WO2014180943A1

WO2014180943A1 - Mcl-1 as critical regulator of foxp3+ regulatory t cell survival, and use thereof to treat severe immune disorders

Info

Publication number: WO2014180943A1
Application number: PCT/EP2014/059423
Authority: WO
Inventors: Adrian LISTON; James Dooley; Daniel Gray
Original assignee: Vib Vzw; Katholieke Universiteit Leuven, K.U.Leuven R&D; The Walter And Eliza Hall Institute Of Medical Research
Priority date: 2013-05-08
Filing date: 2014-05-08
Publication date: 2014-11-13

Abstract

The present application relates to the field of immunology, more particularly to diseases and disorders characterized by insufficient numbers of Foxp3 positive regulatory T cells (T_regs). The mechanisms of the homeostatic processes that regulate the Treg numbers were studied, and it was found that Mcl-expression is critical for Treg survival. As most studies aiming at increasing T_reg numbers to modulate immunity only achieve a transient increase, this finding has important therapeutic consequences: combined Mcl-1 and Foxp3 expression results in a stable population of T_regcells; enhancing T_regnumbers will result in clinical improvement in many severe immune disorders.

Description

Mcl-1 as critical regulator of Foxp3+ regulatory T cell survival, and use thereof to treat severe immune disorders

Field of the invention

The present application relates to the field of immunology, more particularly to diseases and disorders characterized by insufficient numbers of Foxp3 positive regulatory T cells (T_regs) . The mechanisms of the homeostatic processes that regulate the Treg numbers were studied, and it was found that Mcl-1 expression is critical for Treg survival. As most studies aiming at increasing Treg numbers to modulate immunity only achieve a transient increase, this finding has important therapeutic consequences: combined Mcl-1 and Foxp3 expression results in a stable population of Treg cells; enhancing Treg numbers will result in clinical improvement in many severe immune disorders, such as IPEX, Omenn syndrome and GVHD. Thus, vectors are provided for inducing simultaneous expression of Mcl-1 and Foxp3, as well as methods to treat immune disorders by enhancing expression of both Mcl-1 and Foxp3 in a subject in need thereof.

Background

The expression of the transcription factor Foxp3 in T cells results in radical transcriptional rewiring¹ and the consequent functional differentiation into regulatory T cells (T_regs) . The most profound effect is the switch from a pro-immunity potential to a pro-tolerance function that is essential for preventing fatal systemic autoimmunity². In addition to this archetypal characteristic, the transcriptional rewiring also alters the basic cellular properties of Tregs, including differential reliance on cytokines^{3, 4} and TC signalling ⁵ for homeostasis as well as an unusual anergic and apoptotic behavior in vitro. The size of the Treg population is critical for immunological balance; even relatively minor modulation alters immunity⁶. Abnormally low numbers of Tregs have been observed in multiple autoimmune and inflammatory conditions⁷ while, conversely, increased Tregs in the aged is thought to contribute to their partial immunosuppressed state^{8, 9}. In addition, Tregs are postulated to impede effective immunity against cancer¹⁰, with expansion of Tregs after radiotherapy reported to limit lymphoma clearance¹¹.

The pivotal role of human Foxp3⁺ T_Reg cells in maintaining immunological self-tolerance is illustrated by the fatal autoimmune disease IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome), which is caused by mutations in the FOXP3 gene⁴⁶. Patients with IPEX syndrome produce a broad range of autoantibodies and develop insulin-dependent diabetes, thyroiditis, eczema, haemolytic anaemia and inflammatory bowel disease (IBD), and without bone marrow transplantation they die at an early age. Other immune disorders associated with reduced Treg function include Omenn syndrome, an autosomal recessive severe combined immunodeficiency (SCID)⁴⁷, as well as other SCIDs and graft versus host disease (GVHD). GVHD is caused by alloreactive T-cells, and alloreactive T-cell proliferation is normally inhibited by Tregs. Insufficient Treg numbers may account for lethality of GVHD, and expansion of Treg cells has shown promise in GVHD patients without adverse effects^48,49.

In humans, in vitro studies using naive CD4⁺ T cells have shown that CD25 and Foxp3 expression is induced following strong cell activation through the T cell receptor (TCR). However, the upregulation of Foxp3 in response to such experimental activation is transient, only induces low levels of Foxp3 and does not confer suppressive capacities⁵⁰. Stable and high expression of Foxp3 is required in order to maintain the suppressive capacities of human T_Reg cells^{51, 52}.

More studies are required to gain an improved understanding of how an efficient and stable induction of Foxp3⁺ T_Reg cells can be best achieved in autoimmune diseases. Indeed, given the growing interest in manipulating Treg numbers in clinical settings, there is a pressing need to understand the cellular and molecular biology of Treg homeostasis. Apoptotic cell death is a major regulator of hematopoietic cell homeostasis¹². In vertebrates, two distinct, but ultimately converging, pathways control apoptosis. The "extrinsic" or "death receptor" pathway is initiated by ligation of cell-surface death domain-containing members of the TNF-receptor family, while the "intrinsic" or "mitochondrial" pathway is initiated by cellular stressors that alter the balance between pro- and anti-apoptotic members of the Bcl-2 family of proteins, culminating in the activation of Bax and Bak and subsequent release of apoptogenic factors from mitochondria¹³. The two pathways converge upon the activation of "executioner" caspases that demolish the cell. In the intrinsic apoptosis pathway, activation of Bax/Bak represents the "point of no return", and thus requires tight regulation. The prosurvival proteins Bcl-2, Bcl-xL, Mcl-l, Al and Bcl-w restrain the activation of Bax/Bak, while the pro-apoptotic BH3-only proteins (Bim, Puma, Bid, Bmf, Bad, Noxa, Bik, and Hrk) inhibit the pro-survival members and are essential for initiation of apoptosis signaling¹⁴. The relative importance of each varies between cell types and stimuli, and has to be evaluated on a case- by-case basis.

In the case of autoimmune diseases, the path to effective therapeutic targeting is likely to require an understanding of the mechanisms of autoimmunity that underlie the breakdown of the tolerance that is normally maintained by T_Reg cells. The induction of specific T_Reg cells in this context could allow the modulation of the immune response for clinical benefit while limiting long-term general immune suppression. Thus, it would be advantageous to understand Treg homeostasis and be able to specifically manipulate Treg levels.

Summary

Foxp3+ regulatory T cells (Tregs) are a crucial immunosuppressive population of CD4+ T cells. In this study, we seek to determine the homeostatic processes that dictate the size of the Treg pool, a highly proliferative yet stable population. Here we find that Tregs are able to rapidly respond to deficits in their population through an IL-2- and costimulation-dependent process of proliferative expansion. By contrast, excess Tregs are removed by a process of attrition, dependent on the Bim-initiated Bak/Bax- dependent intrinsic apoptotic pathway. The expression of Mcl-1 regulates Treg survival during both expansion and attrition. Loss of this anti-apoptotic Bcl-2 family member resulted in rapid decline in the Treg population and swift onset of fatal autoimmunity, while Bcl-2 and Bcl-xL were redundant.

Thus, increasing Treg numbers in subjects in need thereof can basically be done by a) adding Treg cells (e.g. by differentiating stem cells into Treg cells), b) stabilizing Treg cells by protecting them from apoptosis, or c) a combination of both.

Expression of Mcl-1 is necessary and sufficient for maintaining Treg numbers. Thus, induced (over)expression of Mcl-1 in Treg cells ensures that the Treg population becomes more stable (i.e., is less sensitive to attrition or apoptosis).

