WO2012127240A2 - Essai immunologique - Google Patents

Essai immunologique Download PDF

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WO2012127240A2
WO2012127240A2 PCT/GB2012/050635 GB2012050635W WO2012127240A2 WO 2012127240 A2 WO2012127240 A2 WO 2012127240A2 GB 2012050635 W GB2012050635 W GB 2012050635W WO 2012127240 A2 WO2012127240 A2 WO 2012127240A2
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ctla
cells
cell
gfp
expressing
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PCT/GB2012/050635
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WO2012127240A3 (fr
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Omar Saleem QURESHI
David Michael SANSOM
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The University Of Birmingham
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

Definitions

  • the invention relates to a method of assaying for modulators of the immune system by monitoring the cycling of CTLA-4 in cells.
  • CTLA-4 is an essential regulator of immune responses whose mechanism of action is the subject of debate.
  • the inventors demonstrate herein that a central molecular feature of CTLA-4 is its ability to capture and degrade co-stimulatory ligands from dendritic cells by a process of trans-endocytosis. Accordingly, all cells expressing CTLA-4, including activated and regulatory T cells, can remove and degrade costimulatory molecules resulting in impaired T cell costimulation.
  • acquisition of CD86 from antigen presenting cells can be demonstrated in vivo and is dependent on TCR engagement and CTLA-4 expression.
  • CTLA-4 acts as an effector molecule to inhibit CD28 costimulation by the cell-extrinsic depletion of ligands.
  • This model accounts for many of the key features of the CD28-CTLA-4 system providing an explanation for the sharing of ligands between CTLA-4 and CD28, the phenomenon of CTLA-4 endocytosis and the constitutive expression of CTLA-4 on regulatory T cells.
  • CD80 is another costimulatory molecule that acts in the same way as CD86. The binding of CD86 and CD80 to CD28 act to stimulate the cell.
  • CTLA-4 Cytotoxic T- Lymphocyte Antigen 4
  • CD 152 Cytotoxic T- Lymphocyte Antigen 4
  • CD 152 is also known as CD 152 and in humans is encoded by the human CTLA-4 gene. It comprises an extracellular V domain, a transmembrane domain and cytoplasmic tail. Mutations in the gene have been associated with diseases such as Grave's disease, Hashimoto's disease, celiac and systemic lupus erythematosis.
  • the inventors have now identified that CTLA-4 binding to, for example, CD80 and CD86 ligands, takes the ligands into the cell where they are destroyed, and is then returned to the surface of the cell.
  • the invention provides a method of identifying a modulator of the immune response system, comprising assaying for the effect of a compound on the intemalisation and recycling of CTLA-4 in a cell, wherein an increase in cycling of CTLA-4 compared to a control without the compound indicates that the compound suppresses the immune response and a decrease in the cycling of CTLA-4 compared to a control without the compound, indicates that the compound increases the immune response.
  • the cycling of the CTLA-4 is determined by monitoring the uptake of a CTLA-4 dependent label into the cell.
  • the CTLA-4 dependency may be tested, for example by deleting CTLA-4 from the cell or blocking CTLA-4 with anti-CTLA-4 antibodies which are generally known in the art.
  • Ipilimumab is a monoclonal antibody which is produced by Bristol-Myers Squibb and binds to CTLA-4.
  • CTLA-4 uptake may be dependent on CTLA-4 binding to CD80 or CD86, especially CD86.
  • CD80 and CD86 are generally known in the art and are found on dendritic cells activated B cells and monocytes.
  • the CD80 or CD86 may be directly or indirectly labelled.
  • the label may be, for example, a radiolabel such as 35 S or 111 In and the uptake is measured by increased radioactivity due to the CD80 or CD86 binding CTLA-4 and being taken into the cell.
  • the CD80 or CD86 may be a fusion protein of the whole protein, or the CTLA-4 binding region of the protein with a detectable label, such as a colourimetric label.
  • the colourimetric label may be, for example, green fluorescent protein (GFP) or aquorin. Alternatively, it may be detected using a bound fluorescent label such as FITC. It may also be labelled with biotin and the presence of the CD80 or CD86 may be detected using labelled avidin which binds to the biotin.
  • the uptake of CD80 or CD86 may also be detected using labelled anti-CD80 or anti-CD86 antibodies, which are commercially available.
  • the uptake of coloured labels may be detected using flow cytometry or confocal microscopy to detect the presence or amount of label in the cells. Such labels are generally known in the art.
  • the cell typically expressing CTLA-4 is typically a eukaryotic cell such as a mammalian, such as mouse or hamster, or fish (such as zebra fish) and insect cell.
  • the cell is a human cell. It may be a T cell, for example an activated T cell, such as a regulatory T cell, It may be CD4 + CD25 " or CD4 + CD25 + . It may naturally express CTLA-4. Alternatively, CTLA-4 may be expressed recombinantly in the cell. Methods of expressing proteins such as CTLA-4 are generally known in the art.
  • the cell may be a Jurkat cell of a Chinese Hamster Ovary cell (CHO cell). The cells are preferably in vitro.
  • the CTLA-4-expressing cell may act as a recipient cell.
  • the cell may receive CD86 or CD80 from a donor cell expressing the ligands.
  • the donor cell may be a dendritic cell or CHO cell expressing CD86 or CD80.
  • the cells may be part of a whole organism such as a non-human mammal, fish or insect.
  • the organism may be a rodent such as a mouse.
  • the mouse may be, for example a CTLA-4 +/+ D011 x ripmOV A/Rag-/- mouse.
  • the modulator may be a lysosomal inhibitor which inhibits CTLA-4 cycling.
  • Such inhibitors include bafilomycin A.
  • the modulator may also be a compound that blocks binding of CD80 or CD86 to CTLA-4. These include antibodies or fragments of antibodies against CTLA-4.
