, and a 3' AAAA overhang. Effective loop structures are suggested to be 3-9 nt in length. It is suggested to follow the sense-loop-antisense order in designing the shRNA cassette and to avoid 5' overhangs in the shRNA construct. shRNAs are usually transcribed from vectors, e.g. driven by the Pol III U6 promoter or Hl promoter. Vectors allow for inducible shRNA expression, e.g. relying on the Tet-on and Tet-off inducible systems commercially available, or on a modified U6 promoter that is induced by the insect hormone ecdysone. A Cre-Lox recombination system has been used to achieve controlled expression in mice. Synthetic shRNAs can be chemically modified to affect their activity and stability. Plasmid DNA or dsRNA can be delivered to a cell by means of transfection (lipid transfection, cationic polymer-based nanoparticles, lipid or cellpenetrating peptide conjugation) or electroporation. Vectors include viral vectors such as lentiviral, retroviral, adenoviral and adeno-associated viral vectors.
Ribozymes (ribonucleic acid enzymes) are another type of molecules that can be used to modulate expression of a gene/target gene of interest. They are RNA molecules capable of catalyzing specific biochemical reactions, in the current context capable of targeted cleavage of nucleotide sequences, in particular targeted cleavage of a RNA/RNA target of interest. Examples of ribozymes include the hammerhead ribozyme, the Varkud Satellite ribozyme, Leadzyme and the hairpin ribozyme.
Besides the use of the inhibitory RNA technology, modulation of expression of a gene of interest can be achieved at DNA level such as by gene therapy to knock-out, knock-down or disrupt the target gene/gene of interest. As used herein, a "gene knock-out" can be a gene knockdown or the gene can be knocked out, knocked down, disrupted or modified by a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques such as described hereafter, including, but not limited to, retroviral gene transfer. One way in which genes can be knocked out, knocked down, disrupted or modified is by the use of zinc finger nucleases. Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target a desired DNA sequence/DNA sequence of interest, which enable zinc-finger nucleases to target unique sequence within a complex genome. By taking advantage of the endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.
Other technologies for genome customization that can be used to specifically or selectively knock out, knock down or disrupt a gene/gene of interest are meganucleases and TAL effector nucleases (TALENs, Cellectis bioresearch). A TALEN® is composed of a TALE DNA binding domain for sequence-specific or sequence-selective recognition fused to the catalytic domain of an endonuclease that introduces double strand breaks (DSB). The DNA binding domain of a TALEN® is capable of targeting with high precision a large recognition site (for instance 17bp). Meganucleases are sequence-specific or sequence-selective endonucleases, naturally occurring "DNA scissors", originating from a variety of single-celled organisms such as bacteria, yeast, algae and some plant organelles. Meganucleases have long recognition sites of between 12 and 30 base pairs. The recognition site of natural meganucleases can be modified in order to target native genomic DNA sequences (such as endogenous genes) or DNA sequences of interest. Another recent genome editing technology is the CRISPR/Cas system, which can be used to achieve RNA- guided genome engineering (including knock-out, knock-down or disruption of a gene of interest). CRISPR interference is a genetic technique which allows for sequence-specific or sequence-selective control of expression of a gene of interest in prokaryotic and eukaryotic cells. It is based on the bacterial immune system-derived CRISPR (clustered regularly interspaced palindromic repeats) pathway. Recently, it was demonstrated that the CRISPR-Cas editing system can also be used to target RNA. It has been shown that the Class 2 type Vl-A CRISPR-Cas effector C2c2 (Casl3a; CRISPR-Casl3a or CRISPR-C2c2) can be programmed to cleave single stranded RNA targets carrying complementary protospacers (Abudayyeh et al. 2016 Science353/science.aaf5573). C2c2 is a single-effector endoRNase mediating ssRNA cleavage once it has been guided by a single crRNA guide toward a target RNA/RNA of interest.
Methods for administering nucleic acid-based therapeutic modalities/agents include methods applying non-viral (DNA or RNA) or viral nucleic acids (DNA or RNA viral vectors). Methods for non-viral nucleic acid administration include the injection of naked DNA (circular or linear), electroporation, the gene gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes (e.g. complexes of nucleic acid with DOTAP or DOPE or combinations thereof, complexes with other cationic lipids), dendrimers, viral- like particles, inorganic nanoparticles, hydrodynamic delivery, photochemical internalization (Berg et al. 2010, Methods Mol Biol 635:133-145) or combinations thereof.
Many different vectors have been used in nucleic acid administration. Currently the major groups are adenovirus or adeno-associated virus vectors, retrovirus vectors , naked or plasmid DNA, and lentivirus vectors. Combinations are also possible, e.g. naked or plasmid DNA combined with adenovirus, or RNA combined with naked or plasmid DNA to list just a few. Other viruses (e.g. alphaviruses, vaccinia viruses such as vaccinia virus Ankara) are used in nucleic acid administration and are not excluded in the context of the current invention.
Administration may be aided by specific formulation of the nucleic acid e.g. in liposomes (lipoplexes) or polymersomes (synthetic variants of liposomes), as polyplexes (nucleic acid complexed with polymers), carried on dendrimers, in inorganic (nano)particles (e.g. containing iron oxide in case of magnetofection), or combined with a cell penetrating peptide (CPP) to increase cellular uptake. Organ- or cellular-targeting strategies may also be applied to the nucleic acid (nucleic acid combined with organ- or cell-targeting moiety); these include passive targeting (mostly achieved by adapted formulation) or active targeting (e.g. by coupling a nucleic acid-comprising nanoparticle with any compound (e.g. an aptamer or antibody or antigen binding molecule) binding to a target organ- or cell-specific antigen) (e.g. Steichen et al. 2013, Eur J Pharm Sci 48:416-427).
CPPs enable translocation of their payload of interest across the plasma membrane. CPPs are alternatively termed Protein Transduction Domains (TPDs), usually comprise 30 or less (e.g. 5 to 30, or 5 to 20) amino acids, and usually are rich in basic residues, and are derived from naturally occurring CPPs (usually longer than 20 amino acids), or are the result of modelling or design. A non-limiting selection of CPPs includes the TAT peptide (derived from HIV-1 Tat protein), penetratin (derived from Drosophila Antennapedia - Antp), pVEC (derived from murine vascular endothelial cadherin), signal-sequence based peptides or membrane translocating sequences, model amphipathic peptide (MAP), transportan, MPG, polyarginines; more information on these peptides can be found in Torchilin 2008 (Adv Drug Deliv Rev 60:548-558) and references cited therein. CPPs can be coupled to carriers such as nanoparticles, liposomes, micelles, or generally any hydrophobic particle. Coupling can be by absorption or chemical bonding, such as via a spacer between the CPP and the carrier. To increase target specificity or target selectivity, an antibody binding to a target-specific antigen can further be coupled to the carrier (Torchilin 2008, Adv Drug Deliv Rev 60:548-558). CPPs have already been used to deliver payloads as diverse as plasmid DNA, oligonucleotides, siRNA, peptide nucleic acids (PNA), proteins and peptides, small molecules and nanoparticles inside the cell (Stalmans et al. 2013, PloS One 8:e71752).
Any other modification of the DNA or RNA to enhance efficacy of nucleic acid therapy is likewise envisaged to be useful in the context of the applications as outlined herein. The enhanced efficacy can reside in enhanced expression, enhanced delivery properties, enhanced stability and the like.
A specific or selective inhibitor of a target of interest may exert the desired level of inhibition of the target of interest with an IC50 of 1000 nM or less, with an IC50 of 500 nM or less, with an IC50 of 100 nM or less, with an IC50 of 50 nM or less, with an IC50 of 10 nM or less, with an IC50 of 1 nM or less, with an IC50 between 1 pM and InM, or with an IC50 between 0.1 pM and 10 nM.
Cross-inhibition of more than one target is possible; for clinical development it can e.g. be desired to be able to test an inhibitor in a suitable in vitro model or in vivo animal model before starting clinical testing with the same inhibitor in a human population, which may require the inhibitor to cross-inhibit the animal (or other non-human) target and the orthologous human target.
Specificity or selectivity of inhibition refers to the situation in which an inhibitor is, at a certain concentration (sufficient to inhibit the target of interest) inhibiting the target protein with higher efficacy (e.g. with an at least 2-fold, 5-fold, or 10-fold lower IC50, e.g. at least 20-, 50- or 100-fold or more lower IC50) than the efficacy with which it is possibly (if at all) inhibiting other targets (targets not of interest). Such specificity or selectivity of inhibition is in particular determined within the setting of the target subject (e.g. human patient, or animal model) and thus can encompass/does not exclude inhibition of (at least one) orthologous target. Exclusivity of inhibition refers to the situation in which an inhibitor is inhibiting only the target of interest.
Specificity or selectivity of cell targeting, in particular Treg targeting refers to the situation in which a composition, at a certain concentration, is interacting with the intended target cell (such as binding to, or such as causing inhibition of function or expression of NCLX in the intended target cell) with higher efficacy (e.g. with an at least 2-fold, 5-fold, or 10-fold higher efficacy, or e.g. with at least 20-, 50- or 100- fold higher efficacy) than the efficacy with which the composition is interacting with other cells (not intended as target cell). Exclusivity of cell targeting refers to the situation in which a composition is interacting only with the intended target cell.
Treatment / therapeutically effective amount
The terms therapeutic modality, therapeutic agent, agent, and drug are used interchangeably herein, and likewise relate to the Tregs as described herein (with inhibited function or expression of NCLX). All refer to a therapeutically active compound, or to a therapeutically active composition (comprising one or more therapeutically active compounds).
"Treatment"/"treating" refers to any rate of reduction, delaying or retardation of the progress of the disease or disorder, or a single symptom thereof, compared to the progress or expected progress of the disease or disorder, or single symptom thereof, when left untreated. This implies that a therapeutic modality on its own may not result in a complete or partial response (or may even not result in any response), but may, in particular when combined with other therapeutic modalities (such as other immunosuppressants or therapeutic modalities other than the Tregs of the invention for treating or suppressing autoimmune or inflammatory diseases or transplant, graft or allograft rejection), contribute to a complete or partial. More desirable, the treatment results in no/zero progress of the disease or disorder, or single symptom thereof (i.e. "inhibition" or "inhibition of progression"), or even in any rate of regression of the already developed disease or disorder, or single symptom thereof. "Suppression/suppressing" can in this context be used as alternative for "treatment/treating". Treatment/treating also refers to achieving a significant amelioration of one or more clinical symptoms associated with a disease or disorder, or of any single symptom thereof. Depending on the situation, the significant amelioration may be scored quantitatively or qualitatively. Qualitative criteria may e.g. by patient well-being. In the case of quantitative evaluation, the significant amelioration is typically a 10% or more, a 20% or more, a 25% or more, a 30% or more, a 40% or more, a 50% or more, a 60% or more, a 70% or more, a 75% or more, a 80% or more, a 95% or more, or a 100% improvement over the situation prior to treatment. The time-frame over which the improvement is evaluated will depend on the type of criteria/disease observed and can be determined by the person skilled in the art.