As for the combination of both options, expression of Foxp3 in (e.g. hematopoietic) stem cells would ultimately lead to differentiation to Treg cells, simultaneous expression of Mcl-1 in these cells would assure that the cells not only differentiate into Tregs, but also that the levels of Treg cells are maintained (and that the increase in Treg cells is not transient).

Accordingly, it is an object of the invention to provide means to achieve stable Treg differentiation of cells, while at the same time ensuring that these cells maintain their number (or proliferate sufficiently). Stable Treg differentiation may e.g. be obtained by first allowing the stem cells to differentiate into T cells, thereafter inducing expression of Foxp3 and Mcl-1 to obtain Treg cells. How to differentiate stem cells into T cells is documented in the art, see e.g. Bhandoola and Sambandam, Nature Reviews Immunology 6: 117-126 (2006). Alternatively, stem cells can express both genes immediately. As mentioned, another object of the invention is to provide means to achieve stable Treg differentiation of stem cells, which may be obtained by inducing expression of Foxp3 in (e.g. hematopoietic) stem cells. Still a further object of the invention is to provide means to ensure that Treg cells maintain their number. This may be achieved by inducing expression of Mcll in Tregs. According to a first aspect, vectors are provided containing nucleic acid encoding Foxp3 and Mcl-l; or containing nucleic acid encoding Foxp3 and/or Mcl-l operably linked to a Foxp3 promoter.

According to particular embodiments, these vectors are viral vectors. Most particularly, they are lentiviral vectors.

The vectors will typically contain suitable regulatory nucleic acid elements (e.g. promoters and enhancers) to ensure proper expression of the transgenes Foxp3 and/or Mcl-l. Although expression of each gene may be driven independently by a separate promoter, according to specific embodiments, Foxp3 and Mcl-l are operably linked to a common promoter. This has the advantage of restricting Mcl- 1 expression to those cells expressing Foxp3. According to particular embodiments, the promoter is the Foxp3 promoter or a truncated version thereof. This has the advantage of being a native promoter of the Foxp3 gene, and of being a promoter that responds to cues for Treg differentiation.

According to particular embodiments, the enhancer sequences are also enhancer sequences from the Foxp3 gene, particularly intronic sequences.

According to a further aspect, the vectors described herein are provided for use as a medicament. Particularly, they are provided as a medicament for gene therapy.

According to particular embodiments, the vectors are provided for use in treatment of a disease characterized by Treg deficiency. Particularly envisaged examples of diseases characterized by Treg deficiency include, but are not limited to, immunodysregulation polyendocrinopathy enteropathy X- linked syndrome (IPEX), Omenn syndrome and graft versus host disease (GVHD).

In yet another aspect, methods of treating a disease characterized by Treg deficiency in a subject in need thereof are provided, comprising the steps of:

- introducing a vector containing nucleic acid encoding Foxp3, or Foxp3 and Mcl-l, in stem cells;

- introducing the stem cells in the subject.

These methods are suitable for treatment of diseases including, but not limited to, immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), Omenn syndrome and graft versus host disease (GVHD).

According to particular embodiments, the stem cells wherein the vector is introduced are stem cells isolated from the subject (i.e., are autologous stem cells). According to further particular embodiments, the stem cells are isolated from the blood of the subject. According to alternative embodiments, the stem cells are isolated from the bone marrow of the subject. According to other particular embodiments, when the stem cells are isolated from the subject, these methods include a step of isolating the stem cells from the subject prior to the introduction of the vector. According to these embodiments, the step of introduction of the stem cells thus rather is a reintroduction of said cells.

According to particular embodiments, the introduction of the vector containing nucleic acid encoding Foxp3, or Foxp3 and Mcl-1, in stem cells allows expression of Foxp3 (and Mcl-1) in said cells. According to particular embodiments, the stem cells are first developed into T cells.

By expression of Foxp3, the T cells will develop into Treg cells. According to specific embodiments, it is envisaged that the stem cells are differentiated into Treg cells (i.e. express Foxp3) before introduction of the cells in the subject. However, embodiments where (part of the) differentiation occurs upon or after introduction of the cells in the subject are also particularly envisaged.

According to particular embodiments, the goal is not only to introduce extra Tregs in the subjects, but also to achieve and maintain a stable number of Tregs in said subject. In such embodiments, it is envisaged that Foxp3 and Mcl-1 are expressed (and keep being expressed) in Tregs that develop from introduced stem cells in the subject. It is particularly envisaged that at least part of the reintroduced stem cells differentiates into Treg cells, thereby rescuing the Treg deficiency.

- introducing a vector containing nucleic acid encoding Mcl-1 operably linked to a Foxp3 promoter in T cells, and;

- introducing the T cells in the subject.

These methods are suitable for treatment of diseases including, but not limited to, immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), Omenn syndrome and graft versus host disease (GVHD). However, since IPEX is linked to dysfunction of Foxp3, it is envisaged that these diseases are better suited for treatment of Omenn syndrome, GVHD or other diseases characterized by Treg deficiency but not by Foxp3 dysfunction. According to particular embodiments, the T cells wherein the vector is introduced are T cells isolated from the subject (i.e., are autologous T cells). According to further particular embodiments, the T cells are isolated from the blood of the subject. According to other particular embodiments, when the T cells are isolated from the subject, these methods include a step of isolating the T cells from the subject prior to the introduction of the vector. According to these embodiments, the step of introduction of the T cells thus rather is a reintroduction of said cells. Optionally, the methods may comprise a further step in which the regulatory T cells are first isolated, and introduction of the vector is only in the regulatory T cells.

According to particular embodiments, the introduction of the vector containing nucleic acid encoding Mcl-1 operably linked to a Foxp3 promoter in T cells allows expression of Mcl-1 in said cells. According to particular embodiments, Mcl-1 is only expressed in Treg cells (i.e., the T cell subset in which Foxp3 is activated). Thus, expression of Mcl-1 only occurs in (at least) part of the T cells, most particularly the Treg cells.

By expressing Mcl-1, the introduced Treg cells are protected from apoptosis. Thus, this introduced population of Treg cells will (at least partly) rescue the Treg deficiency.

Brief description of the Figures

Figure 1: Homeostatic expansion of Tregs is driven by increased IL-2 production.

Foxp3Thyl.l/DTR females were depleted of Foxp3DT + Tregs on day 0 and blood leukocytes were assessed on indicated days (n≥4/group). a, Percentages of Foxp3+DTR+ (grey circles), Foxp3+Thyl.l+ (white circles) and total Foxp3+ (black circles) Tregs. b, Proliferation rate (%Ki67+) and c, apoptosis rate (%activated Caspase 3+) of Foxp3+Thyl.l+ Tregs, following DT treatment, d, Foxp3DTR+ Tregs were eliminated from Foxp3Thyl.l/DTR mice during treatment with CTLA4-lg. Peripheral blood was assessed for the percentages of Foxp3+Thyl.l+ Tregs in saline treated (filled circles) or CTLA4-lg-treated (empty circles) mice (n≥3/group). e, Foxp3Thyl.l/DTR females were depleted of Foxp3DTR+ Tregs, and assessed for plasma IL-2 levels (empty squares, left-hand axis) and surface CD25 expression on Foxp3+Thyl.l+ Tregs (filled squares, right-hand axis) from days 0-15 (n≥4/group). f, Foxp3Thyl.l/DTR mice were depleted of Foxp3DTR+ Tregs and injected daily with an IL-2 blocking antibody or an Ig isotype-matched control antibody. Splenocytes were analyzed on day 6 for the percentages of Foxp3+DTR+ (filled) and Foxp3+Thyl.l+ regulatory T cells (unfilled), with or without Treg depletion (DT treatment) and with or without anti-IL-2 treatment (n=4, 4,7,7). g, Proliferation rate (%Ki67+) and h, apoptosis rate (activated Caspase-3+) of Foxp3+Thyl.l+ regulatory T cells, with or without DT treatment and with or without anti-IL-2 treatment (n=4). * p<0.05.