  • inhibitors include inhibitors of CTLA-4 transendocytosis which disrupt removal of ligands. These will prevent CTLA-4 from suppressing immune responses thereby increasing immune responses.
  • Example inhibitors will include those targeting actin recruitment to the CTLA-4 containing vesicles such as Latrunculin, a naturally occuring toxin obtained from sponges.
  • Blocking delivery of CTLA-4 to the plasma membrane by interfering with vesicle trafficking pathways will promote immune activation by disrupting CTLA-4 function.
  • Example compounds for this purpose include inhibitors of phospholipase D (butan-l-ol) or inhibitors of ARF-1 such as brefeldin A.
  • Compounds inhibiting delivery of CTLA-4 or its bound ligands to lysosomes will be effective immunomodulatory compounds.
  • Such compounds will include lysosome neutralising compounds (ammonium chloride), vacuolar ATPase inhibitors (Bafilomycin A) and inhibitors of ubiquitylation.
  • CTLA-4 degradation is controlled by modification of CTLA-4 with ubiquitin.
  • Ubiquitination of CTLA-4 itself directly modifies the stability of CTLA-4, its surface expression and re-cycling pattern, all of which impact on its ability to function by removal and destruction of the ligand.
  • manipulation of the ubiquitin enzyme system has potential therapeutic benefit.
  • Ubiquitin contains 76 amino acid residues, two of which have particular importance (Gly-78 and Lys-48). Following activation of Gly-78 in an ATP-dependent manner ubiquitin is attached to the ⁇ -amino group of lysine on the protein substrate.
  • Lys-45 is located on the surface of a Type III reverse turn and can generate multi-ubiquitin chains since its ⁇ -amino group is exposed such that it can form an additional amide isopeptide bond (Myung, J., et al Med. Res. Rev. (2001) 21, 245-273).
  • ubiquitination modification As their targeting signal.
  • polyubiquitination and proteasomal degradation is not the only pathway followed by a membrane protein.
  • Mono-ubiquitination which involves the addition of an ubiquitin molecule to one lysine
  • multi-ubiquitination which involves the addition of an ubiquitin molecule to several lysine residues, can regulate the internalisation and sorting of target receptors to endocytic compartments (d'Azzo et al 2005 Traffic, 6, 429-441).
  • ATP used by the ubiquitin- activating enzyme charges ubiquitin and forms an ubiquitin C-terminal adenylate.
  • ubiquitin-protein isopeptide ligases (E3) bind to the target protein and attach the C-terminus of ubiquitin to lysine residues of the target.
  • a polyubiquitin chain can be formed through the same ubiquitination conjugation cascade (Myung et al, Supra and Hueng et al, Oncogene (2004) 23, 1958-1971).
  • the ubiquitin pathway provides a number of sites to target to adjust CTLA-4 recycling.
  • the modulator being tested may be a modulator of ubiquitylation.
  • the modulator may be:
  • inhibitors or enhancers to modulate an immune response in vitro or in vivo is also provided.
  • the inhibitor may be MG132 (CAS number 133407-82-6) or UBEI-41 (available from Biogenova).
  • CTLA-4 Compounds capable of keeping CTLA-4 and its bound ligand together in a "locked” state will downregulate CTLA-4 function.
  • Such compounds could include antibodies recognising the complex of CTLA-4 and its bound ligands.
  • Compounds capable of increasing the rate of CTLA-4 recycling to the cell surface will be capable of enhancing CTLA-4 function.
  • FIG. 1 CTLA-4 mediated acquisition of co-stimulatory molecules.
  • A Flow cytometric analysis of CD86-GFP transfer into CTLA-4 expressing cells.
  • CHO cells expressing CD86- GFP (Far Red labeled) were co-cultured with CHO controls or with CTLA-4 + CHO cells in the presence or absence of 10 nM Bafilomycin A.
  • Singlet CTLA-4 expressing cells were analyzed for GFP acquisition by excluding Far Red-i- donor cells from analysis (see fig.
  • CHO-CD86 cultured alone are shown in fig. S7A.
  • D Confocal images of cells expressing wild type (wt) CTLA-4 or CTLA-4 lacking the cytoplasmic domain (del36) (glows red) incubated for 2 hours with CD86-GFP (glows green) expressing cells.
  • E Flow cytometric analysis of CD86 surface expression on CHO-CD86 cells co-incubated with increasing numbers of untransfected (control upper line), wild-type CTLA-4 (lower line) or CTLA-4 del36 cells - middle line at right (expressed as %
  • Fig. 2 Human T cells use CTLA-4 to remove CD86 from dendritic cells.
  • A Typical CD86 expression on a human monocyte-derived dendritic cell (DC) cultured in the absence of T
  • TCR stimulation promotes CTLA-4 trafficking and trans-endocytosis of CD86.
  • CD4 CD25 (Treg) or CD4 CD25 T cells were incubated with DCs and anti-CD3 overnight, fixed, stained using anti-CD86 (glows green), anti-CD3 for T cells (glows blue), and visualized by confocal microscopy. Arrow indicates
  • E Surface levels of CD86 on DCs incubated with CD4 + CD25 +
  • Fig. 4 In vivo capture of CD86 by CTLA-4.
  • Balbc Rag2 ⁇ / ⁇ mice were reconstituted with CD86-GFP transduced Balbc Rag2 ⁇ / ⁇ bone marrow to permit the development of APC expressing CD86-GFP. 3wk later mice were injected with DO11.10 CD4 + T cells and immunized as described in figure S16.
  • FIG. 1 Representative images of CD4 + T cells purified from spleen after treatment in vivo with peptide showing CD4 and CD25 staining.