A "therapeutically effective amount" refers to an amount of a therapeutic agent to treat, inhibit or prevent a disease or disorder in a subject (such as a mammal). Efficacy in vivo can, e.g., be measured by assessing the duration of survival (e.g. overall survival), time to disease progression (TTP), response rates (e.g., complete response and partial response, stable disease), length of progression-free survival (PFS), duration of response, and/or quality of life.
The term "effective amount" or "therapeutically effective amount" may depend on the dosing regimen of the agent/therapeutic agent or composition comprising the agent/therapeutic agent (e.g. medicament or pharmaceutical composition). The effective amount will generally depend on and/or will need adjustment to the mode of contacting or administration. The effective amount of the agent or composition comprising the agent is the amount required to obtain the desired clinical outcome or therapeutic effect without causing significant or unnecessary toxic effects (often expressed as maximum tolerable dose, MTD). To obtain or maintain the effective amount, the agent or composition comprising the agent may be administered as a single dose or in multiple doses. The effective amount may further vary depending on the severity of the condition that needs to be treated; this may depend on the overall health and physical condition of the subject or patient and usually the treating doctor's or physician's assessment will be required to establish what is the effective amount. The effective amount may further be obtained by a combination of different types of contacting or administration.
The aspects and embodiments described above in general may comprise the administration of one or more therapeutic compounds to a subject (such as a mammal) in need thereof or in need of treatment. In general a (therapeutically) effective amount of (a) therapeutic compound(s) is administered to the mammal in need thereof in order to obtain the described clinical response(s).
"Administering" means any mode of contacting that results in interaction between an agent or composition comprising the agent (such as a medicament or pharmaceutical composition) and an object (e.g. cell, tissue, organ, body lumen) with which said agent or composition is contacted. The interaction between the agent or composition and the object can occur starting immediately or nearly immediately with the administration of the agent or composition, can occur over an extended time period (starting immediately or nearly immediately with the administration of the agent or composition), or can be delayed relative to the time of administration of the agent or composition. More specifically the "contacting" results in delivering an effective amount of the agent or composition comprising the agent to the object. In a further aspect, the invention relates to methods of producing the isolated Tregs in which the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited, or substantially inhibited, such methods comprising a step of isolating Treg-cells from peripheral blood, umbilical cord blood, thymus or leukapheresis product obtained from a subject, or a step of reprogramming conventional CD4+ T-cells obtained from a subject into Tregs (or, in other words, a step of producing induced Tregs) (cfr. supra). Such methods may further comprise a step of ex-vivo expanding the isolated or reprogrammed Tregs (cfr. supra). Optionally, such methods may further comprise a step of ex-vivo manipulation to inhibit, or to substantially inhibit, the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in the Tregs by means of pharmacological inhibition or by means of a DNA nuclease specifically knocking out or disrupting NCLX, an RNase specifically targeting NCLX, or an inhibitory oligonucleotide specifically targeting NCLX (cfr. supra).
Yet another aspect of the invention relates to therapeutic or pharmaceutical kits comprising at least one vial comprising at least one of the isolated Treg cell or population of Treg cells, or comprising a composition comprising such Tregs, wherein the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in said Tregs is inhibited, or substantially inhibited. In one particular embodiment, said Tregs are of allogeneic origin (independent of having been isolated, enriched and/or expanded from natural Tregs or from induced Tregs).
Therapeutic or pharmaceutical kits
The invention further relates to (therapeutic/pharmaceutical/medicament) kits comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising a Treg cell according to the invention or comprising a composition comprising a Treg cell according to the invention. Other optional components of such kit include use instructions; one or more containers with sterile pharmaceutically acceptable carriers, excipients or diluents [such as for producing or formulating a (pharmaceutically acceptable) composition of the invention]; one or more syringes; one or more needles; etc. In particular, such kits may be pharmaceutical kits.
Other Definitions
The present invention is described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are nonlimiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.
In referring to genes or proteins herein, no distinction is made in the annotation. Thus, whereas for example the human NCLX gene would be referred to as the NCLX gene, the mRNA as NCLX mRNA, and the protein as NCLX, such distinction is not, or not always, made hereinabove or hereinafter.
In any of the above, a "subject" in general is a mammalian species. The mammalian species in general is a higher species including primates, cattle (e.g. cows, sheep, goats, pigs), horses, and pets (e.g. dogs, cats). In one embodiment the subject is a human subject.
It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims The content of the documents cited herein are incorporated by reference. EXAMPLES
EXAMPLE 1. Materials and methods
Cell isolation and cell sorting
Human Tregs were isolated as described before (Arroyo-Hornero et al. 2022, Allergy 77:2818-2821; Van Zeebroeck et al. 2021, Front Immunol 12:655122; Farh et al. 2015, Nature 518:337-343) from the peripheral blood of healthy subjects or isolated from buffy coats of healthy donors purchased from the Belgian Red Cross in compliance with institutional review board protocols (CME2019/042 and CME2016/629). In brief, CD4+ T cells were isolated by incubating whole blood with RosetteSep™ Human CD4+ T Cell Enrichment Cocktail (15062, Stemcell) followed by Ficoll-Paque PLUS (GE17-1440-02, Sigma) gradient centrifugation or by using EasySep™ Human CD4+ T Cell Isolation Kit (17952, StemCell). CD25- enriched and -depleted CD4+ cells were isolated with CD25 microbeads II (130-097-044, Miltenyi Biotec). Regulatory T cells (Tregs) were FACS sorted with a FACS Aria II (BD Biosciences) using CD4 APC-Cy7 (clone RPA-T4), CD25 PeCy7 (clone M-A251) and CD127 PerCP-Cy5.5 (clone A019D5). T effector (Teff) cells were isolated by untouched CD4+ T cell isolation (17952, Stemcell) or were FACS sorted for CD25-CD127+ cells. Tregs were isolated according to the following parameters: CD4+CD25hlCD127low to a FOXP3 purity >96%.
Cell culture, Treg suppression assay and pre-incubation
Human T cells or PBMC were cultured if not otherwise stated in 96-well round-bottom plates (Costar) at 5xl04 cells per well in serum-free XVIVO15 (BE02-060F, LONZA) or XVIVO15 medium supplemented with 5% Fetal Bovine Serum (FBS) (S1400, BioWest). Where indicated, T effector cells (Teff) were labelled with CellTrace CFSE (C34554, ThermoFisher Scientific) at lpM and cultured with fresh or pre-treated Tregs (as indicated in Figure legends) in 96-well round-bottom plates in XVIVO15 medium (LONZA) supplemented with 5% Fetal Bovine Serum. In some cases, Tregs were stained with cell trace violet (CTV) (C34557, ThermoFisher Scientific) at a final concentration of 2.5pM. Cells were stimulated with Treg Inspector beads (aCD2/aCD3/aCD28-coated beads) at 1 bead/cell ratio (130-092-909, Miltenyi Biotec) for 4 to 5 days before analyzed by flow cytometry. When stated, medium was supplemented with additional +40mM NaCI. Tregs (5xl04 cell/well) were stimulated in XVIVO15 medium supplemented with 5% FBS for a period of 6-, 12-, 24-, 72 hours or 4 days in the presence of lpg/mL of plate-bound aCD3 (clone UCHT1), 1 or 5pg/mL of soluble aCD28 (clone 28.2) and 25U/mL of IL-2 (11147528001, Sigma) in the presence or absence of +40mM NaCI. Where indicated, Tregs were also incubated in the presence of lOpM antimycin A (AA) (A8674, Sigma Aldrich) or lOpM of CGP-37157 (220005, EMD Millipore). After the incubation period, Tregs were removed from culture, washed and used on the different specified assays. DMSO (D2650, Sigma Aldrich) was included as solvent control if necessary. Generation of monocyte-derived dendritic cells (moDCs)
For monocyte-derived DCs, CD14+ monocytes were magnetically bead-isolated from PBMCs (17858, StemCell Technologies) and cultured with 50U/ml IL-4 (11340045, Immunotools) and 50ng/ml GM-CSF (300-03, Peprotech) in X-VIVO15 supplemented with 10% FBS. After 5 days of incubation, DCs were harvested and stored in liquid nitrogen for later use.
Treg suppression assay using DCs
Treg suppression assays in the presence of DCs were performed as before (Arroyo-Hornero et al. 2022, Allergy 77:2818-2821) . Briefly, CD4+CD25‘ Tconvs (referred as Teff) were stained with lpM Cell Trace CFSE Cell Proliferation Kit (C34554, ThermoFisher Scientific) and cultured with autologous Tregs in 96- well U-bottom plates in X-VIVO15 supplemented with 5% FBS. Cells were stimulated for 4 days using allogenic moDCs in the presence of 0.5ug/mL of soluble aCD3 (555329, BD Bioscience).
Mixed lymphocyte reaction (MLR)
MLR was performed as described before (Kleinewietfeld et al. 2009, Blood 113:827-836). Briefly, human Treg cells were preincubated under CTRL or HS (+40mM NaCI) conditions with plate bound aCD3, soluble aCD28 and IL-2 for a period of 24 hours. Autologous PBMCs (100000 cells/well) were stained with CFSE (C34554, ThermoFisher Scientific) and later stimulated with irradiated (3000 rad) allogeneic PBMCs (100000 cells/well) in RPMI + 10% FCS for 5 days. For suppression of proliferation, pre-incubated autologous Treg cells were added at the indicated ratio.