Figure 2: The intrinsic apoptosis pathway is required to restrain Treg numbers to homeostatic levels, a, Representative flow profiles (TCRfi vs Foxp3 gated on CD4+ cells, Ki67 histograms gated on Foxp3+ CD4+ cells) for wildtype, Foxp3Cre Bak-/- Baxfl/fl mice and control littermates at 6-8 weeks of age. b, Average percentages and absolute numbers of splenic Foxp3+ Tregs in wt, Foxp3CreBak-/-Baxfl/fl mice and control littermates at 6-8 weeks of age. Data from one experiment representative of three are shown, with n≥3/group.

Figure 3. Regulatory T cell survival is independent of Bcl-2 and BclxL.

a, C57BL/6.Lv5.1 (Ly5.1) chimeras reconstituted with a 50:50 mixture of hematopoietic precursors from wt Ly5.1 and either Bcl-2+/+ or Bcl-2-/- mice then analyzed 8-12 weeks later for average percentages (mean ± sd) of Ly5.2+CD4+Foxp3+ cells recovered from the thymus or spleen (n=4, 6). b, Average percentages (mean ± sd) of Ly5.2+CD4+Foxp3+ cells recovered from the thymus and spleen of the same mixed hematopoietic chimeras reconstituted with precursors from either wt (Bcl-2+/+) or Bcl-2-/- mice (n=4, 6). c, Representative flow profiles of CD4 vs. FoxP3 gated on CD4+ cells from Foxp3CreBclxwt/wt and Foxp3CreBclxfl/fl mice, d, Average percentages (mean ± sd) of CD4+Foxp3+ Tregs in the thymus and spleen of Foxp3CreBclxwt/wt and Foxp3CreBclxfl/fl siblings at 6-8 weeks of age (n=6,9). * p<0.05

Figure 4: Spontaneous fatal immunopathology following regulatory T cell-specific deletion of Mcl-1. a, Mcl-1 reporter expression was measured in lymphocyte subsets in CD127Cre Mcllwt/fl-huCD4 mice. Average huCD4 reporter MFI in double negative (DN), double positive (DP) and single positive (SP) thymocytes, the latter subdivided into CD8 SP, conventional CD4 SP and CD4+ Foxp3+ SP (n=3/group). b, Average huCD4 reporter MFI in CD19+ B cells, naive conventional CD4+ (CD4+ nTc), activated conventional CD4+ (CD4+ actTc), Foxp3+ Treg, naive conventional CD8+ (CD8+ nTc) and activated conventional CD8+ T cells (CD8+ actTc) (n=3/group). c, Representative histogram of huCD4 reporter MFI in naive conventional CD4+ (CD4+ nTc), Foxp3+ Treg and naive conventional CD8+ (CD8+ nTc), with control huCD4 staining in wildtype Foxp3+ Treg. d, Weights of Foxp3CreMcl-lwt/wt, Foxp3CreMcl- lwt/fl and Foxp3CreMcl-lfl/fl littermates at 6-8 weeks of age (n≥7/group ). e, Survival curve for Foxp3CreMcl-lwt/wt, Foxp3CreMcl-lwt/fl and Foxp3CreMcl-lfl/fl littermates (n>15/group). f, Plasma IgE levels in Foxp3CreMcl-lwt/wt and Foxp3CreMcl-lfl/fl littermates. g, Average disease score and h, representative histology (scale bar of 200 μιτι) of the lungs and small intestine of Foxp3CreMcl-lwt/wt, Foxp3CreMcl-lwt/fl and Foxp3CreMcl-lfl/fl littermates. i, Average percentage of CD44+CD62Llow activated cells within CD4+ and CD8+ splenic T cells in Foxp3CreMcl-lwt/wt, Foxp3CreMcl-lwt/fl and Foxp3CreMcl-lfl/fl littermates at 6-8 weeks of age (n=8,10,6,6,4,3).

Figure 5: Mcl-1 is required for regulatory T cell survival.

a, Average percentages of Foxp3+ Tregs within splenic CD4+ T cells in Foxp3CreMcl-lwt/wt, Foxp3CreMcl-lwt/fl and Foxp3CreMcl-lfl/fl littermates at 6-8 weeks of age (n≥7/group). b, Average percentages of ΚΪ67+ cells within splenic Foxp3+ Tregs in Foxp3CreMcl-lwt/wt, Foxp3CreMcl-lwt/fl and Foxp3CreMcl-lfl/fl littermates at 2.5 weeks of age (n≥3/group). c, Expression of the huCD4 reporter for Mcl-1 recombination on Foxp3+ and Foxp3- cells in Foxp3CreMcl-lfl/fl mice, d, Schematic of experimental protocol for analysis of inducible deletion of Mcl-1 or Bclx alleles in mixed hematopoietic chimeras. Inset dot plot shows expression of the huCD4 Mcl-1 reporter on leukocytes 2 days following tamoxifen treatment, e, Ly5.1 versus Foxp3 expression on lymph node cells from mixed chimaeras with Mcllf\/f\ or Sc/xfl/fl Ly5.2 donor compartments analyzed 3 days following treatment with tamoxifen or vehicle control. Plots are representative of 3 experiments, each with n=3-4 mice per group, f, Ratios of Ly5.1+ to Ly5.1- CD4+Foxp3+ lymph node cells in mixed chimaeras with the indicated Ly5.2+ donor compartment 3 days following treatment with tamoxifen or vehicle.

Figure 6: Regulation of Mcl-1 in regulatory T cells by Bim and IL-2.

a, Representative flow cytometric profiles (CD4 vs Foxp3 gated on CD4+ cells, Ki67 histograms gated on Foxp3+CD4+ cells) for Foxp3wtBimfl/fl and Foxp3CreBimfl/fl littermates at 6-8 weeks of age. b, Average percentages and absolutes number of CD4+Foxp3+ Tregs from pooled lymph nodes of Foxp3wtBimfl/fl and Foxp3CreBimfl/fl littermates (n=8, 5). c, Western blot analysis of Mcl-1 expression in ex vivo isolated Tconv or Tregs, or Treg cultured overnight with or without IL-2, in the absence or presence of QVD-OPH (a broad spectrum caspase inhibitor used to prevent apoptosis in the absence of IL-2). d, CD127Cre Mcllwt/fl-huCD4 Foxp3wt/DTR females were depleted of Foxp3DTR+ Tregs on day 0 and huCD4 Mcl-1 reporter expression was measured in Foxp3+ Tregs during the expansion phase. MFI was normalized to Tregs from CD127Cre Mcllwt/fl-huCD4 Foxp3wt mice (n=4/group). * p<0.05

Figure 7. Enhanced proliferative kinetics of Foxp3+ regulatory T cells.

6-8 week-old C57BL/6 mice were treated with either a single injected pulse of BrdU or continuous exposure to BrdU in the drinking water. Mice were analyzed 2 hours, 16 hours and 1-10 days after treatment, a, BrdU incorporation in CD4 Foxp3+, CD4 Foxp3- and CD8 T cells following a single injected BrdU pulse, b, BrdU incorporation in CD4 Foxp3+, CD4 Foxp3- and CD8 T cells following continuous BrdU exposure. n≥4/group, mean ± sd.