  • CTLA-4- expressing cells and Far Red labelled CD86-GFP-expressing CHO cells (donor cells) were co-cultured for 3h. Following FACS acquisition of the total mixed population (i) cells were gated on low pulse width (ii) to exclude cell clusters. Far Red negative cells were then gated to exclude CD86-GFP donor cells from the analysis (iii). The resultant singlet CTLA-4 expressing cells were then analysed for GFP content (iv).
  • CD86-GFP expressing CHO cells were co-cultured for 3h with control CHO or CTLA-4-expressing CHO cells. Compared with control CHO cells (upper panel), cells expressing CTLA-4 (lower panel, Far Red negative) acquire GFP. This is associated with a concomitant decrease in GFP signal on the CD86 donor cells (Far Red positive cells).
  • Control CHO cells or CTLA-4 expressing CHO cells were incubated with CD86-GFP expressing CHO cells (Far Red labelled) for 3 hours.
  • CD86 was then surface labelled by anti- CD86 PE at 4°C and gated using the strategy in Fig. 5. Whilst 36% of CTLA-4 positive cells capture CD86-GFP only 0.5% label for surface CD86.
  • the CD86 staining of the donor cells following incubation with CTLA-4 + cells is shown in the lower panel, revealing a marked reduction in CD86 surface expression. Representative of 2 independent experiments.
  • CTLA-4 expressing CHO cells were incubated alone (left panels) or with Far Red labelled CD86-GFP expressing CHO cells for 3 hours (right panels) in the presence or absence of the lysosomal inhibitor Bafilomycin A. Cells were then fixed and stained with anti-CTLA-4. Plots are gated on CHO-CTLA-4 (Far Red negative) cells. CD86 transfer does not reduce CTLA-4 expression in GFP+ cells. Additionally, in the presence of Bafilomycin A the CD86-GFP signal increases (due to blockade of lysosomal degradation) however the expression of CTLA-4 is not affected (lower right panel). This suggests CTLA-4 is not degraded along with ligand.
  • B Histogram overlays of the data in A showing CTLA-4 expression in the presence of absence of bafilomycin. Data are representative of 2 experiments.
  • Both CD80 and CD86 are acquired by CTLA-4 expressing cells.
  • CTLA-4 expressing CHO cells were incubated with Far Red-labelled donor CHO cells expressing either CD80-GFP or CD86-GFP. Cells were co-cultured for the times indicated in the presence or absence of ammonium chloride and analysed by FACS as shown in figure 5. Data are representative of at least 3 independent experiments.
  • CD86 APC or CTLA-4 APC antibodies were titrated on to beads that have known antibody binding capacity (ABC) (Quantum Simply Cellular anti-mouse IgG).
  • ABSC antibody binding capacity
  • the geometric mean fluorescence was determined for both CD86 and CTLA-4 antibodies generating a standard curve of MFI vs number of antibody molecules bound. This allows an estimation of the number of molecules expressed per cell for CD86 (A) or CTLA-4 (B).
  • Arrows intersecting standard curve show fluorescence values of CD86 on CHO-CD86 transfectants or mature DC (A) and the values for CTLA-4 expressing Jurkat or CHO-CTLA-4 cells (B).
  • CTLA-4 expressing Jurkat cells were incubated with CD86-expressing CHO cells overnight at various ratios and reduction in CD86 expression measured (C). At a ratio of 1 Jurkat cell to 1 CHO cell which corresponds to a ratio of approximately 8 CD86 molecules to 1 CTLA-4 molecule we observe a 35% reduction in CD86 expression. This reduction was sufficient to observe a functional inhibition in T cell proliferation when these cells were used to stimulate CFSE-labelled responder T cells (D). Data are representative of 2 experiments.
  • CD86 expression pattern is punctate in cells cultured with CTLA-4. Confocal micrograph of adherent CHO-CD86 cells cultured overnight either alone or with CHO-CTLA-4 cells. Cells were fixed, permeabilised and stained with goat anti-human CD86 (CI 9) green. Dici are representative of more than 20 independent experiments.
  • CHO-CD86 expressing cells were labelled with the lipophilic dye PKH26 to label the plasma membrane and then incubated with CHO-CTLA-4 cells. Transfer of membrane dye was monitored by flow cytometry to assess the level of plasma membrane exchange between CTLA-4 and CD86 cells.
  • CTLA-4 + recipents were gated on those that acquired CD86-GFP(red box) and those which did not (black box). The PKH transfer associated with these populations was then analysed. Data are representative of 2 experiments.
  • CHO cells stably expressing either wild type CTLA-4 or CTLA-4 del36 (lacking the cytoplasmic domain) were initially incubated with an unlabelled anti-CTLA-4 antibody at 37°C for 30 minutes to allow labelling of internalising CTLA-4.
  • Surface receptors were then stained red (anti-mouse Ig Alexa 594) at 4°C.
  • Cells were then fixed and permeabilized and stained with alexa 488 conjugated anti-mouse Ig. Cells that internalise the unlabelled CTLA- 4 antibody at 37°C antibody stain green.
  • Bar chart shows quantification of plasma membrane (PM) to internalized (I) protein ratios of WT and mutant CTLA-4 plus SEM.
  • CTLA-4 specifically downregulates CD80 and CD86 but not CD40 on dendritic cells
  • Dendritic cells were incubated with human CD4 + CD25 " T cells and anti-CD3 (0 ⁇ g/ml) for 4 days to allow T cell expression of CTLA-4. Cultures were carried out in the presence or absence of anti-CTLA-4 (20 ⁇ g/ml). Dendritic cells were then gated using anti-CD 11c and the levels of CD80, CD86 and CD40 analysed by flow cytometry. CD80 and CD86 levels were specifically increased by anti-CTLA-4 treatment whereas there was no change in CD40 levels.