Murine Treg suppression assay
For murine Treg cell suppression assays, spleens were harvested from 15-week-old C57BL/6J mice. CD4+CD25+ Treg and CD4+CD25‘ T effector cells (Teff) were isolated using the mouse CD4+CD25+ Regulatory T Cell Isolation Kit according to the manufacturer's instructions (Miltenyi Biotec #130-091- 041, Bergisch Gladbach, Germany). CD4+CD25‘ Teff cells were stained with eF450 Proliferation Dye (Thermo Fisher, Schwerte, Germany). Cells were then cultured at a 1:2, 1:4, 1:8 and 1:16 ratio with 100.000 CD4+CD25‘ Teffs in 96-well plates (Sarstedt, Numbrecht, Germany) together with 50.000 Treg expansion beads (130-092-909, Miltenyi Biotec) in RPMI1640 medium (Thermo Fisher) in the presence or absence of +40mM NaCI for 4 days.
Flow Cytometry
Cells were analyzed by flow cytometry (FACS) as described before14 if not specified elsewhere. Duplicates were performed for the different experiments and analysis. Cells were first stained with LIVE/DEAD cell kit (L34972, Invitrogen) or ef780 viability dye (Thermo Fisher) to exclude dead cells. For surface staining, cells were labelled with respective antibodies for 15 minutes in MACS buffer (0.5% BSA, 2mM EDTA) at 4°C. For intracellular stainings, cells were first fixed and made permeable using eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (00-5523-00, Invitrogen) according to manufacturer's instructions and later labelled with respective intracellular antibodies in Perm buffer for 30 minutes at 4°C, washed and assayed in MACS buffer. For cytokine detection, cells were stimulated with 50 ng/ml phorboll2-myristatel3-acetate (PMA) (P1585, Sigma) and 250ng/ml lonomycin (10634, Sigma) in the presence of GolgiPlug (555029, BD) for 5 hours. Data was acquired on a BD LSR Fortessa II, BD FACS Calibur or BD FACS Canto II and analyzed with FlowJo software (TreeStar).
CRISPR/Cas in human Tregs
CRISPR/Cas technology was applied as done before (Van Zeebroeck et al. 2021, Front Immunol 12:655122). Briefly, Tregs were purified by FACS-sorting and expanded for a period of 6 days on 24 wellplates in the presence of lOpg/mL of plate-bound aCD3, lpg/mL of soluble aCD28 and 300U/mL of IL-2 in XVIVO15 medium supplemented with 5% FBS. On day 6, cells were resuspended in XVIVO15 medium supplemented with 5% FBS and lOOU/mL of IL-2 and further incubated in 6 well-plates for a period of 24 hours. For transfection, cells and RNP mixture was pipetted to a well of a 16-well strip nucleovette (Amaxa™ P3 Primary cell 4D-Nucleofector™ X Kit S V4XP-3032, LONZA) and placed on a 4D- Nucleofector™ (LONZA) using EO115 program. Afterwards, cells were plated in 24-well plates with XVIVO15 medium supplemented with 5% FBS and lOOU/mL IL-2. Western blotting was done as described before (Kleinewietfeld et al. 2013, Nature 496:518-522) by using UQCRFS1 (PA5-21420, ThermoFisher Scientific) and 6-Actin (3700, Cell signalling) specific antibodies and DNA was isolated as described above for KO determination using Sanger sequencing. Viable KO and control cells were divided into different functional assays. Cells were diluted in RLT buffer for RNA isolation and qRT-PCR analysis, performed as described above. For measuring of suppressive capacity, control (Mock CTRL) and RISP KO Tregs were co-cultured with CFSE-labelled allogeneic total PBMCs on a 1 to 3 ratio in the presence of Treg Inspector beads (aCD2/aCD3/aCD28-coated beads) (130-092-909, Miltenyi Biotec) at 1 bead/cell ratio for 4 days. After incubation, cells were stained and PBMC proliferation was assessed by flow cytometry using a LSR Fortessa II (BD).
Seahorse assays
The metabolic profile was evaluated in 24- and 72 hours cultured Tregs stimulated with aCD3, aCD28 and IL-2, in the presence or absence of +40mM NaCI. Real-time measurements of oxygen consumption rate (OCR) were made using an XF-96 Extracellular Flux Analyzer (Agilent). After incubation times, cells were collected, counted and plated in Cell-tak (354240, Corning) coated XF-96 plates (102416-100, Agilent) at the concentration of 2 x 105 cells/well. Cells were let to incubate for 60 min at 37°C, without CO2. A mitochondrial stress test designed to repeat 4 cycles (of 3 minutes mixing followed by 3 minutes measuring) per phase was used to measure OCR under basal conditions and in response to 5pM oligomycin (75351, Sigma Aldrich), 1.5pM of carbonylcyanide-4- (trifluoromethoxy) -phenylhydrazone (FCCP) (C2920, Sigma Aldrich) and l .M of antimycin A (A8674, Sigma Aldrich) and Rotenone (R8875, Sigma Aldrich).
Phosphorylation analysis
For phosphorylation analysis, Tregs were stimulated as described above for a period of 4 hours in control medium or media supplemented with +40mM NaCI, lOpM antimycin A (AA), 5pM or 200pM of Etomoxir. Controls were incubated in the presence of lOOU/mL IL-2 for 30 minutes of stimulation. Cells were fixed with BD Cytofix™ Fixation Buffer (554655, BD Biosciences) for 10 minutes at 37°C and permeabilized with Perm Buffer III (BD Biosciences, 58050) for 30 minutes on ice. Cells were stained with anti-pSTAT5 antibody (560311, BD Biosciences) for 30 minutes at 4°C and were acquired on a BD LSR Fortessa II.
Determination of intracellular ATP
For ATP rate assays, Tregs were cultured as mentioned before for a period of 6 hours in the presence or absence of +40mM NaCI. Oligomycin A (5pM) was used as a positive control for ATP decrease. ATPIite Luminescence Assay System (6016941, Perkin Elmer) was used for ATP measurements according to the manufacturer's instructions. Briefly, after incubation time, cells were lysed using a cell lysis solution for 5 minutes in constant shaking followed by the addition of a substrate solution. Cells were further incubated for 15 minutes in dark-adapted conditions and luminescence was measured on a FLUOstar OPTIMA reader (BMG Labtech).
Mitochondrial membrane potential measurements
For mitochondrial membrane potential, TMRE-Mitochondrial membrane potential assay kit (abll3852, Abeam) was used according to the manufacturer's instructions. Briefly, Tregs were cultured and activated as described above for a period of 6 hours in the presence or absence of +40mM NaCI and AA (lOpM). FCCP (lOpM) was used as a positive control and added to the cells 10 minutes before the end of incubation period. After incubation, TMRE was added to the cells and incubated for 20 minutes at 37°C. After, cells were washed and acquired by flow cytometry on a BD Calibur (BD Bioscience).
MitoTracker analysis
To determine mitochondrial content, MitoTracker Green (M7514, ThermoFisher Scientific) was used at a final concentration of lOOnM directly into cell culture medium and let to incubate for 60 minutes at 37°C. After, cells were washed in ice-cold PBS (17-516F, LONZA) and further acquired by flow cytometry using a BD LSR Fortessa II.
Electron Transport Chain Complex Assays
The activity of mitochondrial electron transport chain complexes was detected by using different kits. To measure Complex II activity, a Complex II Enzyme activity microplate assay kit (abl09908, Abeam) was performed according to the manufacturer's instructions. Briefly, Tregs were isolated by FACS sorting. Protein extraction was carried using a detergent solution and final concentration was adjusted to recommended dilution for plate loading. Samples were loaded to a 96-well microplate coated with antiComplex II monoclonal antibody and let to incubate for 2 hours at RT. After, plate was washed, and an activity solution control buffer or buffer containing +4mM NaCI was added to the corresponded wells. Optical density was measured at OD600nm in an iMark Microplate Reader (BIORAD) under kinetic mode for 60 minutes allowing interval measures of 20 seconds. Complex ll/lll activity was assessed using the MitoTox Complex ll+lll OXPHOS Activity Assay Kit (abl09905, Abeam) according to the manufacturer's instructions. Activity solution was mixed with increasing concentrations of NaCI (serial dilutions from +64mM to +0.0625mM NaCI) or with AA as positive control (serial dilutions from 352nM to 0.3438nM). Bovine heart mitochondria were added and absorbance at 550nm was measured in kinetic mode on a Spectramax 190 plate reader. Complex ll/lll activity was calculated relative to the solvent control (water). In some experiments lOpM of CGP-37157 (220005, EMD Millipore) was added.
Mitochondrial ROS measurements
ROS production was assessed on the mitochondria of Tregs using MitoSOX™ Red Mitochondrial Superoxide Indicator kit (M36008, ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, after Tregs incubation for 6 hours in the presence or absence of +40mM NaCI, cells were washed of medium and MitoSOX™ reagent working solution was added and incubated for 10 minutes at 37°C. After, cells were washed and fluorescence was measured by flow cytometry on a BD LSR Fortessa II.
Metabolite extraction and quantification
For metabolite analysis, Tregs were cultured as described above for a period of 12 hours in the presence or absence of +40mM NaCI. Metabolites were extracted on both cells and medium fractions and identified using Liquid chromatography-mass spectrometry (LC-MS). For metabolite extraction on cells, medium was removed and cells washed with ice cold 0.9% NaCI solution. After, a cellular extraction buffer (80% methanol, containing 2pM d27 myristic acid) was added to the cells and let to incubate for 2-3 minutes on ice. Cells were later centrifuged and supernatant was used for further metabolite identification. Protein pellets were kept in order to determine protein concentration by BCA assay. For medium extraction, 990pL of medium extraction buffer was added to lOpL of medium and stored overnight at -80°C. After incubation, medium samples were centrifuged and supernatants were used for metabolites analysis. Following extraction, the complex mixture of metabolites was separated prior to MS measurement using a Dionex UltiMate 3000 LC System (ThermoFisher Scientific) coupled to a Q Exactive Orbitrap mass spectrometer (ThermoFisher Scientific) operating in negative ion mode. Practically, lOpI of the sample was injected on a C18 column (Aquility UPLC®HSS T3 1.8pm 2.1x100mm). A gradient using solvent A (H2O, lOmM Tributyl-Amine, 15mM acetic acid) and solvent B (100% Methanol) was applied as follows: 0 minutes, 0%B; 2 minutes, 0%B; 7 minutes, 37%B; 14 minutes, 41%B; 26 minutes, 100%B; 30 minutes, 100%B; 31 minutes, 0%B; 40 minutes, 0%B. The flowrate was kept constant at 0.250ml/min and the column was kept at 40°C throughout the analysis. The MS operated in full scan mode (range 70-1050 Th in negative normalized mode) using a spray voltage of 3.2kV, a capillary temperature of 320°C, sheath gas at 50.0, auxiliary gas at 10.0. The AGC target was set at 3e6 using a resolution of 140.000, with a maximum IT of 512ms. Data collection was performed using the Xcalibur software, version 4.2.47 (ThermoFisher Scientific).