Figure 8. Niche-filling behavior of regulatory T cells in secondary lymphoid organs. Foxp3Th_yi.i/DTR heterozygous females were treated with DT on days 0 and 1 to eliminate FOXP3DT regulatory T cells. Spleens were ana lyzed on days 0, 5, 9, 14, 22 (n≥4/group). a, Percentage of Foxp3+DTR+ (gray circles), Foxp3+Thyl.l+ (white circles) a nd total Foxp3+ (black circles) regulatory T cells (mea n ± sd). b, Absolute numbers of Foxp3+DTR+ (grey circles), Foxp3+Thyl.l+ (white circles) and total Foxp3+ (black circles) regulatory T cells (mea n ± sd). c, CD25 expression, d, proliferation rate (%Ki67+) a nd e, apoptosis rate (activated Caspase 3+) of Foxp3+Thyl.l+ regulatory T cells, following DT treatment (mean ± sd). f, From the same mice, the percentage of Foxp3+DTR+, Foxp3+Thyl.l+ and total Foxp3+ regulatory T cells was measured in pooled lymph nodes (mea n ± sd). g, The percentage of effector-memory CD44+CD62Liow cells within the CD4+Foxp3- population in the blood following DT injection. * p<0.05.

Figure 9. Treg niche-filling and retraction is independent of thymic function and deconversion, a,

Proportion of Foxp3+ Tregs within CD4 single positive thymocytes (SP), splenic recent thymic emigrants (RTE, CD4+GFP+) and splenic mature T cells (Mature, CD4+GFP-) in the spleen of Foxp3nyi.i Rag2-GFP-Tg mice (n≥9/group). b, Foxp3+Thyl.l+ splenocytes from Foxp3nyi.i/DTR Rag2- GFP-Tg treated with DT on days -1 and 0 to eliminate FOXP3DT regulatory T cells were assessed for GFP expression on days 0, 5, 14 (n≥3/group). c, Percentage of Foxp3+ cells in 1 month-old mice and 12-month-old mice thymectomized or sham-operated at 1 month of age (n≥3/group). d, Splenocytes from Foxp3cre/DTR Rosa26-stopfi-YFP mice treated with DT on days -1 and 0 to eliminate FOXP3DTR regulatory T cells were assessed for Foxp3-YFP+ ex-Tregs on days 0, 5, 9, 14 (n≥4/group).

Figure 10. Homeostatic expansion of Tregs is independent of dendritic cells. Foxp3wt/DTR heterozygous females and Foxp3wt/DTR CDllcCre iDTR heterozygous females were treated with DT daily to eliminate FOXP3DTR+ Tregs with or without also eliminating CDllc+ dendritic cells. Addition of DT to Foxp3wt/DTR CDllcCre iDTR heterozygous females efficiently eliminated dendritic cells as measured by CDllc+MHCII+ cells, a, representative flow cytometry plots and b, percentage (mean ± sd) of peripheral blood dendritic cells in Foxp3wt/DTR CDllcCre iDTR, with or without DT infection, c, Percentage of Foxp3+ Tregs in the peripheral blood of Foxp3wt/DTR heterozygous females and Foxp3wt/DTR CDllcCre iDTR heterozygous females on days 0, 1, 4 and 5 of DT injection (mean ± sd). Figure 11. Treg-specific deletion in Bak/Bax results in elevated Treg numbers in the thymus and lymph nodes. Wildtype, Foxp3cre Bakwt/wt Baxwt/wt, Foxp3cre Bakwt/wt Baxfi/fi , Foxp3cre Bak-/- Baxwt/wt and Foxp3ae Bak-/- Baxfi/fi siblings were assessed for Treg number at 6-8 weeks of age. a, Representative flow profiles from the thymus (TCF^ vs Foxp3 gated on CD4+ SP cells, Ki67 histograms gated on Foxp3+ CD4+ SP cells), b, Average percentage (mean ± sd) and absolute number (mean ± sd) of thymic Foxp3+ Tregs. c, Representative flow profiles from the lymph nodes (TCF^ vs Foxp3 gated on CD4+ cells, Ki67 histograms gated on Foxp3+ CD4+ cells), d, Average percentage (mean ± sd) and absolute number (mean ± sd) of Foxp3+ Tregs in the lymph nodes. n≥3/group. * p<0.05.

Figure 12. CD8 proliferation and helper T cell polarization in Mcll-deficient mice, a, Average percentage (mean ± sd) of splenic CD8 cells in Foxp3cre Mcllwt/wt, Foxp3cre Mcllwt/fi and Foxp3cre Mcllfi/fi siblings at 6-8 weeks of age (n=8,10,6). b, Fold-increase in Thl (CD4+IFNy+), Th2 (CD4+IL-4+) and Thl7 (CD4+IL-17+) splenocytes in Foxp3cre Mcllfi/fi compared to heterozygous siblings (mean ± sd, n≥3/group).

Figure 13. Regulatory T cell differentiation and homeostasis in young Mcll-deficient mice, a,

Representative flow profiles and b, average percentage (mean ± sd) of thymic Foxp3+ Tregs within the CD4 SP compartment in Foxp3cre Mcllwt/wt, Foxp3cre Mcllwt/fi and Foxp3cre Mcllfi/fi siblings at 2.5 weeks of age (n=3,4,3). c, Representative flow profiles and d, average percentage (mean ± sd) of Foxp3+ Tregs within splenic CD4 T cells in Foxp3cre Mcllwt/wt, Foxp3cre Mcllwt/fi and Foxp3cre Mcllfi/fi siblings at 2.5 weeks of age (n=3,4,3). * p<0.05.

Figure 14. Poor survival of Mcll-deficient regulatory T cells in a competitive environment. Mixed bone-marrow chimeras were generated by reconstituting irradiated Rag-deficient mice with 50% FOXP3DTR Ly5.1 bone-marrow and either 50% Ly5.2 Foxp3cre Mcllwt/wt (n=4) or Foxp3cre Mcllfi/fi Ly5.2 bone-marrow (n=7), then analyzed at 8 weeks after reconstitution. a, Schematic of experimental setup, b, Reconstitution of Ly5.2 bone-marrow among total lymphocytes (mean ± sd). c, Total Treg numbers within bone-marrow chimera recipients (mean ± sd). d, Proportion of Tregs that are Ly5.2 (mean ± sd). * p<0.05.

Figure 15. Molecular control over Treg homeostasis. The main findings of this paper are the molecular mechanisms by which Treg number is homeostatically controlled. Induced partial deficiency of Tregs results in a loss of suppression over conventional T cells, which become activated in a costimulation-dependent manner and increase production of IL-2. IL-2 then increases expression of Mcl-l, allowing reduced apoptosis and increased proliferation within remaining Tregs, to fill the niche. Once the niche is filled, Treg control over conventional T cells is re-established and IL-2 production returns to baseline. In the converse situation, when excess Tregs are present, Bim sequestration of Mcl-l allow atrophy of surplus Tregs until the homeostatic level is achieved.

Detailed description

Definitions

The present invention will be 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 non-limiting. 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.

The following terms or definitions are provided solely to aid in the understanding of the invention. 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, 2^nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), 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.

The term 'vector' as used in the application refers to nucleic acid molecules, usually double-stranded DNA, which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a cDNA molecule. The vector is used to transport the insert nucleic acid molecule into a suitable host cell. A vector may contain the necessary elements that permit transcribing the insert nucleic acid molecule, and, optionally, translating the transcript into a polypeptide. The insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated. The term 'vector' may thus also be defined as a gene delivery vehicle that facilitates gene transfer into a target cell. This definition includes both non-viral and viral vectors. Non-viral vectors include but are not limited to cationic lipids, liposomes, nanoparticles, PEG, PEI, etc. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno- associated viral, adenoviral, herpesviral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector.

Typically, the vector will serve as a 'nucleic acid expression cassette', i.e. as nucleic acid molecules that include one or more transcriptional control elements (such as, but not limited to promoters, enhancers and/or regulatory elements, polyadenylation sequences, and introns) that direct (trans)gene expression in one or more desired cell types, tissues or organs.