  • A WT control or CTLA-4-transduced (right peak) Jurkat cells were stained for total CTLA- 4 expression and analysed by flow cytometry. Isotype control staining is shown in the filled histogram and compared with wild-type untransduced (dotted line) and transduced CTLA-4 staining (red line) demonstating that WT Jurkat cells do not express CTLA-4.
  • B Acquisition of CD86-GFP by CTLA-4 transduced Jurkat cells as measured by FACS compared to control Jurkat. Lower panels show the effect of Bafilomycin (BafA)
  • C Electron micrograph showing CD86-HA transfer to a CTLA-4 expressing Jurkat cell. 70nm thick cryosections were stained using anti-HA antibody and detected using rabbbit anti-mouse antibody followed by protein A gold. Data are representative of 3 experiments.
  • CTLA-4 expressing Jurkat cells were incubated for 3 hours with CD86-GFP expressing CHO cells in the presence of NH 4 C1. Cells were analysed as outlined in figure SI. Anti- CTLA-4 ( ⁇ ), CTLA-4 Ig ( ⁇ ) and CD80-Ig ( ⁇ ) inhibited the transfer of CD86.
  • Anti-CD28 does not block CD86 transfer.
  • CTLA-4 expressing Jurkat cells were incubated for 3 hours with CD86-GFP expressing CHO cells in the presence of NH 4 C1. Cells were analysed as outlined in figure SI. The effect of anti-CTLA-4 ( ⁇ ), anti-CD28 ( ⁇ ) or anti-CD45RO ( ⁇ ) on transfer of CD86-GFP was assessed. Data are representative of at least 3 experiments.
  • TCR stimulation drives CTLA-4 trafficking in T cells and Treg.
  • CTLA-4 cycling pool (C) Total expression of CTLA-4 in resting human peripheral blood T cells gated on CD25 expression. Levels of CTLA-4 were determined by intracellular staining of CD4 + T cells along with FoxP3. (D) Resting or activated (anti-CD3 and anti-CD28 beads) Tregs were stained for surface or cycling CTLA-4. Surface staining was carried out at 4 °C and cycling of CTLA-4 was detected by anti-CTLA-4-PE labelling at 37°C. "Activated" is right peak. Data are representative of at least 3 experiments.
  • CTLA-4 downregulation of costimulatory molecules is functionally relevant.
  • A Proliferation of CFSE-labelled CD4 + CD25 T cells 4d after stimulation by DC re-isolated from culture with Tregs in the presence or absence of anti-CTLA-4 antibody (anti-CTLA-4 upper trace).
  • B CFSE-labelled CD25 " T cells were stimulated with DC previously incubated with Treg. Cultures were supplemented with either control CHO cells or those transfected with CD86 CD86 CHO left peak.
  • C T cell blasts expressing CTLA-4 were used as suppressor cells. Dendritic cells were incubated with T cell blasts with or without anti-CTLA- 4 and their capacity to stimulate proliferation of CD25 " CFSE labelled responder CD4 cells was assessed. T cell responses to DC in the absence of blasts (left panel) and presence of blasts (right panel) are shown. Data are representative of 3 experiments. CTLA-4 upper trace of blasts traces.
  • Mouse CD4+ CD25+ T cells can effectively capture CD86 in vitro.
  • A T cells from either WT or CTLA-4 -deficient mice (Ctla4 "/_ ) were incubated overnight with anti-CD3 (O ⁇ g/ml) and murine CD86-GFP expressing CHO cells. Transfer of CD86 was observed into wildtype T cells but not CTLA-4 knockout T cells. Wildtype cells did not acquire control GFP protein (B) DO 11 T cells were incubated with CHO cells expressing I- A d and CD86-GFP either with or without OVA peptide and analysed by confocal microscopy. Cells were analysed for CD4 (blue) CD25 (red) and CD86 (green) expression. Data are representative of 2 experiments.
  • Figure 21 illustrates the workflow for in vivo analysis of ligand trans-endocytosis.
  • FIG. 4 FACS analysis showing Foxp3 staining of T cell populations shown in figure 4B and 4C. Plots are gated on CD4+CD25+ cells.
  • B Quantification of confocal images from Fig. 4 shows that the mean GFP fluorescence of the CD86-GFP transferred to T cells approaches that of cells that were originally transduced with CD86-GFP indicating a high level of transfer. Fluorescence values were generated by measuring CD86-GFP content of recipient T cells and donor CD86-GFP+ cells using Image J software. Each circle represents an individual cell. Left column - recipient T cells, right column donor cells.
  • C Quantification of in vivo experiments shown in Figure 4.
  • CTLA-4 internalises and directs its ligands for lysosomal degradation.
  • Large arrows indicate targets for inhibition including internalisation of ligand (1) sorting to MVB (2) inhibition of lysosomal degradation (3), delivery of CTLA-4 to the cell surface (4) and re-cycling of CTLA-4 following ligand capture (5).
  • Cytoplasmic sequences determine the rate of CTLA-4 internalisation (Step 1 of Figure 22).
  • Human CTLA-4 rate of internalisation Human CTLA-4 transfected CHO cells were incubated at 4°C for 30 minutes with CTLA-4-PE and washed. Cells were either incubated at 4°C or raised to 37°C for times shown. Remaining surface CTLA-4 was then detected using an Alexa 647 coupled secondary Ab at 4°C. The use of different CTLA-4 species variants reveals that the cytoplasmic domain sequence controls the rate of internalisation.
  • cytoplasmic lysines prevent CTLA-4 degradation (Steps 2 and 3 of Figure 22).
  • CHO-CTLA-4 wild type or mutant were exposed to drug treatment (cycloheximide, NH 4 C1, or both) for 3 h CTLA-4 protein was then measured by microscopy (A and C) or by FACS (B and D). Data shows that removal of cytoplasmic lysine residues stabilised the CTLA-4 protein (C and D). Data also show that inhibiting lysosomes with ammonium chloride prevents CTLA-4 degradation (A and B).