Intracellular Na+ quantification
Mouse (C57BL/6) CD4+ T cells were enriched using CD4+ T cell Isolation Kit (Miltenyi 130-104-454) and incubated for 30 minutes with HS (+10mM or +40mM NaCI) and washed with iso-osmolal sucrose solution. The pellet was lysed with 0.1% Triton and total Na+ was quantified using an iCE 3000 Series atomic absorption spectrometer (Thermo Scientific) as described before (Neubert et al. 2020, PLoS Biol 18:e3000722). Animal care and use followed the regulations of the German Animal Welfare Act. The procedures followed were approved by the Umweltamt der Stadt Regensburg and performed in accordance with institutional guidelines.
Transmission electron microscopy
Tregs were cultured as described above in the presence or absence of +40mM NaCI for a period of 6 hours. After incubation, cells were fixed with specific fixative (8% formaldehyde (FA) (EM-grade), 5% glutaraldehyde (GA) (EM-grade), 0.1M Cacodylate buffer) on an equal volume as cells. Cells were fixed for 30 minutes RT in constant rotation. After incubation, fixative was removed and cells were washed 3 times for 30 minutes at 4°C in 0.1M Cacodylate buffer in constant rotation. Once washes were complete, equal volume of 1% Osmium tetroxide (OsO4) was added to the cells and samples were shaken for 1 hour at 4°C. Afterwards, samples were washed by constant shaking for 20 minutes in ddH2O. This step was repeated 4 times. After last washing step, samples were left for 1 hour in 1% Uranyl Acetate (UrAc) under cold and dark conditions for bulk staining. After staining incubation, samples were once again washed by constant shaking in ddl-120 for 20 minutes. Washing step was done at 4°C and repeated 4 times. After, samples were dehydrated at 4°C in constant shaking, infiltrated at 4°C and embedding was performed using Spurr's resin. Final step of polymerization was done at 70°C. For the sample sectioning, ultrathin sections of gold interference color made with an ultra-microtome (Leica EM UC6) was performed and post-stained in a Leica EM AC20 for 40 minutes in UrAc at 20°C and for 10 minutes in lead citrate at 20°C. Generated sections were examined and imaged with a JEM 1400plus transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kV. The 2D transmission electron microscopy images were manually annotated for mitochondria. Cells that were partially visible in the images were omitted from the study. Individual mitochondria were identified with connected component analysis. Morphological features (area, eccentricity, major/minor axis length, and perimeter) were extracted from each mitochondrion. The dimensionality of this feature set was then reduced with U-MAP (https://doi.org/10.21105/joss.00861), which allowed for 2D visualization. The distributions of each feature were compared with kernel density estimation and visualization in violin plots.
RNA isolation and quantitative polymerase chain reaction with reverse transcription (qRT-PCR)
Cells were lysed in RLT buffer (Qiagen) and stored at -80°C until RNA was extracted. For RNA isolation, the Rneasy plus Micro Kit (74034, Qiagen) was used according to the manufacturer's instructions and further converted to cDNA using qScriptTM cDNA SuperMix kit (95048, QuantaBio) according to manufacturer's instructions. Real Time PCR was performed on a Step ONE Plus RT-PCR machine (Applied Biosciences) using the TaqMan Fast Universal PCR Master Mix (4367846, ThermoFisher Scientific). Primers were purchased from ThermoFisher Scientific (IL10- Hs00961622_ml; CTLA4- Hs01011591_ml; IFNG- Hs00989291_ml; FOXP3- Hs01085834_ml) with the exception of /32M (4326319E-1402015, Applied Biosystems). Fold-changes in expression were calculated using the AACT method using human 02M as endogenous control for mRNA expression as described before (Kleinewietfeld et al. 2013, Nature 496:518-522). All fold-changes in the graphs are expressed normalized to the control group.
RNA sequencing (RNAseq)
For RNA sequencing analysis, RNA was isolated as described above. Sequencing libraries were prepared with the NEB Next Ultra DNA Library Prep Kit for Illumina (version 6.0-2/18), according to the manufacturer's protocol including a size selection to 250bp insert size. Sequence-libraries of each sample were equimolarly pooled and sequenced on 4 NextSeq500 v2 flow-cell at 1 x 75 bp (76-8-8-0). Quality of raw sequence reads was checked using FastQC version 0.11.8, and nucleotide calls with a quality score of 28 or higher were considered high quality. Due to low read quality, one sample from the 6 hours control group was removed from analysis. Adapters were removed using cutadapt v.2.4. Reads were aligned to the hgl9 genome reference, using STAR (2.5.0e) and a maximum of five mismatches were allowed. Gene counts were retrieved using htseq-count using the "union" option. After removing absent features (zero counts in all samples), the raw counts were imported to R/Bioconductor package DESeq2 v.3.9 to identify differentially expressed genes among samples. The default DESeq2 options were used, including log fold change shrinkage. Differentially expressed genes were considered only when the Benjamini-Hochberg adjusted p-value (false discovery rate [FDR]) was < 0.05. Heatmaps and bar plots were created using the gplots::heatmap.2() and barplot() function respectively on transformed raw counts.
Reanalysis of published transcriptomic datasets
EGAS00001004470 (Alissafi et al. 2020, Cell Metab 32:591-604. e7) were downloaded from the European Genome-phenome Archive (EGA). The quality of raw sequence reads was checked using FastQC version 0.11.8, and the data was analyzed as described above. Droplet-based single cell (sc)RNAseq preparation
For single cell RNA sequencing (scRNAseq), Tregs were incubated for a period of 6 hours in the presence and absence of +40mM NaCI. After incubation, cells were collected and converted to barcoded scRNAseq libraries by using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (lOx Genomics), aiming for an estimated 5,000 cells per library and following the manufacturer's instructions. Samples were processed using a kit associated to V2 barcoding chemistry of lOx Genomics. Single samples were processed in a single well of a PCR plate, allowing all cells from a sample to be treated with the same master mix and in the same reaction vessel. scRNAseq profile
RNA-Seq profiling of single Treg cells was performed with an average sequencing saturation metric of >80%, as calculated by Cell Ranger. Aggregation of sample conditions was done using the Cell Ranger Aggr software from lOx Genomics. Digital gene expression matrices were pre-processed and filtered using the SCRAN and ScaterR packages. Outlier cells were first identified based on three metrics (library size, number of expressed genes and mitochondrial proportion); cells were tagged as outliers when they were four median absolute deviations distant from the median value of each metric across all cells. Secondly, a principal component analysis plot was generated based on the following metrics: 'pct_counts_in_top_100_features', 'total_features_by_ counts', 'pct_counts_feature_control', 'total_features_by_counts_feature_control', 'loglO_total_counts_endogenous' and
'loglO_total_counts_feature_control'. Outlier cells in this principal component analysis plot were identified using the Rpackage mvoutlier. Low-abundance genes were removed using the 'calcAverage' function and the proposed workflow. The raw counts were normalized and Iog2 transformed by first calculating 'size factors' that represented the extent to which counts should be scaled in each library. Highly variable genes were detected using the proposed workflow of the scranR package and by applying false discovery rate<0.05 and var.out$bio>0.01 as cutoffs. Highly variable genes were subsequently used for unsupervised dimensionality reduction techniques and principal component analysis. Unsupervised clustering of the cells was performed using graph-based clustering based on SNN-Cliq and PhenoGraph as implemented in the Seurat v.2.3Rpackage (default parameters). Clustering was visualized in two-dimensional scatter plots (via tSNE) using the Seurat v.2.3Rpackage.
Modelling of metabolic pathways based on scRNAseq data
The metabolic landscape of Tregs was modeled using the Compass method (version 0.9.5) (Wagner et al. 2021, Cell 184:4168-4185) by leaving the standard settings unaltered. The gene expression matrix of Tregs single cell data was used as input. The Compass output data was concatenated and transformed as described (Wagner et al. 2021, Cell 184:4168-4185). To determine which reactions and metabolites were significantly different between groups (CTRL and +40mM NaCI), a Wilcoxon rank sum tests on Compass scores was performed.
Pathway analysis
Ingenuity Pathway Analysis (IPA; Ingenuity Systems/Qiagen) was used to map lists of significant genes (FDR <0.05) to gene ontology groups and biological pathways. The functional and canonical pathway analysis was used to identify the significant biological functions and pathways. Functions and pathways with p-values less than 0.05 (Fischer's exact test) were considered to be statistically significant.
Gene set enrichment analysis
The Gene set enrichment analysis (GSEA) was done using GSEA software (1,2), which uses predefined gene sets from the Molecular Signatures Database (MsigDB). For the present study, we used all the H: hallmark gene sets for GSEA analysis and list of ranked genes based on a score calculated as loglO of p- value multiplied by sign of fold-change. The minimum and maximum criteria for selection of gene sets from the collection were 10 and 500 genes, respectively (Subramanian et al. 2005, PNAS USA 102:15545- 15550).
Salt intake in healthy individuals
Daily salt intake in healthy individuals was assessed as described before (Mahler et al. 2022, Nutrients 14:253). HS and NS intake was defined by a cutoff of >7g/day or <7g/day, respectively. The cohort was comprised of 8 male and 10 female participants with an age range of 35.8±10.4yrs. PBMCs of participants were isolated from peripheral blood and analyzed by FACS as described above.