The term 'operably linked' as used herein refers to the arrangement of various nucleic acid molecule elements relative to each such that the elements are functionally connected and are able to interact with each other. Such elements may include, without limitation, a promoter, an enhancer and/or a regulatory element, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a gene of interest to be expressed (i.e., the transgene). The nucleic acid sequence elements, when properly oriented or operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression of the transgene. By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements may be expressed in terms of the 5' terminus and the 3' terminus of each element, and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements. As understood by the skilled person, operably linked implies functional activity, and is not necessarily related to a natural positional link. Indeed, when used in vectors, regulatory elements or a promoter will typically be located immediately upstream of the transgene (although this is generally the case, it should definitely not be interpreted as a limitation or exclusion of positions within the vector), but this needs not be the case in vivo. In vectors, transgenes can be operably linked to (or used in conjunction with) their natural promoter, as well as with another promoter.

As used in the application, the term 'promoter' refers to nucleic acid sequences that regulate, either directly or indirectly, the transcription of corresponding nucleic acid coding sequences to which they are operably linked (e.g. a transgene or endogenous gene). A promoter may function alone to regulate transcription or may act in concert with one or more other regulatory sequences (e.g. enhancers or silencers). In the context of the present application, a promoter is typically operably linked to regulatory elements to regulate transcription and/or expression of a transgene.

The term 'transgene' as used herein refers to particular nucleic acid sequences encoding a polypeptide or a portion of a polypeptide to be expressed in a cell into which the nucleic acid sequence is inserted. It is particularly envisaged herein that a vector may contain more than one transgene, which may be operably linked to the same or a different promoter. How the nucleic acid sequence is introduced into a cell is not essential to the invention, it may for instance be through integration in the genome or as an episomal plasmid. Of note, expression of the (at least one) transgene may be restricted to a subset of the cells into which the nucleic acid sequence is inserted. The term 'transgene' is meant to include (1) a nucleic acid sequence that is not naturally found in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence naturally found in the cell into which it has been introduced ; (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or a similar nucleic acid sequence naturally occurring in the cell into which it has been introduced ; or (4) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been introduced. By 'mutant form' is meant a nucleic acid sequence that contains one or more nucleotides that are different from the wild- type or naturally occurring sequence, i.e., the mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and/or insertions. According to a particular embodiment, the nucleic acid expression cassette is used for gene therapy. According to this embodiment, the promoter may be homologous (i.e. from the same species as the animal (in particular mammal) to be transfected with the nucleic acid expression cassette) or heterologous (i.e. from a source other than the species of the mammal to be transfected with the expression cassette). As such, the source of the promoter may be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, or may even be a synthetic promoter (i.e. having a non-naturally occurring sequence), provided that the promoter is functional, i.e. able of driving expression of the (at least one) transgene. It goes without saying that, if more than one transgene is used, more than one promoter may be used. In fact, the number of promoters will typically vary from one (driving expression of all transgenes) to the number of transgenes (wherein expression of each transgene is driven by a separate promoter operably linked to it).

The term "Foxp3" as used herein refers to the Forkhead box P3 gene (official symbol FOXP3, Gene ID: 50943 in humans), as well as the m NA and protein encoded by this gene. The protein encoded by this gene is a member of the forkhead/winged-helix family of transcriptional regulators. Defects in this gene are the cause of immunodeficiency polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), also known as X-linked autoimmunity-immunodeficiency syndrome. Alternatively spliced transcript variants encoding different isoforms have been identified, and, unless specified otherwise, are envisaged within the term Foxp3.

The term "Mcl-1" as used herein refers to the myeloid cell leukemia sequence 1 gene (official symbol MCL1, Gene ID: 4170 in humans) and its encoded mRNA and protein. This gene encodes an anti- apoptotic protein, which is a member of the Bcl-2 family. Alternative splicing results in multiple transcript variants, which are envisaged under the term Mcl-1. According to particular embodiments however, the term Mcl-1 particularly refers to the longest gene product (isoform 1). With "functional expression" of a (trans)gene, it is meant the transcription and/or translation of functional gene product. In the context of functional expression of Foxp3 or Mcl-l, it means these genes are expressed so that the encoded protein exerts its normal function: transcriptional regulation in case of Foxp3, and anti-apoptotic function in case of Mcl-l. The phrase "regulatory T cells" or "Treg(s)" as used herein refers to a subpopulation of T cells which modulate the immune system, maintain tolerance to self-antigens, and abrogate autoimmune disease. They were formerly sometimes referred to as suppressor T cells, as this pool of T cells forms a component of the immune system that suppresses immune responses of other cells, a regulatory mechanism to prevent excessive immune reactions. Particularly envisaged within the definition of Tregs are Foxp3-positive Tregs, i.e. regulatory T cells that express Foxp3.

A "disease or disorder characterized by Treg deficiency" refers to any disease or disorder wherein lack of appropriate Treg response (either by reduced Treg number and/or by reduced Treg functionality) leads to excessive (auto-) immune response. Note that disorder is used herein in the medical sense of diseases not caused by infectious organisms. The prototypical example of a disease characterized by Treg deficiency is IPEX, but also different forms of severe combined immunodeficiency (SCID) are characterized by the absence of functional T-lymphocytes (including Tregs), such as for example Omenn syndrome. Standard treatment for diseases or disorders characterized by Treg deficiency, especially when the disorder is a genetic disorder, typically is bone marrow transplantation. A typical example of a non-genetic disease characterized by Treg deficiency is graft-versus-host disease (GVHD), a common complication following an allogeneic tissue transplant, wherein immune cells of the graft recognize the recipient (the 'host') as foreign. This excessive immune response can be fatal, but it has been shown that donor Tregs can suppress GVHD.

A "subject" as used herein refers to vertebrate organisms which possess Tregs. It is particularly envisaged that the subject is a mammal; more particularly the subject is a human. According to a first aspect, vectors are provided containing nucleic acid encoding Foxp3 and Mcl-l; or containing nucleic acid encoding Foxp3 and/or Mcl-l operably linked to a Foxp3 promoter. The vectors can be episomal vectors (i.e., that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome. Examples of episomal vectors include (extrachromosomal) plasmids and so-called mini-circles, which are composed of the expression cassette only and are devoid of bacterial sequences, and examples of vectors that integrate into the host cell genome including viral vectors. Representative plasmid vectors include pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles). Some of the plasmid vectors can be adapted to incorporate elements that enhance episomal plasmid persistence in the transfected cells. Such sequences include S/MARs that correspond to scaffold/matrix attached region modules linked to a transcription unit (Jenke et al., 2004; Manzini et al., 2006).

Representative viral vectors include vectors derived from adeno-associated virus, adenovirus, retroviruses and lentiviruses. Alternatively, gene delivery systems can be used to combine viral and non-viral components, such as nanoparticles or virosomes (Yamada et al., 2003).

Retroviruses and lentiviruses are RNA viruses that have the ability to insert their genes into host cell chromosomes after infection. Retroviral and lentiviral vectors have been developed that lack the genes encoding viral proteins, but retain the ability to infect cells and insert their genes into the chromosomes of the target cell (Miller, 1990; Naldini et al., 1996). The difference between a lentiviral and a classical Moloney-murine leukemia-virus (MLV) based retroviral vector is that lentiviral vectors can transduce both dividing and non-dividing cells whereas MLV-based retroviral vectors can only transduce dividing cells.