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs Peripheral blood mononuclear cells
  • CD4 + T cells were isolated by incubating PBMC with human CD4 + T cell- enrichment cocktail and magnetic colloid according to the manufacture's instruction (Stem Cell Technologies).
  • CD4 + cells were incubated with anti-CD25- microbeads (Miltenyi Biotech, Auburn, CA) at 4°C for 30 minutes.
  • CD4 + CD25 ⁇ T cells which did not bind to the column were collected from the flow-through and washed before use.
  • CD4 + CD25 + T cells were subsequently retrieved from the column.
  • Human monocytes were purified from PBMC by negative selection using human monocyte enrichment mixture and magnetic colloid according to the instructions of the manufacturer (StemCell, Meylan, France).
  • DCs dendritic cells
  • monocytes (2 x 10 6 cells/ml) were cultured in RPMI 1640 medium containing 10% FCS and antibiotics with GM-CSF (PeproTech, Rocky Hill, NJ; 800 U/ml) and IL-4 (PeproTech; 500 U/ml). Additional medium containing GM- CSF and IL-4 was added every 2 days.
  • SEB-specific T cell blasts PBMCs were incubated with ⁇ g/ml SEB for 6 days. Live CD4 + cells were then negatively purified as above.
  • DCs were re-isolated from T cell cultures using anti-CD2 microbeads (Miltenyi) to deplete T cells.
  • DNA constructs and Transfectants
  • CTLA-4 cDNA was cloned into a CMV expression vector pCDNA3.1 as previously described (25).
  • a CTLA-4 construct with the C-terminal 36 amino acids deleted was generated by PCR and cloned into the same vector to generate CTLA-4 del36.
  • GFP- tagged CD80 and CD86 were generated by PCR to remove the stop codon and cloned into the EGFP-N1 vector (Clontech).
  • Mouse CD86-GFP fusion was cloned into modified (IRES-GFP deleted) MIGR1 vector (a gift from Dr. W.S. Pear, University of Pennsylvania, Philadelphia, PA).
  • CHO cell lines expressing either CD80, CD86 or CTLA-4 were generated by electroporation of human cDNAs cloned into a CMV expression vector.
  • Cells were grown in DMEM containing 10% FBS as previously described (25).
  • Cells expressing the plasmid were selected using G418 (500 ⁇ g/ml) treatment and FACS. Cultures were maintained at 37°C in a humidified incubator containing 5% C0 2 and were passaged by trypsinization.
  • CHO cells expressing IAd were obtained from Prof. Gordon Freeman (Dana Faber Cancer Institute) and transfected with mouse CD80-GFP using Amaxa nucleofection.
  • CTLA4 was cloned into the MP71 retrovirus vector. Retroviral supernatants were obtained by transfecton of Phoenix-A packaging cells with retroviral vectors containing CTLA4 in combination with pCLampho, using the FUGENE6 transfection reagent (Roche Molecular Biochemical). Retroviral supernatants were harvested 48h after transfection and used for transduction of Jurkat cells.
  • RetroNectin TaKaRa
  • 2%BSA RetroNectin
  • 1 ml of retroviral supernatants were added to RetroNectin pre-coated wells and centrifuged at 1500 rpm at room temperature for 15 minutes.
  • 2xl0 6 Jurkat cell were added and centrifuged at 2000 rpm at 30°C for 60 minutes.
  • 24 hours post- infection media was changed to fresh RPMI 1640 with 10% FCS.
  • Jurkat cells were maintained in RPMI containing 10% FBS.
  • 72 hours post-infection cells were sorted by staining with CTLA4-PE (BD) using MoFlow.
  • CD86-GFP fusion sequence was cloned into modified (IRES-GFP deleted) MIGR1 vector (a gift from Dr. W.S. Pear, University of Pennsylvania, Philadelphia, PA).
  • Retroviral superntants were prepared by transfecting retroviral vector into the packaging cell line Plat-E (32) using Lipofectamine 2000 (Invitrogen).
  • Plat-E packaging cell line
  • Retrovirus infection of cells was performed as described previously (33). Fresh BM cells were cultured in IMDM supplemented with 20% FCS, L-glutamine, sodium pyruvate, nonessential amino acids, penicillin, streptomycin, 50ng/ml stem cell factor, 50ng/ml IL-6, and lOng/ml IL-3. After 48, 72, and 96 hr, cells were spin-infected with retrovirus by centrifuging the culture plate in the presence of lOug/ml polybrene for 90 min.
  • Retrovirally transduced bone marrow was intravenously injected into irradiated (450 Rad) Rag -/- mice.
  • CD4+ cells were isolated from DOl l mice or from CTLA-4+/+ or CTLA-4-/- DOl lxRip-mOV A/Rag-/- mice and adoptively transferred I.V into Balb/c Rag- /- mice at l-5xl0 6 cells per recipient.
  • mice received 100 ⁇ g of OVA/alum i.p.
  • mice were given 100 ⁇ g of OVA peptide I.V followed by 600 ⁇ g of chloroquine I.P, 3 hours later.
  • spleens were harvested and digested with 5mg/ml collagenase dispase (Roche) prior to staining for confocal.
  • CD86 transfer into CTLA-4 expressing cells was carried out by labeling CD86 GFP expressing CHO cells with CellTrace Far Red DDAO-SE (Invitrogen). Cells were then incubated together with CTLA-4 expressing CHO cells for 3 hours in the presence or absence of 10 nM Bafilomycin A or 10 ⁇ NH 4 C1. To identify GFP transfer to single, CTLA-4 expressing cells and exclude clusters containing CD86-GFP, cells were gated for low pulse width and negative for Far Red staining as shown in Figure 5. CD80 GFP transfer was assayed in a similar way. Where used, PKH 26 (Sigma) labelling was carried out by incubating CHO-CD86 cells according to maufacturers instructions.