Experimental autoimmune encephalomyelitis (EAE)
CD4+CD25+Treg cells were isolated using the mouse CD4+CD25+ Regulatory T Cell Isolation Kit according to the manufacturer's instructions (Miltenyi Biotec #130-091-041, Bergisch Gladbach, Germany) from C57BL/6J mice. Cells were plated for 24hrs in 48-well plates (Sarstedt, Numbrecht, Germany) at 750 thousand cells per well in RPMI 1640 with 10% FCS (both Thermo Fisher) and 2.000 lU/ml recombinant murine IL-2 (Miltenyi Biotec) in the presence or absence of 40mM NaCI or in the presence of 40mM NaCI plus lOpM of CGP-37157 (220005, EMD Millipore). Subsequently, cells were harvested, washed with PBS and adjusted to one million cells/ml in PBS. Cells were injected intravenously (i.v) (500 thousand cells/mouse) at day 6 of EAE. Active EAE was induced by immunizing 12-week-old female, in house bred C57BL/6J mice with 200 pg MOG (35-55, Charite, Berlin, Germany) in 200 pg CFA (BD) subcutaneously at the flanks and tail base. 200 ng of pertussis toxin (PTX) (List, Campbell, CA) were applied intraperitoneally (i.p) on days 0 and 2 post immunization. Animals were weighted and scored on a daily basis. Scores were as follows: 1- limp tail, 2- gait ataxia, 3- paraparesis, 4- tetraparesis, 5- moribund or death. All animal experiments were performed in accordance with the local animal welfare regulations and approved by the respective authorities (AZ: 55.2.2-2532-2.1293-29). Xenogeneic graft versus host disease (xGvHD)
Six- to ten-week-old male NSG mice were purchased from Charles River and housed randomly on different IVC cages at the in-house animal care facility. Mice received autoclaved chow and tap water ad libitum for 9 days before induction. For xGvHD induction, each animal received 1.3xl07 CD25-depleted PBMCs alone (PBMC group) or together with 0.2x107 CD25-enriched Tregs pre-activated with aCD3 (lpg/mL), aCD28 (lpg/mL) and IL-2 (25U/mL) for 6 hours in control media or media supplemented with +40mM NaCI (HS group) or in media containing lOpM of antimycin A (AA group) or in media supplemented with +40mM NaCI containing lOpM of CGP-37157 (CGP group). After thorough washing, cells were resuspended in PBS and injected through the tail vein. The weight and clinical symptoms of the mice were monitored during the entire course of the experiment. Clinical symptoms were scored according to general appearance and mobility as described before (Hernandez et al. 2015, J Clin Invest 125:4212-4222). At sacrifice, organs were collected, processed and cells were used for FACS. Engraftment of human cells was monitored by FACS analysis of peripheral blood.
Quantification and statistical analysis
Statistical analyses were performed with GraphPad Prism Version 8. All data were presented as mean ± standard error of the mean (SEM), unless stated otherwise. Value of n is always displayed in the Figure as individual data point, referring to an independent biological replicate, more information about absolute n numbers can be found in the Figure legends. All statistical test used were also indicated in the respective Figure legends. Normality of the data was tested by Shapiro-Wilk normality test. Significance between two groups was analyzed by t-test (when normal distributed) or Wilcoxon matched-pairs signed rank test (for non-normal distributed). For more than two groups with one variable only, one-way ANOVA with Tukey's post-hoc test (for normal distributed data) was used. Matched data with more than two groups (or time points) was analyzed by Friedman test and FDR-correction was performed via Benjamini-Hochberg procedure or Holm Sidak's multiple comparison tests. For the survival analysis of the in vivo data, a curve comparison test with Mantel-Cox and Gehan-Breslow- Wilcoxon test was applied. Exact statistical tests used are described in the respective Figure legends. Respective p-values were depicted in the figures and legends.
Study approval
Human studies were conducted in compliance with the institutional review board protocols from Hasselt University (CME2019/042, CME2016/629 and UH-SALTMS-P1). If not mentioned elsewhere, animal studies were approved by the ethics committee of animal studies at the University of Hasselt (ID 201739). Table I. Key resources table
Figure imgf000039_0001
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EXAMPLE 2. High salt induced expression signature resembles dysfunctional autoimmune Tregs
Perturbed function of Tregs is associated to autoimmunity, however, factors contributing to this phenotype still remain elusive. Raising the extracellular salt concentration by adding extra +40mM NaCI compared to control conditions to co-cultures of human Tregs and CFSE labelled CD4+ T effector cells, mimicking physiologically increased Na+ concentrations found in interstitial tissues after HS diets (Muller et al. 2019, Nat Rev Immunol 19:243-254; Wiig et al. 2013, J Clin Invest 123:2803-2815; Machnik et al. 2009; Nat Med 15:545-552) or during inflammation (Jantsch et al. 2015, Cell Metab 21:493-501), significantly inhibited the suppressive capacity of human Tregs in vitro (Figure 1A). The effect could already be observed at lower NaCI concentrations starting from +10mM NaCI and it was neither due to changes in osmotic pressure, since addition of mannitol as a tonicity control did not affect suppressive function of Tregs, nor by acting directly on T effector cells (data not shown) (Hernandez et al. 2015, J Clin Invest 125:4212-4222). Furthermore, the HS effect on Tregs could also be observed in more physiological in vitro assays. Salt-treated Tregs were unable to efficiently suppress allogeneic activation in mixed lymphocyte reactions (MLR) (Kleinewietfeld et al. 2009, Blood 113:827-836) and displayed diminished suppressive capacity on T effector cells in the presence of dendritic cells (DCs) (Arroyo-Hornero et al. 2022, Allergy 77:2818-2821). In line with previous reports (Hernandez et al. 2015, J Clin Invest 125:4212- 4222; Sumida et al. 2018, Nat Immunol 19:1391-1402), high sodium conditions induced IFN-y expression in Tregs (Figure IB) and a higher proliferation of Tregs in suppression assays (Figure 1C), characteristic for dysfunctional Tregs observed in autoimmunity and indicative of Treg metabolic reprogramming under HS exposure.
In order to better understand salt-induced molecular changes and to investigate a potential overlap between HS-treated Tregs and dysfunctional autoimmune Tregs, we performed bulk-RNA sequencing (RNAseq) of Tregs that were cultured under HS and control conditions for a period of 72 hours, and compared the expression signature to published RNAseq datasets of Tregs isolated from patients with different autoimmune diseases, more in particular with multiple sclerosis (MS), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) (Alissafi et al. 2020, Cell Metab 32:591-604. e7). Of note, gene set enrichment analysis (GSEA) revealed 'Hallmarks of Interferon gamma response' as one of the top upregulated pathways after HS exposure with 69 genes being enriched and a normalized enrichment score (NES) of 1.63 and a false discovery rate (FDR) of 0.007. In line with this, the expression pattern of Tregs isolated from 5 healthy individuals was strongly indicative of a pro-inflammatory Thl- like phenotype, with the upregulation of key Thl-associated genes like the chemokine receptor CXCR3, the transcription factor TBX21/T-bet and IFNG and other signature genes and activation markers upon HS exposure.
Importantly, GSEA analysis (more detailed description below) and investigation of Th 1-like signatures of Tregs from patients with MS, SLE and RA (Alissafi et al. 2020, Cell Metab 32:591-604.e7) showed a similar strong and top enrichment of 'Hallmarks of Interferon gamma response' with 70 genes being enriched in the MS cohort (NES=1.44; FDR=0.031), 129 genes in the SLE cohort (NES=2.62; FDR=0.000) and 108 genes in the RA cohort (NES=1.54; FDR=0.022), indicating highly overlapping and common functional features between the autoimmune and HS datasets. This observation goes well in line with our results on a higher IFN-y expression after HS treatment (Figure IB), as well as with previously studies on the acquisition of a Thl-like phenotype in Tregs of patients with MS (Sumida et al. 2018, Nat Immunol 19:1391-1402) and Tregs exposed to HS environments (Hernandez et al. 2015, J Clin Invest 125:4212- 4222). Thus, our data show that HS induces a dysfunctional Thl-like phenotype in Tregs that closely mimics key gene expression signatures and functional features of Tregs isolated from patients with autoimmunity. Importantly, besides the previously noted similarities to Tregs of people with MS (Sumida et al. 2018, Nat Immunol 19:1391-1402), comparable features seem also be shared with Tregs from other autoimmune diseases like SLE and RA.
In more detail, enrichment plots (GSEA) of Interferon gamma response of bulk RNA-seq from Tregs activated under HS (+40mM NaCI n=5) or control conditions (CTRL n=5) for 72 hours, and of isolated Tregs from peripheral blood of healthy individuals (n=14) and subjects with multiple sclerosis (MS n=10), subjects with systemic lupus erythematosus (SLE n=8) and subjects with rheumatoid arthritis (RA n=9) were constructed. The RNA sequencing data from healthy individual- and patient derived Tregs were downloaded from the European Genome-phenome Archive (EGAS00001004470) and reanalyzed accordingly. Such enrichment plots comprise an enrichment score (ES) curve and a ranked list metric curve. The ES curve represents the running sum of the weighted enrichment score obtained from the GSEA algorithm. The ranked list metric curve represents the degree of correlation of genes with the depicted pathway (red for positive and blue for negative correlation). Furthermore the normalized enrichment score (NES) and the corresponding FDR are reported within the plot.
NaCI/salt group: NES=1.63 with FDR=0.007; MS patient/control group: NES=1.44 with FDR=0.031; SLE patient/control group: NES=2.62 with FDR=0.000; RA patient/control group: NES=1.54 with FDR=0.022
EXAMPLE 3. Salt induced Treg dysfunction is associated with severe changes in immunometabolism
Since we observed highly overlapping signatures between 72 hour HS exposed Tregs and Tregs isolated from patients with autoimmunity (Alissafi et al. 2020, Cell Metab 32:591-604.e7) and both showed indications for immunometabolic remodeling, we hypothesized that salt effects cellular immunometabolism of Tregs particular at early stages. Thus, in order to investigate the functional consequences of HS on Tregs in more detail, we analyzed changes in gene expression under HS exposure over time. Human Tregs were activated in vitro for 6 hours and compared to 72 hours in the presence or absence of HS and gene expression was analyzed by bulk RNA-seq in comparison to ex vivo isolated Tregs without prior activation (Figure 2A). Of note, the impact of HS on gene expression was much more pronounced at 6 hours post activation as compared to 72 hours, indicating that HS induces particularly profound changes in gene expression of human Tregs at early stages of activation (Figure 2A). When analyzing the 6 hour time point in more detail, we identified 1250 upregulated and 1380 downregulated genes (FDR p<0.05) under HS exposure (Figure 2A). Amongst highest regulated genes under HS were candidates belonging to the catenin pathway (CTNN1, DACT3 and TCF7), previously shown to be critically affected by HS (Sumida et al. 2018, Nat Immunol 19:1391-1402) as well as key effector molecules for Tregs such as IL10 (Figure 2B). While, further in agreement with previous reports, the expression of IFNG was not majorly affected at 6 hours and only developed in line with the HS-induced dynamic induction of a Thl-like Treg signature at later time points, resulting in an increased IFNG/IL10 ratio (Figure 2C) (Hernandez et al. 2015, J Clin Invest 125:4212-4222; Sumida et al. 2018, Nat Immunol 19:1391-1402). Interestingly, the early expression analysis of HS-treated Tregs also indicated severe changes in cellular metabolism. Further amongst highest regulated genes we particularly found genes localized in the mitochondria or associated to mitochondrial function (0MA1, ETFB and BCAT2) (Figure 2B). To investigate the consequences of HS at 6 hours post activation in more detail, we applied single cell RNA- seq (scRNA-Seq) on highly pure isolated human Tregs treated under HS or control conditions. The tSNE projections were performed on 4785 cells and 10 clusters were identified based on the expression of key signature genes. Of note, HS-treated Tregs were highly differentially represented within the clusters. While in clusters 0 and 3 there was an overrepresentation of HS-treated Tregs; clusters 1, 2 and 4 depicted an underrepresentation of these cells (Figure 2D).