Adenoviral vectors are designed to be administered directly to a living subject. Unlike retroviral vectors, most of the adenoviral vector genomes do not integrate into the chromosome of the host cell. Instead, genes introduced into cells using adenoviral vectors are maintained in the nucleus as an extrachromosomal element (episome) that persists for an extended period of time. Adenoviral vectors will transduce dividing and nondividing cells in many different tissues in vivo including airway epithelial cells, endothelial cells, hepatocytes and various tumors (Trapnell, 1993).

Adeno-associated virus (AAV) is a small ssDNA virus which infects humans and some other primate species, not known to cause disease and consequently causing only a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, although the cloning capacity of the vector is relatively limited.

Another viral vector is derived from the herpes simplex virus, a large, double-stranded DNA virus. Recombinant forms of the vaccinia virus, another dsDNA virus, can accommodate large inserts and are generated by homologous recombination. According to particular embodiments, the vectors are viral vectors. Most particularly, they are lentiviral vectors. The vectors will typically contain suitable regulatory nucleic acid elements (e.g. promoters and enhancers) to ensure proper expression of the transgenes Foxp3 and Mcl-l. Although expression of each gene may be driven independently by a separate promoter, according to specific embodiments, Foxp3 and Mcl-l are operably linked to a common promoter. This has the advantage that the genes are expressed simultaneously. According to particular embodiments, the promoter is the Foxp3 promoter. This has the advantage of being a native promoter of the Foxp3 gene, and of being a promoter that responds to cues for Treg differentiation. According to particular embodiments, the enhancer sequences are also enhancer sequences from the Foxp3 gene, particularly intronic sequences (see Example 5).

It is particularly envisaged that kits are provided containing the vectors as described herein, optionally together with a pharmaceutically acceptable excipient, a manual, or other common kit contents. According to a further aspect, the vectors described herein are provided for use as a medicament. Particularly, they are provided as a medicament for gene therapy. Gene therapy protocols, intended to achieve therapeutic gene product expression in target cells, in vitro, but also particularly in vivo, have been extensively described in the art. These include, but are not limited to, intramuscular injection of plasmid DNA (naked or in liposomes), interstitial injection, instillation in airways, application to endothelium, intra-hepatic parenchyme, and intravenous or intra-arterial administration (e.g. intrahepatic artery, intra-hepatic vein). Various devices have been developed for enhancing the availability of DNA to the target cell. A simple approach is to contact the target cell physically with catheters or implantable materials containing DNA. Another approach is to utilize needle-free, jet injection devices which project a column of liquid directly into the target tissue under high pressure. These delivery paradigms can also be used to deliver viral vectors. Another approach to targeted gene delivery is the use of molecular conjugates, which consist of protein or synthetic ligands to which a nucleic acid-or DNA-binding agent has been attached for the specific targeting of nucleic acids to cells (Cristiano et al., 1993). According to particular embodiments, the vectors are provided for use in treatment of a disease characterized by Treg deficiency. Particularly envisaged examples of diseases characterized by Treg deficiency include, but are not limited to, immunodysregulation polyendocrinopathy enteropathy X- linked syndrome (IPEX), Omenn syndrome and graft versus host disease (GVHD). In yet another aspect, methods of treating a disease characterized by Treg deficiency in a subject in need thereof are provided, comprising the steps of:

introducing a vector containing nucleic acid encoding encoding Foxp3, or Foxp3 and Mcl-1, in stem cells;

- introducing the stem cells in the subject.

According to particular embodiments, the stem cells wherein the vector is introduced are stem cells isolated from the subject. According to further particular embodiments, the stem cells are isolated from the blood of the subject. According to alternative embodiments, the stem cells are isolated from the bone marrow of the subject. According to other particular embodiments, when the stem cells are isolated from the subject, these methods include a step of isolating the stem cells from the subject prior to the introduction of the vector. According to these embodiments, the step of introduction of the stem cells thus rather is a reintroduction of said cells.

After isolation of the stem cells, they may be differentiated into T cells first, prior to expression of the Foxp3 and Mcl-1. They may be differentiated prior to introduction of the vector, or after the introduction of the vector, but before expression. The latter may be achieved e.g. by using an inducible promoter. Expression of Foxp3 in T cells will differentiate them into Treg cells. According to specific embodiments, the stem cells are differentiated into T cells (and most particularly, into Treg cells) prior to (re)introduction of the cells in the subject. According to alternative embodiments, however, the transduced stem cells will be introduced as such, and differentiate into T cells in the subject.

Thus, according to these specific embodiments, methods are provided for treating a disease characterized by Treg deficiency in a subject in need thereof, comprising the steps of:

Optionally obtaining stem cells from a subject (e.g. from a sample of blood from the subject); - Introducing a vector (e.g. a viral vector, such as a lentiviral vector) containing nucleic acid encoding Foxp3, or Foxp3 and Mcl-1, in the stem cells from the subject;

Reintroducing the transduced stem cells in the subject.

It is particularly foreseen that the vector is an expression vector, and that the transduced stem cells will express Foxp3 and Mcl-1.

According to particular embodiments, the introduction of the vector containing nucleic acid encoding Foxp3 and Mcl-1 in stem cells allows expression of Foxp3 and Mcl-1 in said cells. According to particular embodiments, the stem cells are first developed into T cells. By expression of Foxp3, the T cells will develop into Treg cells. According to specific embodiments, it is envisaged that the stem cells are differentiated into Treg cells (i.e. express Foxp3) before introduction of the cells in the subject. However, embodiments where (part of the) differentiation occurs upon or after introduction of the cells in the subject are also particularly envisaged. Indeed, at least part of the stem cells engrafted (or injected) in a subject will naturally go to the thymus and develop into T cells. Expression of Foxp3 then ensures further differentiation in Treg cells. This is particularly envisaged for treatment of pediatric diseases (e.g. IPEX), since the thymus is most active during the neonatal and pre-adolescent periods. As the goal is to achieve and maintain a stable number of Tregs in said subject, it is envisaged that Foxp3 and Mcl-1 are expressed (and keep being expressed) in Tregs that develop from introduced stem cells in the subject. It is particularly envisaged that at least part of the reintroduced stem cells differentiates into Treg cells, thereby rescuing the Treg deficiency.

- introducing the T cells in the subject.

These methods are suitable for treatment of diseases including, but not limited to, immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), Omenn syndrome and graft versus host disease (GVHD). However, since IPEX is linked to dysfunction of Foxp3, it is envisaged that these diseases are better suited for treatment of Omenn syndrome, GVHD or other diseases characterized by Treg deficiency but not by Foxp3 dysfunction.

Contrary to the methods that transduce stem cells, where the primary goal is to differentiate the stem cells into Treg cells (and then optionally providing further resistance to apoptosis by expressing Mcl-1), the primary goal of the methods transducing T cells is to develop Treg cells with higher resistance to apoptosis. Whereas it is particularly envisaged that this is done using expression of Mcl-1, expression of other anti-apoptotic proteins in this pathway such as Bcl-2 or IL-2 is also envisaged (again, expression restricted to regulatory T cells, e.g. by using the Foxp3 promoter). Likewise, the knockdown of pro-apoptotic proteins in the Treg pathway such as Bim, Noxa, Puma can be envisaged (particularly, but not exclusively, using sh NA against these proteins, again under control of the Foxp3 promoter). Combinations of these strategies are also envisaged. By way of non-limiting example, a vector encoding Bcl-2 and shRNA against Puma, both operably linked to the Foxp3 promoter, can be used in these methods. By extension, this strategy can also be used in transduction of stem cells.