  • responder CD4 + CD25 ⁇ T cells were labeled using 5-(and- 6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE, Molecular Probes).
  • CD4+ CD25- T cells were washed twice in PBS and resuspended at 2 x 10 cells/ml and an equal volume of CFSE in PBS added such that the final concentration was 2.5 ⁇ . Cells were then incubated at RT for 10 minutes with gentle agitation and washed twice in normal growth medium.
  • stimulating with CD86 transfectants CHO cells were fixed using 0.025% glutaraldehyde in PBS for 2-3 min and washed extensively.
  • DC was re-isolated by depleting CD2 positive cells with anti-CD2 microbeads (Miltenyi Biotech). Where indicated T cell blasts were used as CTLA4+ suppressor cells. Blasts were generated by CD3/CD28 bead stimulation for 5 days and added to along with CFSE-labelled responder T cells. Cells were stimulated with DC and anti-CD3 (0 ⁇ g/ml) for 5 days in the presence or absence of anti-CTLA-4 (20 ⁇ g/ml) before analysis by flow cytometry.
  • CD86 surface downregulation For surface analysis of DC phenotypes, cells were collected into cold PBS and surface labeled at 4°C with antibodies against CD86, CD80, CD40 or CD 11c directly conjugated to FITC, PE or APC (BD PharMingen, San Diego, CA). DCs were gated based on forward and side scatter, and CD1 lc labeling. DCs were incubated with T cells at the ratios indicated.
  • CTLA-4 Surface staining of CTLA-4 was carried out on ice for 30 min. Staining of cycling CTLA-4 was carried out by incubation at 37°C for 30min. Total CTLA-4 was carried out by fixing cells with 3.0% formaldehyde solution in PBS and permeabilizing with 0.1% saponin and then stained with anti-CTLA-4 PE (BNI3-Pharmingen) or anti-CTLA-4 C-19 antibody (Santa Cruz) followed by an appropriate secondary antibody. Where stated T cells were stimulated using anti-CD3 anti-CD28 beads (Invitrogen) for 2h prior to staining. To study CTLA-4 cycling in Treg, cells were incubated overnight with anti-CD3 anti-CD28 beads and CTLA-4 staining carried out at 37°C. Where blocking anti-CTLA-4 was used unconjugated anti- CTLA-4 was used at 20 ⁇ g/ml.
  • FOXP3 staining was performed using a FOXP3 staining kit (Ebioscience) according to the manufacturer's instructions.
  • Imaging was carried out using a Zeiss LSM 510 or a Zeiss LSM 780 inverted laser scanning confocal Microscope using a lOOx oil immersion objective with excitation at 488nm, 543nm and 633nm. Constant laser powers and acquisition parameters were maintained throughout individual experiments for analysis. Digital images were prepared using ImageJ (Wayne Rasband, NIH). For quantitation, cells were outlined and mean fluorescence intensity measured using ImageJ. Movies were prepared using ImageJ. All confocal images shown are representative of at least thirty micrographs taken from at least three independent experiments.
  • CTLA-4 expressing cells were labelled with DDAO-SE and incubated with CD86-GFP CHO cells for 3h.
  • CTLA-4 expressing CHO cells were identified by brief labelling with anti-CTLA-4 APC (BD) and incubated with CHO cells stably expressing CD86-GFP.
  • Z-stacks were acquired as a time-series approximately every 3 minutes for 90 minutes or every 1 minute. Projections were then generated using Image J and converted into movies or single images.
  • CHO cells were incubated together for 16 hours on a poly-L-lysine coated coverslip in a 24 well plate. Cells were then fixed with methanol at -20°C for 20 minutes. Non-specific binding sites were blocked by incubation in blocking solution (BS) which consisted of 5% donkey serum (Sigma) in PBS at room temperature (RT). Cells were incubated with anti-CD86 primary antibody (B7-2, C19 antibody, Santa Cruz, CA) and anti-CTLA-4 (BNI3 Pharmingen), washed and then labeled with donkey anti- goat Alexa 546 and anti-mouse Alexa 488 secondary antibodies (Molecular Probes).
  • BS blocking solution
  • RT room temperature
  • Dendritic cells and T cells were added at a 1:3 ratio to poly-L-lysine coated coverslips in a 24 well plate.
  • Cells were activated by the addition of anti-CD3 (Clone OKT3) at a concentration of 1 ⁇ and were incubated for 72h (CD4 + CD25 ⁇ ) or overnight (CD4+CD25+) at 37 °C and 5 % C0 2 .
  • CD4 + CD25 ⁇ CD25 + CD25 ⁇
  • CD4+CD25+ CD4+CD25+
  • DC were pulsed with ⁇ g/ml SEB for 2h and washed prior to use.
  • Cells were centrifuged in a swing out rotor at 200 g for 10 minutes and fixed with methanol at -20°C for 20 minutes. Non-specific binding sites were blocked by incubation in blocking solution (BS).
  • BS blocking solution
  • coverslips were dried and mounted with Vectashield (Vector Laboratories, UK) prior to visualization by confocal microscopy.
  • DO 11.10 TCR transgenic mice and BALB/C mice were purchased from The Jackson Laboratory.
  • RAG-2-/- mice were purchased from Taconic Farms.
  • RIP-mOVA mice on a BALB/c background that express a membrane-bound form of OVA under the control of the rat insulin promoter (from line 296- IB) were a gift from W. Heath (Walter and Eliza Hall Institute, Melbourne, Australia).
  • DO11.10 mice, RIP-mOVA mice and RAG-/- mice were crossed as previously described.
  • CTLA-4-/- mice on a BALB/c background were a generous gift from A. Sharpe (Brigham and Women's Hospital, Boston, USA). All mice were housed in the University of Birmingham Biomedical Services Unit and used according to Home Office and institutional regulations.