In order to explore the metabolic features of HS-treated Tregs, we used the recently developed Compass algorithm (Wagner et al. 2021, Cell 184:4168-4185), which utilizes flux balance analysis to infer metabolic states of cells based on scRNA-seq. Compass algorithm analysis indicated shifts in several metabolic pathways, pointing towards a severely altered metabolic state of HS-exposed human Tregs. Importantly, the analysis revealed a downregulation of key nodes for Treg pyruvate metabolism and tricarboxylic acid (TCA) cycle after HS exposure, whereas the urea cycle was detected as being upregulated. Within the TCA cycle, several reactions and enzymes were found to be affected by salt such as succinate dehydrogenase (SDH), malate dehydrogenase and isocitrate dehydrogenase. SDH drives the enzymatic conversion of succinate to fumarate playing a pivotal role in OXPHOS as the electron acceptor complex II. Inhibition of this enzyme leads to the accumulation of succinate, diminishing electron passage through the ETC complexes resulting in a downregulation of OXPHOS capacity (Nastasi et al. 2021, Sci Rep 11:1458). Furthermore, accumulation of succinate and downregulation of SDH was shown to impair T cell activation and production of key anti-inflammatory cytokines such as IL-10 and IL-2 in CD4+ T cells (Nastasi et al. 2021, Sci Rep 11:1458).
In order to investigate potential changes on the metabolite levels after HS exposure, we applied Liquid chromatography-mass spectrometry (LC-MS) of Tregs that had been cultured under control or HS conditions. Interestingly, our data revealed an upregulation of succinate levels under HS conditions, corroborating with the detected downregulation of SDH through Compass analysis. Moreover, an accumulation of fumarate was observed under these conditions, which can potentially be linked with the observed upregulation of the urea cycle. Furthermore, we also detected an upregulation of malate alongside a downregulation of NADH and NADPH. These results go well in line with the Compass predictions, where a downregulation of malate dehydrogenase inhibits the conversion of malate to oxaloacetate resulting in higher levels of malate and a diminished production of NADH. In line with the above findings, GSEA analysis on the scRNA-Seq data showed 'Hallmark of oxidative phosphorylation' as the most downregulated pathway with 149 genes affected (NES=2.4; FDR=0.001), identified after HS- exposure for 6 hours. This was further independently corroborated by IPA analysis, where unbiased analysis of the scRNA-Seq data recognized OXPHOS as being the top downregulated pathway after HS exposure. In summary, our data demonstrate that salt exposure induces massive shifts in gene expression particularly at early time points, which are indicative for a severe deregulation in Treg immunometabolism.
EXAMPLE 4. High salt inhibits oxidative phosphorylation of Tregs
Since the RNA-seq and scRNA-seq data pointed towards a disturbed mitochondrial metabolism of HS- treated Tregs, we investigated the respiratory capacity of HS-treated human Tregs by bioenergetic analysis assessing their oxygen consumption rate (OCR) using seahorse technology. Both the seahorse profile at 72 hours and 24 hours post activation revealed that Treg maximal respiration was significantly reduced after salt exposure (Figure 3A), which was not due to alterations in cell viability (Figure 3B). To independently confirm the data, we next analyzed ATP production of human Tregs directly. In line with the previous results and the downregulation of ATP under HS conditions detected by LC-MS, Tregs cultured under HS conditions showed a dramatic reduction of ATP production, even to a similar extend as compared to oligomycin exposure, a potent inhibitor of mitochondrial complex V of the ETC at 6 hours post activation (Figure 3C). To examine whether changes in the mitochondrial mass could account for the observed effects on Tregs metabolism, we analyzed mitochondria of human Tregs by FACS using Mitotracker. No changes were observed between HS and control conditions, indicating that the previously observed effects were not due to changes in mitochondrial mass. In line with these findings, analysis of HS and control activated human Tregs by transmission electron microscopy (tEM) did not reveal any major morphological changes.
We next tested if HS led to changes in mitochondrial membrane potential (Tm) by tetramethylrhodamine ethyl ester (TMRE) staining. As controls we used carbonyl cyanide p- (trifluoromethoxy)phenylhydrazone (FCCP), a potent uncoupler of m and antimycin A (AA), a specific inhibitor of complex III. Of note, HS led to a collapse of the mitochondrial membrane potential and significantly reduced TMRE signal in Tregs even to a comparable extend to AA at 6 hours post activation (Figure 3D). Furthermore, we observed increases in mitochondrial reactive oxygen species (mtROS) after HS exposure in human Tregs (Figure 3E). Based on the above results, we thus hypothesized that salt may interferes with cellular metabolism by disturbance of mitochondrial respiration at the level of the ETC. To investigate the effect of HS on mitochondrial respiration in more detail, we analyzed the transcriptional profile of activated human Tregs after 6 hours under inhibition of complex III by AA. Interestingly, hierarchical clustering analysis of RNA-seq data revealed a highly overlapping gene expression signature of top significant genes upon HS and AA treatment (FDR<0.05) indicating the activation of a similar expression program. Previous data showed that the deletion of RISP induced a distinct gene expression pattern that was mimicked closest by treatment of murine Tregs by AA (Weinberg et al. 2019, Nature 565:495-499). Strikingly, by comparing our RNA-seq data from human Tregs (HS vs CTRL, AA vs CTRL) with the published murine expression data on AA treatment and chimeric RISP KO (AA vs CTRL in murine Tregs, RISP KO vs wildtype (WT) murine Tregs) (Weinberg et al. 2019, Nature 565:495-499), we found highly overlapping gene signatures with the OXPHOS pathway being the top hit (IPA z-score < -6; blue), indicating an inhibition of OXPHOS through down regulation of similar genes related to this pathway in all 4 data sets. In summary, our results demonstrate that HS severely disturbs Treg mitochondrial respiration. The similarities of data sets between HS treatment and interfering with ETC complex III indicate that sodium affects Treg mitochondrial metabolism on a similar level.
EXAMPLE 5. Perturbation of mitochondrial metabolism severely alters the function of human Tregs
To dissect the mechanistic consequences of HS perturbed mitochondrial function of Tregs, we analyzed the expression data for functional changes on Tregs treated under HS or AA. I ntriguingly, the scRNA-seq data at 6 hours post activation identified the transcription factor FOXP3 as one of the most downregulated genes after HS exposure. Downregulation of FOXP3 expression after HS-exposure for 6 hours on activated Tregs was independently confirmed by real-time qPCR (Figure 4A) and by FACS at the protein level, measured after 24 hours of activation under similar conditions (Figure 4B). In line with this, the incubation of T cells with AA for 24 hours, led also to a significant reduction of FOXP3 expression (Figure 4C). Since FOXP3 is essential for Treg stability and function and mutations in the gene can lead to the development of severe autoimmunity (Kleinewietfeld & Hafler 2014, Immunol Rev 259:231-244; Sakaguchi et al. 2010, Nat Rev Immunol 10:490-500; Arroyo Hornero et al. 2020, Front Immunol 11:253; Kleinewietfeld et al. 2013, Nature 496:518-522), its intriguing to speculate that the rapid downregulation of FOXP3 expression after HS exposure could account for the observed loss of function in Tregs. In the light of these findings, it is of interest that recent data revealed a direct link between FOXP3 expression and OXPHOS (Howie et al. 2017, JCI Insight 2:e89160). In line with the downregulation of F0XP3, the expression pattern of other essential Treg genes resembles that of non-functional Tregs (Dominguez- Villar et al. 2018, Nat Immunol 19:665-673). Downregulation of genes for key functional effector molecules like IL10 and CTLA4 indicated a strong pro-inflammatory bias of HS or AA on Tregs and were independently confirmed by qRT-PCR (Figure 4D) and protein expression analysis for CTLA4 and IL-10 by FACS. Further, and in line with the gene expression signature in RISP KO in murine Tregs (Weinberg et al. 2019, Nature 565:495-499), STAT5 phosphorylation, indicative for IL-2 responsiveness, was significantly hampered under HS and AA conditions (Figure 4E).
To investigate if the inhibition of complex III abrogates suppressive function of human Tregs as compared to HS treatment (Figure 1A), we analyzed the effects of AA in in vitro suppression assays. To exclude any direct impact on effector T cells, Tregs were pre-activated for 72 hours in the presence of AA or HS and subsequently tested for their suppressive function under standard conditions. Importantly, similar to HS pre-activation, the inhibition of complex III by AA significantly abrogated human Treg function, indicating that the interference by either pharmacological inhibition or salt-mediated perturbation of mitochondrial function leads to equal functional consequences in vitro (Figure 4F). To further confirm these results, we applied CRISPR/Cas mediated genome editing to knock-out (KO) the RISP gene in primary human Tregs (Van Zeebroeck et al. 2021, Front Immunol 12:655122). To analyze if the ablation of UQCRFS1 in human Tregs induces a similar phenotype as the pharmacological inhibition of complex III or the mitochondrial perturbation by HS, RISP KO Tregs were tested in in vitro suppression assays. As expected, based on our IPA comparison analysis and previous findings in mice (Weinberg et al. 2019, Nature 565:495-499), the deletion of the RISP gene in human cells resulted in lack of suppression in vitro, closely resembling the dysfunctional phenotype of HS- and AA- treated human Tregs (Figure 4G). In line with the above results, RISP KO Tregs further displayed an increased IFNG/IL10 ratio (Figure 4H).