According to particular embodiments, the T cells wherein the vector is introduced are T cells isolated from the subject (i.e., are autologous T cells). However, it is also particularly envisaged that the donor of the T cells is another subject than the acceptor. According to further particular embodiments, the T cells are isolated from the blood of the subject. According to other particular embodiments, when the T cells are isolated from the subject, these methods include a step of isolating the T cells from the subject prior to the introduction of the vector. According to these embodiments, the step of introduction of the T cells thus rather is a reintroduction of said cells. Optionally, the methods may comprise a further step in which the regulatory T cells are first isolated, and introduction of the vector is only in the regulatory T cells.

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. Examples

Summary

In this study we take advantage of the location of Foxp3 on the X chromosome and the effect of X inactivation on a diphtheria toxin receptor knock-in construct to study the dynamics of Treg response to homeostatic perturbation in a highly controlled manner. We find that modulation of the Treg population creates a TC costimulation- and IL-2-dependent feedback loop, which increases Treg proliferation and reduces apoptosis to drive rapid restoration of Treg numbers. Furthermore, we find that the Bak/Bax-dependent intrinsic apoptotic pathway naturally limits the Treg population, with Treg accumulation observed in their absence. The survival of Tregs is safeguarded by the pro-survival Bcl-2 family member Mcl-1; deletion of Mcl-1 causes a rapid loss of Tregs and onset of fatal autoimmunity. Mcl-1 expression is regulated by IL-2, increasing during the Treg expansion phase following in vivo depletion. Finally, the BH3-only protein, Bim, is the primary antagonist of Mcl-1 in Tregs, as conditional deletion of Bim led to the equivalent accumulation of excess Tregs as observed with loss of Bak/Bax.

Example 1. Foxp3+ regulatory T cells demonstrate IL-2-dependent niche-filling behavior

To determine the homeostatic characteristics of Tregs we compared the proliferative behavior of Foxp3+ Tregs in BrdU labeling experiments. Despite the prior characterization of Tregs as semi-anergic, quiescent cells in vitro, Tregs showed substantially higher levels of proliferation than conventional T cells in vivo, with ~50% turnover every 10 days in unmanipulated hosts during homeostatic conditions (Fig. 7). As these results suggest a highly dynamic - yet stable - population, we developed a 50% depletion system to examine responses to such perturbations in the Treg niche.

Female mice heterozygous for both Foxp3Thyl.l and Foxp3DTR alleles (Foxp3Thyl.l/DTR mice) have two distinct populations of Tregs due to random X inactivation of Foxp3 alleles. Half express the marker Thyl.l, while the other half express the diphtheria toxin receptor (DT ). Upon injection of diphtheria toxin (DT), the 50% of DTR+ Tregs will be rapidly eliminated and the response of the DTR- Thyl.l+ compartment to this overall 50% drop in Treg numbers can be tracked. DT addition efficiently eliminated DTR+ Tregs, however, the overall proportion of Foxp3+ cells was rapidly restored by the expansion of DTR-Thyl.l+ Tregs (Fig. la). Indeed, the 6-fold increase of Thyl.l+ Treg by day 5 caused an initial overshoot of ~200% in total Treg, followed by a slow decline to basal levels (Fig. la, Fig. 8). During the niche-filling process, Thyl.l+ Tregs increased their proliferation rate to ~70% and decreased their rate of apoptosis (Fig. lb-c). We conclude that this expansion of existing Tregs must be the major driver of niche-filling, as there was no evidence of any substantial contribution by recent thymic emigrants (Fig. 9a-c) and peripheral conversion should have resulted in equal contribution by DTR+ and Thyl.l+ Tregs (the injected DT is cleared within hours).

We observed an increase in effector-memory CD44highCD62lowCD4+ T cells after partial Treg depletion (Fig. 8); therefore, we sought to determine whether Treg nichef illing depended upon such T cell activation. Activation of conventional T cells was indeed crucial for Treg niche-filling, as costimulatory blockade with CTLA4-lg prevented restoration of Treg numbers (Fig. Id), although dendritic cell (DC)-mediated activation was not required (Fig. 10). Synchronous with Treg niche-filling was an increase in plasma IL-2 levels and internalization of the high affinity subunit of the IL-2 receptor, CD25, by Tregs (Fig. le), identifying IL-2 as a potential mediator of feedback from conventional T cell activation to Treg.

Indeed, antibody mediated IL-2 blockade in Foxp3Thyl.l/DTR mice during the 50% Treg depletion impaired niche-filling by partially inhibiting their increased proliferation rate and by completely blocking their reduction in apoptosis (Fig. lf-h). Collectively, these data demonstrate that Tregs actively maintain homeostasis by swiftly responding to partial insufficiency via a feedback loop involving activation of conventional T cells, increased IL-2 production and altered Treg proliferation/apoptosis balance.

Example 2. Foxp3+ regulatory T cell numbers are constrained by the intrinsic apoptotic pathway

The cellular dynamics of Tregs following ablation highlighted the potential importance of Treg apoptosis during two phases: (i) the reduction of apoptosis during the expansion response to a numerical deficit and (ii) mediating the Treg decline following numerical overshoot. By contrast, proliferation rates during Treg surplus did not drop below homeostatic baseline (Fig. lb) and no evidence for "de-conversion" of excess Tregs into conventional T cells was observed when the Thyl.l marker was replaced with a fate-mapping tracker in Foxp3Cre/DTR osa26-flstopfl-YFP mice (Fig. 9d). Despite this (indirect) evidence for modulation of apoptosis being crucial for Treg homeostasis, defects in the "death receptor" pathway have not been associated with increased Treg numbers^{15, 16}. By contrast, the marked expansion of Treg observed in mice lacking the pro-apoptotic multi-BH domain Bcl-2 proteins Bax and Bak or those lacking the BH3-only protein Bim implicates the intrinsic pathway of apoptosis in regulating Treg numbers^{4, 17} , although these experiments could not distinguish increased conversion into the Treg lineage due to defective thymocyte deletion from elevated peripheral homeostasis.

To analyze the impact of the intrinsic pathway of apoptosis specifically on peripheral Treg homeostasis, we generated Foxp3CreBak-/-Baxfl/fl mice. Due to the redundancy of Bak and Bax¹⁸, this results in a Treg-specific knockout of the entire intrinsic apoptotic pathway. These mice exhibited normal thymic Treg development (Figure 11), but peripheral accumulation of Foxp3+ Tregs to ~100% above normal numbers (Fig. 2). This accumulation was not due to excess proliferation (indeed, turnover was reduced), indicating that apoptosis is a critical regulator of peripheral Treg homeostasis.

Example 3. Foxp3+ regulatory T cells depend on Mcl-1 for survival during homeostasis and proliferation Bak and Bax are tightly regulated by pro-survival members of the Bcl-2 family, of which Bcl-2, Bcl-xL and Mcl-1 have been reported to be expressed in Tregs¹. The leading pro-survival candidate for maintaining Treg survival was Bcl-2, due to the dynamic expression of Bcl-2 observed in Tregs^19"21 and the Treg accumulation that occurs in mice with forced Bcl-2 overexpression²¹.

However, lethally irradiated mice reconstituted with a 50:50 mixture of C57BL/6.Ly5.1:6c/2-/- hematopoietic precursors exhibited a normal proportion of Tregs derived from the 6c/2-deficient compartment, demonstrating that Bcl-2 is dispensable for their survival (Fig. 3a-c). Analysis of the second candidate, Bcl-xL, could not be performed using conventional knockouts (and hematopoietic reconstitution), as it is required for cell survival at the double positive (DP) thymocyte stage^{22, 23}. We therefore created mice with Treg-specific deletion of Bcl-xL by crossing a Foxp3Cre strain24 with Bcl-xfl mice²⁵. The resulting Foxp3CreBcl-xfl/fl mice had normal numbers of Tregs in both the thymus and periphery with no obvious immunological or pathological phenotype, demonstrating no role for this anti-apoptotic protein in Treg homeostasis (Fig. 3d-f).