  • splenocytes were labelled in glass-bottom dishes (MatTek) at 4°C with anti-CD25 PE and CD4 APC for 15 minutes before imaging by confocal microscopy. Quantitation of GFP fluorescence was carried out by outlining cells and measuring mean fluorescence in ImageJ. Electron Microscopy
  • Cryo-immuno-EM was performed on cells fixed with 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, supported in 10% gelatin, and infused with 2.3M sucrose. Sections (70 nm) were cut at - 120°C and picked up in 1 : 1 2.3M sucrose:2% methylcellulose.
  • Mouse anti-HA HA.11, Covance
  • rabbit anti-mouse intermediate antibody Dako
  • lOnm protein A gold and then contrast stained/dried in 1 :9 4% uranyl acetate:2% methyl cellulose.
  • Samples were viewed on a JEOL 1010 TEM, and images gathered with a Gatan OriusSClOOB CCD camera.
  • the T cell protein CTLA-4 is essential to the prevention of autoimmune disease (1-3). Although the molecular basis for CTLA-4 action has been suggested to be a cell-intrinsic inhibitory signal (4) possibly mediated by the cytoplasmic domain (5), a cell-extrinsic function for CTLA-4 is clearly evident from in vivo models (6-13). Therefore a molecular explanation of CTLA-4 function compatible with such a cell-extrinsic mechanism is needed. The intercellular transfer of a ligand from one cell to its receptor on a different cell is observed in both immune settings and elsewhere (14-19).
  • CTLA-4 + CTLA-4 + CHO cells with donor CHO cells expressing a C-terminally tagged CD86 protein (CD86-GFP).
  • CD86-GFP C-terminally tagged CD86 protein
  • CTLA-4 molecules and CD86 molecules expressed by our cell lines to determine the stoichiometry of CD86 depletion (fig. 10). This showed that a ratio of approximately 1 :8 (CTLA-4:CD86) molecules was sufficient for functionally relevant depletion.
  • CHO-CD86 cells were cultured alone or with CHO-CTLA-4 cells then stained for CD86 and CTLA-4 using antibodies (Fig. 1C).
  • CD86-expressing cells display a characteristic plasma membrane staining pattern (fig. 11 A), however in the presence of CTLA-4, CD86 containing vesicles were observed inside CTLA-4 recipient cells (Fig. 1C, fig. 11 A).
  • CD86 was stained using an antibody against the cytoplasmic domain this indicated that the whole ligand had been transferred into the CTLA-4 + cell.
  • CD86 was robustly expressed on the surface of dendritic cells (Fig. 2A). In the presence of activated T cells, however, CD86 on the plasma membrane of DCs was reduced and instead found in a punctate pattern that co-localized with CTLA-4 (Fig. 2B). Importantly, incubation with a blocking anti-CTLA-4 antibody prevented the down-regulation of CD86 on the DC as well as
  • CTLA-4 Jurkat cells fig. 14C
  • CTLA-4-Ig and anti-CD28 confirmed the specificity of CD86-GFP transfer to Jurkat cells (fig. 15 and fig. 16) and demonstrated that CD28 was not capable of trans-endocytosis.
  • CTLA-4-transfected (but not mock transfected) resting T cells exhibited specific sequestration and internalisation of CD86, from the DC (Fig. 2, D and E), but had no effect on HLA-DR expression. Taken together, these data demonstrated CTLA-4 expression by T cells was sufficient to confer the ability to remove CD 86 from DCs.
  • CD86 acquisition To test this, human CTLA-4 T cell blasts were incubated in the presence of CD86-GFP-expressing CHO cells with or without anti-CD3. TCR stimulation increased the acquisition of CD86-GFP in a manner that was blocked by anti-CTLA-4 and enhanced by bafilomycin (Fig. 3, A). Similarly, staphylococcal enterotoxin B (SEB)-reactive T cell blasts incubated with dendritic cells also showed enhanced acquisition of CD86 (Fig. 3, B and C).
  • SEB staphylococcal enterotoxin B
  • CD86 was downregulated from the APC surface and observed in intracellular puncta inside the Treg.
  • CD86 remained on the plasma membrane of DC in the presence of CD4 CD25 T cells that lacked CTLA-4 (Fig. 3, D and E).
  • CTLA-4 a blocking anti-CTLA-4 antibody.
  • suppression could be overcome by restoring co- stimulation using CD86-expressing transfectants (fig. 18B).
  • T cell blasts could act as suppressor cells in a CTLA-4-dependent manner (fig. 18C) again indicating that depletion of costimulatory molecules by CTLA-4 has functional consequences.
  • mice endocytosis in mice.
  • mice CD4 T cells stimulated in vitro could acquire CD86-GFP from CHO cell targets (fig. 19A and B).
  • CHO cell targets fig. 19A and B.
  • DOl l. lO TCR transgenic T cells specific for a peptide fragment of chicken ovalbumin (OVA) presented by I-A d
  • OVA ovalbumin
  • mice were transferred into Balbc Rag2 ' mice, which 3 weeks prior, had been reconstituted with CD86-GFP-transduced Rag2 ⁇ / ⁇ bone marrow.
  • Recipient mice therefore lacked lymphocytes, except the adoptively transferred DOl l.lO T cells, and expressed CD86-GFP on their antigen presenting cells.