EXAMPLE 6. Salt induced inhibition of mitochondrial function disrupts T reg fitness and function in vivo. To investigate, if high sodium intake may affect the phenotype of human Tregs in vivo, we analyzed the phenotype of human Tregs in respect to individual salt intake of healthy volunteers (Figure 5A-C). Daily salt consumption of participants was calculated based on a specifically designed comprehensive dietary questionnaire covering individual food habits as described before (Mahler et al. 2022, Nutrients 14:253) and volunteers were grouped into 'low salt' (LS) and 'high salt' (HS) groups based on a cutoff of 7g/day NaCI intake (Figure 5A). PBMCs isolated from individual participants were then used to assess the cytokine expression of Tregs by FACS. Strikingly, the analysis of Tregs revealed a significant reduction of IL-10 expression in FOXP3+ Tregs from the HS group in comparison to the LS group (Figure 5B) and the percentage of IL-10 expression in Tregs negatively correlated with salt intake in a significant manner (Figure 5C). However, we only observed tendencies for decreases in FOXP3 and increases in IFN-0 expression in the HS group (data not shown).
To ultimately examine, if salt mediated disturbance of mitochondrial respiration of Tregs affects also their function in inflammatory settings in vivo, we tested this experimentally in two independent animal models for murine and human Tregs. Previous reports have shown that HS also inhibits the function of murine thymic derived Tregs (Hernandez et al. 2015, J Clin Invest 125:4212-4222; Luo et al. 2019, Cell Rep 26:1869-1879) and murine in vitro suppression assays under HS conditions, similar to previous experiments with human cells (Figure 1A), independently confirmed these findings (Figure 5D). In order to directly dissect the impact of HS on murine Treg fitness and function, we performed an adoptive transfer experiment of murine Tregs to contain experimental autoimmune encephalomyelitis (EAE). In this model the adoptive transfer of Tregs was shown to significantly ameliorate EAE, presumably in dependence of Treg-derived IL-10 production (Zhang et al. 2004, Int Immunol 16:249-256; Kohm et al. 2002, J Immunol 169:4712-4716). EAE was induced in 12-week-old C57BL/6 recipient mice and Tregs were isolated from naive donor mice and pre-incubated in either control or HS conditions for a period of 24 hours and subsequently injected into recipient mice after 6 days of EAE induction and mice were scored daily to monitor EAE development (Figure 5E). In stark contrast to mice that received control Tregs (CTRL), animals receiving salt pre-treated Tregs (+40mM NaCI) failed to contain the development of EAE, resulting in significantly higher disease scores (Figure 5F).
To investigate, if the short-term disturbance of mitochondrial function of human Tregs by HS or AA would similarly have long-term consequences in vivo, we used the model of xenogeneic graft versus host disease (xGvHD) in immunodeficient NOD-SCID I L-2Ry_/“ (NSG) mice, one of the few available models for a robust analysis of human Treg function in vivo (Hernandez et al. 2015, J Clin Invest 125:4212-4222; Kleinewietfeld et al. 2009, Blood 113:827-836; Hahn et al. 2015, Front Immunol 6:623). To this end, we pre-incubated human Tregs under HS, AA or control conditions for 6 hours before co-injection with CD25-depleted PBMCs into NSG mice to monitor their in vivo potential to contain xGvHD. Control animals solely received CD25" PBMCs to elicit xGvHD (Figure 5G). Injected effector cells and Tregs of all 4 groups engrafted equally well as verified by FACS analysis of human cells and the majority consists of CD3+T cells in blood at 15 days post injection (p.i.) (Figure 5H). Weights of the mice together with survival rates were monitored over a period of 60 days. A loss in weight in controls occurred around day 25 p.i. (Figure 51). Importantly, only mice receiving CD25-depleted PBMC together with untreated control Tregs were significantly able to prevent severe xGvHD. HS or AA pre-treated Tregs were unable to restrain disease in CD25" PBMC receiving animals (Figure 5J). In line with the survival curves, the analysis of splenocytes on sacrifice showed that only animals receiving functional Tregs displayed a lower percentage of human CD8+ T cells, known as major drivers of xGvHD (Guichelaar et al. 2013, Clin Cancer Res 19:1467-1475; Mutis et al. 2006, Clin Cancer Res 12:5520-5525) (data not shown). In summary, our data demonstrates that even the short-term perturbation of mitochondrial respiration in Tregs by treatment of either HS or AA, similarly lead to long-term disruption of human and murine Treg function in vivo.
EXAMPLE 7. Salt-induced mitochondrial dysfunction depends on mitochondrial NCLX
Recent reports have shown that extracellular HS conditions lead to subtle increases of intracellular Na+ in macrophages (Neubert et al. 2020, PLoS Biol 18:e3000722; Neubert et al. 2019, Front Immunol 10:599). To examine, if this phenomenon is also observed in adaptive immune cells, we tested if the incubation under HS conditions leads to increases of the intercellular Na+ content in CD4+ T cells (Figure 6A). Indeed, extracellular HS conditions raised intracellular Na+ levels to a similar range as observed in macrophages (Geisberger et al. 2021, Circulation 144:144-158; Neubert et al. 2020, PLoS Biol 18:e3000722; Neubert et al. 2019, Front Immunol 10:599) indicating that extracellular HS led to increased intracellular Na+ content also in T cells and this effect was already observed at lower HS conditions (+10mM NaCI), although to a lesser extent. In order to directly examine the impact of HS conditions on the ETC, we analyzed effects of increased Na+ on the activity of complex II and complex III. We tested complex II activity in the presence of +4mM NaCI, reportedly in the physiological range of the cytosol under HS conditions (Neubert et al. 2020, PLoS Biol 18:e3000722; Neubert et al. 2019, Front Immunol 10:599). Complex II function derived directly from human Tregs, was diminished by almost 50% in a cell- and mitochondrion-free assay, indicating that increased intracellular Na+ could directly affect late complex II activity of human Tregs (Figure 6B). We next investigated the combined complex ll/lll function in intact purified mitochondria upon increased NaCI concentrations. Of note, NaCI dose- dependently inhibited complex ll/lll activity to a similar extend as AA. Thus, our data indicate that HS, similarly to a blockade of complex III by AA, induces a disturbance of mitochondrial respiration in Tregs at the level of complex ll/lll of the ETC.
A recent study has demonstrated that Na+ influx into mitochondria under hypoxic conditions is regulated by the mitochondrial Na+/Ca2+ exchanger NCLX, interfering with OXPHOS function (Hernansanz-Agustin et al. 2020, Nature 586:287-291), seemingly mirroring our previously observed effects of Tregs under HS conditions. Considering HS-induced increases in intracellular Na+ and direct effects on complex ll/lll activity, we tested the effects of a specific inhibitor of NCLX (CGP-37157; CGP) on complex ll/lll activity in intact mitochondria. Of note, CGP significantly reversed salt-induced decrease of complex ll/lll activity, indicating that mitochondrial influx of Na+ mediates the HS-induced effect on the ETC (Figure 6C). To examine, if this effect is also directly observed in Tregs, we analyzed the impact of NCLX inhibition under HS conditions in suppression assays. Human Tregs were pre-activated under HS conditions in the presence or absence of CGP and were tested for their function to inhibit effector T cell proliferation. Notably, pharmacological inhibition of NCLX in Tregs under HS environments, led to a significant recovery of Treg suppressive function in vitro (Figure 6D). Furthermore, in line with increases of F0XP3, Treg incubation with CGP was able to significantly reduce the IFNG/IL10 ratio (Figure 6E, 6F), indicating that CGP inhibits the Th 1-1 ike proinflammatory phenotype conferred by HS treatment. CGP further partially restored HS-perturbed Treg function in vivo. The inhibition of NCLX in Tregs led to increased potency to contain EAE and xGvHD (Figure 6G, 6H). Thus, mechanistically, mitochondrial Na+ influx induced by extracellular HS conditions and its direct effects on the ETC in Tregs through NCLX seem to be key for the observed immunometabolic disturbance and dysfunction of HS-treated human Tregs, closely mirroring phenotype and function of autoimmune Tregs.
EXAMPLE 7. Inhibition of NCLX/SLC8B1 expression in human Tregs
A highly efficient guide (g)RNA for the human SLC8B1 gene was designed and selected as described before (Van Zeebroeck et al. 2021, Front Immunol 12:655122). A schematic representation is given in Figure 7. For KO calculation, isolated DNA from the different conditions alongside specific primers targeting the gene of interest were investigated by Sanger sequencing (LCG Genomics). KO score is generated by ICE analysis tool (Synthego) that indicates the percentage of KO on the genomic level.
The sequence of the gRNA is included in the Table 2.
Table 2
Figure imgf000050_0001
EXAMPLE 8. Discussion
Here we have demonstrated for the first time that HS severely affects cellular metabolism and suppressive function of Tregs. We observed that HS leads to a pro-inflammatory Th 1-1 ike profile which closely overlaps with the transcriptomic features of Tregs isolated from patients with autoimmune disorders such as MS, SLE and RA. Our data demonstrates that raised physiological HS conditions, similar to Na+ concentrations that can be reached inside inflamed tissues in vivo, perturbs the function of Tregs by metabolic reprogramming through the interference with mitochondrial respiration.
Mechanistically, Na+ is taken up to the intracellular compartment and subsequently via NCLX to the mitochondria, where it directly blocks mitochondrial respiration on the level of complex ll/lll of the ETC, leading to significantly decreased OCR and mitochondrial membrane potential and consequently to lower ATP levels. This energy obstruction based on HS-induced mitochondrial dysfunction seems to be the initial step how sodium alters Treg function and leads to a swift downregulation of FOXP3 and massive changes in gene expression, in line with significant phenotypic and functional alterations, closely mimicking dysfunctional Tregs in autoimmunity (Dominguez-Villar et al. 2018, Nat Immunol 19:665-673; Hernandez et al. 2015, J Clin Invest 125:4212-4222; Sumida et al. 2018, Nat Immunol 19:1391-1402). A similar phenotype can be induced by pharmacological blockade or interference with complex III by AA and our data demonstrates that the HS-induced effect on Treg dysfunction could be reversed by inhibition of mitochondrial Na+ influx through blocking of NCLX. Hallmarks of the phenotype induced by the interference with mitochondrial respiration in human Tregs are a lack of IL-2 responsiveness by hampered STAT5 phosphorylation and decreased F0XP3, IL-10 and CTLA-4 levels as well as increases in mtROS and IFN-y production and activation of the WNT/beta-catenin pathway, consequently leading to a loss of Treg suppressive function.
Importantly, our data provide evidence that even the short-term disruption of mitochondrial function can have long-lasting consequences for the fitness and suppressive capacity of human and murine Tregs in vivo. Similar processes may occur in humans in vivo, linking dietary salt intake to immune function and defining it as a potential risk factor contributing to disease. By now, already a few studies reported that even moderate changes in salt intake could impact human immunophenotypes (Muller et al. 2019, Nat Rev Immunol 19:243-254). Our data show a significant negative correlation of IL-10 expression in FOXP3+ Tregs in relation to higher salt intake in healthy subjects.
Our data go well in line with recent data that indicated that Tregs of people with MS display a disturbed metabolic phenotype and defects in mitochondrial respiration (Duscha et al. 2020, Cell 180:1067-1080; La Rocca et al. 2017, Metabolism 77:39-46). Moreover, the RNA expression data set used here for reanalysis of autoimmune Tregs, revealed a signature of metabolic reprogramming and mitochondrial dysfunction (Alissafi et al. 2020, Cell Metab 32:591-604. e7). It is thus tempting to speculate that perturbed mitochondrial function elicited by HS encounter, copying hallmarks of dysfunctional Tregs in autoimmunity, further provides a piece of evidence linking environmental factors and shifts in the ionic microenvironment to the observed deregulation of the immune cell balance (Arroyo Hornero et al. 2020, Front Immunol 11:253).
Tregs not only play a role in autoimmunity, but have also been shown to be important mediators to contain chronic inflammation in cardiovascular diseases (CVD) such as atherosclerosis (Ait-Oufella et al. 2006, Nat Med 12:178-180), hypertensive target organ damage (Kvakan et al. 2009, Ciruculation 119:2904-2912) and myocardial infarction (Weirather et al. 2014, Circ Res 115:55-67). Interestingly, reduced Treg numbers and a dysfunctional Treg phenotype have also been reported in CVD and were associated with progression of disease (Meng et al. 2016, Nat Rev Cardiol 13:167-179; Baardman & Lutgens 2020, Metabolites 10:279; Saigusa et al. 2020, Nat Rev Cardiol 17:387-401; Tang et al. 2010, N Engl J Med 363:2385-2395). Since Tregs in CVD also displayed a pro-inflammatory Thl-like phenotype (Butcher et al. 2016, Circ Res 119:1190-1203; Bansal et al. 2019, Circulation 139:206-221), it would be of interest to investigate if excess sodium may further contribute to chronic inflammation and CVD by disabling Tregs.
In this respect, it is notable that HS-perturbed Tregs seem to rather resemble 'fragile' Tregs than 'ex- Tregs'. While exhibiting decreases in F0XP3, HS-perturbed Tregs still largely maintained demethylation of the FOXP3 locus and showed a Th-like phenotype and further expression of characteristic markers for Treg fragility (data not shown). Although this becomes a detrimental scenario in autoimmunity or CVD, it can be beneficial in cancer, promoting more efficient anti-tumor immunity (Hatzioannou et al. 2021, Front Immunol 12:731947; Overacre-Delgoffe & Vignali 2018, Cancer Immunol Res 6:882-887; Overacre- Delgoffe et al. 2017, Cell 169:1130-1141), indicating that the mechanistic insights how HS acts on Tregs may of further interest in the field of cancer. However, further research is needed to investigate this in more detail.
It is tempting to speculate that the discovery of the metabolic effect of salt on Tregs also applies for other salt-sensitive immune cell types like Thl7 cells (Wu et al. 2013, Nature 496:513-517; Kleinewietfeld et al. 2013, Nature 496:518-522) or myeloid cells (He et al. 2020, Nat Commun 11:1732; Willebrand et al. 2019, Front Immunol 10:1141) and therefore may have a general impact on regulating immune reactions in response to changes in the ionic microenvironment. Of note, earlier studies in the murine system have already indicated that HS affects cellular metabolism of macrophages (Binger et al. 2015, J Clin Invest 125:4223-4238; Ip et al. 2015, Nat Commun 6:6931). Further, Hernansanz-Augustin et al. (2020, Nature 586:287-291) demonstrated in endothelial cells and fibroblasts that hypoxia activates the mitochondrial NCLX, resulting in an accumulation of cytosolic Ca2+ and subsequent Na+ accumulation in mitochondria (Hernansanz-Augustin et al. 2020, Nature 586:287-291) . Intriguingly, mitochondrial Na+ accumulation resulted in lower membrane fluidity and diminished complex ll/lll activity, ultimately leading to a burst of superoxide and reduced ATP production. These findings go well in line with our data in human Tregs, where increases in Na+ seem to directly interfere with late complex II and complex III activity leading to the described phenotypic and functional consequences for human Tregs even in the absence of hypoxic conditions, suggesting a common cell type-independent mechanism how Na+ could affect OXPHOS. Since the inhibition of NCLX reversed the salt-induced Treg dysfunction in vitro and in vivo, our findings indicate that this mechanism may represent a potential target to restore Treg function under conditions of altered ionic microenvironments or hypoxia. However, since CGP as an inhibitor of the Na+/Ca2+ exchanger NCLX (Boyman et al. 2013, J Mol Cell Cardiol 59:205-213) showed limited specificity by effecting also the plasma membrane Na+/Ca2+ antiporter SLC8A1 (Czyz & Kiedrowski 2003, Biochem Pharmacol 66:2409-2411; Tanaka et al. 2002, Br J Pharmacol 135:1096-1100), further research is needed to overcome these issues in the future. Although CGP was successfully used to prevent heart failure (HF) and sudden cardiac death in an animal model (Liu et al. 2014, Circ Res 115:44-54), tamoxifen induced cardiomyocyte-specific deletion oi Slc8bl (encoding for NCLX) in adult mouse hearts resulted in sudden death associated with left ventricular remodeling and HF (Luongo et al. 2017, Nature 545:93-97). These reports indicate on the complexity of potentially targeting NCLX by using CGP in vivo. However, several attempts have already been made to optimize its specificity and pharmacokinetic profile by the generation of new CGP analogues, which might be superior for potential future therapeutic usage (Martinez-Sanz et al. 2016, Eur J Med Chem 109:114-123; Viejo et al. 2021, Molecules 26:4473). Moreover, novel targeted approaches and strategies may further allow cell type and mitochondria specific applications in the future (Liew et al. 2021, Angew Chem Int Ed Engl 60:2232-2256).
In summary, we have demonstrated for the first time that HS perturbs mitochondrial function of human Tregs by inhibition of mitochondrial respiration on the level of ETC complex ll/lll. The critical disturbance of energy production induces metabolic reprogramming in Tregs and goes in line with severe changes in gene expression and functional disturbances including swift alterations in F0XP3 expression. The HS effect could be mimicked by pharmacological inhibition or genetic disruption of mitochondrial complex III, leading to a similar phenotype observed in dysfunctional Tregs of patients with autoimmunity and our data indicates that salt intake could impact functional features of Tregs in humans. Since even the short-term engagement in HS environments could lead to long-term loss of function of human and murine Tregs in vivo, our findings have important implications for autoimmunity. The interference with this pathway by targeting mitochondrial NCLX offers novel treatment options for salt-sensitive diseases.

Claims

1. An isolated regulatory ? cell (Treg cell) or population of isolated Treg cells characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited in the Treg cell or cells.
2. The isolated Treg cell or population of isolated Treg cells according to claim 1 wherein the function of NCLX is inhibited by a pharmacological compound.
3. The isolated Treg cell or population of isolated Treg cells according to claim 1 wherein the expression of NCLX is inhibited by a DNA nuclease specifically knocking out or disrupting NCLX, an RNase specifically targeting NCLX, or an inhibitory oligonucleotide specifically targeting NCLX.
4. The isolated Treg cell or population of isolated Treg cells according to any one of claims 1 to 3 wherein the Treg cells are polyclonal Treg cells, in vitro amplified or expanded Tregs, antigen-specific Tregs, engineered T cell receptor (TCR)-Tregs, engineered chimeric antigen receptor (CAR)-Tregs, monospecific CAR Tregs, dual CAR Tregs, universal CAR Tregs, modular CAR Tregs, B-cell-targeting antibody receptor (BAR) Tregs, design Tregs, or chimeric cytokine receptor (CCR) Tregs.
5. A composition comprising the isolated Treg cell or population of isolated Treg cells according to any one of claims 1 to 4.
6. The composition according to claim 5 which is a pharmacological composition.
7. The isolated Treg cell or population of isolated Treg cells according to any one of claims 1 to 4 or the composition according to claim 5 or 6 for use as a medicament.
8. The isolated Treg cell or population of isolated Treg cells according to any one of claims 1 to 4 or the composition according to claim 5 or 6 for use in treating an auto-immune disease; for use in treating an inflammatory disease; for use in suppressing transplant, graft or allograft rejection; or for use in treating a cardiovascular disease.
9. The isolated Treg cell or population of isolated Treg cells or the composition for use according to claim 7 or 8 wherein the Treg cell or cells, or the composition comprising them, are adoptively transferred in a subject.
10. The isolated Treg cell or population of isolated Treg cells or the composition for use according to any one of claims 7 to 9 wherein the Treg cells are autologous Treg cells, allogeneic Treg cells, or induced Treg cells.
11. A method of producing the isolated Treg-cell according to any of claims 1 to 4, the method comprising a step of isolating Treg-cells from peripheral blood, umbilical cord blood, thymus or leukapheresis product obtained from a subject, or comprising a step of reprogramming conventional CD4+ T-cells obtained from a subject into Tregs.
12. The method according to claim 11 further comprising a step of ex-vivo expanding the isolated or reprogrammed Tregs.
13. The method according to claim 11 or 12 further comprising a step of ex-vivo manipulation to inhibit the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX in the Tregs by means of pharmacological inhibition or by means of a DNA nuclease specifically knocking out or disrupting NCLX, an RNase specifically targeting NCLX, or an inhibitory oligonucleotide specifically targeting NCLX.
14. A pharmaceutical kit comprising at least one vial comprising at least one of the isolated Treg cell or population of Treg cells according to any of claims 1 to 4, or the composition according to claim 5 or 6.
15. An isolated TCR-engineered Treg-cell, CAR-engineered Treg-cell, or BAR-engineered Treg cell characterized in that the function or expression of the mitochondrial Na+/Ca2+ exchanger NCLX is inhibited in the engineered Treg cell.
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