  • mice Seven days after OVA immunization, mice were re-challenged with OVA peptide in the presence of the lysosomal inhibitor, chloroquine. T cells were then harvested and immediately analyzed by confocal imaging. This revealed CD86-GFP in endosomal compartments of antigen- stimulated, but not unstimulated, T cells (Fig. 4A). Moreover, internalized CD86-GFP was + +
  • mice bred to mice that express OVA under the control of the rat insulin promo tor (Rip-mOVA). These mice were useful because they develop OVA-specific Treg cells and we have shown that those deficient in
  • CTLA-4 fail to regulate diabetes (13). After in vivo re-challenge with OVA peptide, CD86-
  • CTLA-4 The CTLA-4 molecule plays a critical role in suppressing autoimmunity and maintaining immune homeostasis; however, its precise mechanism of action has been a subject of debate. Recent data have provided evidence that CTLA-4 can perform a non-redundant effector function for Treg, requiring a cell extrinsic mechanism of action (9, 13). Here we demonstrate a cell-extrinsic model of CTLA-4 function which operates by the removal of co- stimulatory ligands from APCs via trans-endocytosis. Importantly, using both human and mouse T cells we establish that trans-endocytosis of ligand occurs in precisely the same settings where we have demonstrated CTLA-4-dependent regulation (8, 13).
  • CTLA-4 Whilst not excluding other mechanisms of CTLA-4 action, we suggest that CTLA-4 carries out the same molecular functions whether expressed by T cells or by Treg; a concept which has significant implications for our understanding of immune homeostasis. Together these data provide a new framework for studies of CTLA-4 and should facilitate our understanding of its immunoregulatory role in the key settings of cancer, HIV infection and autoimmune disease.
  • Figure 22 shows possible targets including internalisation of ligand (1). Sorting to MVB (2), inhibition of lysosomal degradation (3), delivery of CTLA-4 to the cell surface (4) and recycling of CTLA-4 following ligand capture (5).
  • Figure 23 shows the effect of cytoplasmic domain sequences on CTLA-4 internalisation ("Step 2").
  • CTLA-4 was transfected into CHO (Chinese Hamster Ovary) cells and incubated at 4°C or 37°C for the times shown. Remaining surface CTLA-4 was detected using labeled antibody (labeled with Alexa 647) at 4°C. The amount of internalisation increased at 37°C.
  • the graph shows different CTLA-4 species showing that the cytoplasmic domain sequence controls the rate of internalisation.
  • Figure 24 shows that mutant CTLA-4 without lysines does not degrade as much as wild type CTLA-4 (steps 2 and 3).
  • Wild Type CTLA-4 has the following lysine residues:
  • lysines 165 and 175 are the predominant ones in controlling degradation of CTLA-4, however, all lysines are potentially modification targets
  • cyclohexamide (CHX) treated cells did not stain for CTLA-4 showing that the protein was degraded.
  • KLESS was not degraded to the same extent.
  • ammonium chloride inhibited CTLA-4 degradation.
  • Figure 25 shows that bafilomycin also inhibits lysosomal degradation of wild type CTLA-4 re-cycling (step 3) and can be used to modify CTLA-4 re-cycling.
  • KLESS mutant was shown to be internalised from the plasma membrane equally compared to WT CTLA-4 in CHO cells (data not shown). However when cells were stained with antibodies to detect surface or intracellular pools, then qualified by confocal microscopy, KLESS was observed to have a higher steady state plasma membrane to internalised ratio than WT. Endocytosis rates were similar from the plasma membrane.
  • Figure 26 shows that KLESS re-cycles faster than WT CTLA-4 using antibody labelling.
  • Inhibitory ubiquitination using UBEI-41, a ubiquitin ⁇ inhibitor (Biogenova) caused WT CTLA-4 to re-cycle more quickly (Figure 27) when used at 20 micromolar.
  • MG132 is a specific, potent proteosome inhibitor - it also depletes the availability of free cellular ubiquitin and thereby reduces degradation of ubiquitin conjugated proteins in mammalian and yeast cells.
  • Figure 28 shows that it reduced WT CTLA-4 degradation and increased CTLA-4 expression when used at 5 micromolar.

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Abstract

L'invention concerne un procédé d'identification d'un modulateur du système de réponse immunitaire, comprenant le dosage de l'effet d'un composé sur l'internalisation et le recyclage de CTLA-4 dans une cellule, une augmentation du nombre de cycles de CTLA-4 par comparaison avec un témoin sans le composé indiquant que le composé inhibe la réponse immunitaire et une diminution du nombre de cycles de CTLA-4 par comparaison avec un témoin sans le composé indiquant que le composé augmente la réponse immunitaire. Le nombre de cycles de CTLA-4 peut être déterminé par la surveillance de CTLA-4 ou la surveillance de l'absorption d'un marqueur CTLA-4 dans une cellule.
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WO2014179491A2 (fr) 2013-04-30 2014-11-06 La Jolla Institute For Allergy And Immunology Modulation de la fonction régulatrice des cellules t par l'intermédiaire d'une protéine kinase c-η
CN108504692A (zh) * 2018-03-26 2018-09-07 安徽大学 一种基因敲除cho细胞株的构建方法及其在治疗性重组蛋白表达中的应用
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WO2014179491A2 (fr) 2013-04-30 2014-11-06 La Jolla Institute For Allergy And Immunology Modulation de la fonction régulatrice des cellules t par l'intermédiaire d'une protéine kinase c-η
EP2992086B1 (fr) * 2013-04-30 2020-09-30 La Jolla Institute for Allergy and Immunology Modulation de la fonction régulatrice des cellules t par l'intermédiaire d'une protéine kinase c-eta
EP3746122A4 (fr) * 2018-02-02 2021-11-24 OncoC4, Inc. Procédés de sélection et de conception d'anticorps anti-ctla-4 plus sûrs et plus efficaces pour la thérapie du cancer
CN108504692A (zh) * 2018-03-26 2018-09-07 安徽大学 一种基因敲除cho细胞株的构建方法及其在治疗性重组蛋白表达中的应用
CN108504692B (zh) * 2018-03-26 2021-07-23 安徽大学 一种基因敲除cho细胞株的构建方法及其在治疗性重组蛋白表达中的应用

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