To investigate the role of Mcl-l in regulating Treg apoptosis, we utilized a Cre-induced huCD4 reporter of Mcl-l expression²⁶. When crossed to the CD127Cre knock-in mice²⁷ in a heterozygous form, this reporter allows the relative quantification of Mcl-l expression in all lymphocyte subsets with a greater dynamic range than previous profiling²⁸. During thymic development, Mcl-l reporter expression peaked at the DP stage, before an ~80% reduction in conventional CD4+ single positive (SP) thymocytes (Fig. 4a). In contrast to conventional CD4+ SP cells, Foxp3+CD4+ SP maintained elevated Mcl-l reporter expression (Fig. 4a), and likewise peripheral Tregs expressed the Mcl-l reporter at ~50% higher levels than conventional T cells (Fig. 4b,c).

To assess the function of Mcl-l in Tregs (while circumventing the impact of its loss on the early thymocyte stage²⁹), we created mice with Treg-specific deletion of Mcl-l. In contrast to the redundancy of Bcl-2 and Bcl-xL in Treg survival, we found that Mcl-l was essential. Remarkably, Foxp3CreMcllfl/fl mice succumbed to a fatal immunopathology, surviving to only 4-8 weeks of age (Fig. 4d,e). Pathology was associated with: immunological dysregulation, inflammatory infiltrate, hyper-lgE, elevated anti-dsDNA antibodies, abnormally increased proliferation of CD8+ T cells, increased activation of CD4+ T cells and spontaneous differentiation into Thl, Th2 and Thl7 effector cells (Fig. 4f- i, Fig. 12 and data not shown), all hallmarks of the Foxp3-deficient scurfy phenotype.

In young Foxp3CreMcl-lfl/fl mice, thymic Treg development was relatively undisturbed and initially there was only a ~60% reduction in peripheral Tregs (Fig. 13). However, unlike the Foxp3Thyl.l/DTR model, this Treg deficit could not be corrected by peripheral expansion in Foxp3CreMcl-lfl/fl mice, with a further reduction in their numbers observed (Fig. 5a). Indeed, the loss of Mcl-l-deficient Tregs was even more extreme in a competitive environment (Fig. 14). Nevertheless, the deficit-sensing mechanism appeared intact, as the remaining Tregs in young Foxp3CreMcl-lfl/fl mice demonstrated a compensatory increase in proliferation (Fig. 5b). Importantly, there was no evidence of an outgrowth of Mcl-lintact Tregs, nor was Mcl-l excision observed in Foxp3- cells (Fig. 5c). In order to measure the kinetics of Treg loss following Mcl-l ablation, we utilized mixed hematopoietic chimeras taking advantage of an inducible knockout system (Fig. 5d). Remarkably, punctual deletion of Mcl-l in this system using tamoxifen-induced activation of CreERT2 recombinase caused Treg numbers to collapse within 2 days (Fig. 5e,f), revealing an acute necessity for Mcl-l in Treg survival.

Example 4. Upstream regulation of Mcl-l in Foxp3+ regulatory T cells by Bim and IL-2

The importance of Mcl-l expression for Treg survival suggests that regulation of Mcl-l may be important in setting the homeostatic balance of Tregs. Several of the pro-apoptotic BH3-only members of the Bcl-2 family can overcome the pro-survival function of Mcl-l and thereby initiate apoptosis³⁰; however, elevated Tregs have been observed only in S/Vn-deficient mice^{4, 17} ' ¹⁹' ²¹. In these mice with germline deletion of Bim, this effect has been ascribed to additional T cells entering the Treg lineage due to defective negative selection¹⁷, and secondary effects due to low-grade inflammation³¹.

To circumvent these issues, we used mice bearing a floxed Bim allele and crossed them with Foxp3Cre mice to create a Treg-specific deletion of Bim, where Bim is lost only after Treg development. The Foxp3CreBimfl/fl mice exhibited normal thymic differentiation, with substantial peripheral expansion of Foxp3+ Tregs (Fig. 6a,b). Notably, the scale of peripheral Treg expansion in Foxp3Cre Bimfl/fl mice was not as great as that observed in Foxp3Cre Bak-/- Baxfl/fl mice (Fig. 2), indicating that, although Bim is the primary initiator of homeostatic Treg apoptosis, additional BH3-only proteins (possibly Puma³²) may contribute a minor role. In addition to the negative regulation of Mcl-l by Bim, the IL-2-dependent reduction in apoptosis during niche filling (Fig. lh) suggested that IL-2 might act as a positive regulator of Mcl-l. Using an in vitro culture system, we observed that stimulation of Tregs with IL-2 increased Mcl-l protein levels (Fig. 6c). We therefore crossed the Mcl-l reporter system described above (CD127Cre McllhuCD4/wt reporter) to the Foxp3DTR/wt partial depletion system, in order to determine whether the increased IL-2 availability during Treg expansion (Fig.l) was linked to in vivo changes in Mcl-l expression. We found a rapid increase in Mcl-l reporter expression in Tregs following partial depletion (Fig. 6d), coinciding with the IL-2-dependent decrease in apoptosis, suggesting a mechanism for the ability of IL-2 to control Treg apoptosis during expansion.

Discussion

Far from being the semi-anergic lineage first described in in vitro experiments, dissection of the homeostatic features of Foxp3+ Tregs demonstrates a highly dynamic and responsive cellular compartment. While entry into the Foxp3+ lineage is a gated event³³, peripheral proliferation and apoptosis constitute the primary determinants of compartment size, with a ~10 day half-life of individual cells prior to proliferation or apoptosis under homeostatic conditions. Several explanations present themselves for the necessity of a high turn-over Treg pool: i) chronic proliferation caused by TC stimulation via self-antigen stimulation³⁴ may necessitate compensatory apoptosis; ii) a pro- apoptotic effect of Foxp3 expression leads to increased basal apoptosis levels in Tregs³⁵, which may require compensatory proliferation; or Hi) high turnover itself may be required for sufficient regulatory function³⁶. Assessment of Treg responsiveness to perturbations from this basal state requires both swift contraction and a mechanism to leave the remaining cells unaffected. The natural chimeric state of female Foxp3Thyl.l/DTR mice used here fulfills both of these conditions, unlike models which rely on partial antibody-mediated depletion³⁷ or escape of abnormal DT-resistant Treg clones³⁸. This approach revealed a much more rapid and dynamic Treg response to perturbation than previous models demonstrated^{37, 3S}, characterized by promptly increased proliferation with concomitant reduction of apoptosis. Furthermore, this system revealed an "overfilling" effect followed by the slower attrition of Treg via apoptosis to re-establish homeostatic levels. The contrast between rapid expansion in the face of Treg-deficiency and gradual contraction during Treg-excess is commensurate with the more severe physiological consequences of suboptimal immune suppression. Importantly, within the range of induced variation studied here, modulation of the proliferation/apoptosis balance was sufficient to drive homeostatic correction, with any substantial involvement of thymic production, peripheral conversion or "de-conversion" all excluded through experiments under these conditions.

Dissection of the molecular mediators of Treg homeostatic responsiveness characterized the roles of both well-known participants in Treg biology and previously unappreciated factors. We found a direct correlation between modulation of Treg numbers and IL-2 production by conventional T cells in a costimulation-dependent manner. Interestingly, elevated expression of IL-2 by conventional T cells occurs prior to the attainment of a typical activated cellsurface profile, a phenomenon which may be related to the lower threshold of TC signaling required for cytokine production³⁹, and which may serve to reduce the time-lag of Treg expansion in response to depletion. Consistent with previous reports identifying the importance of IL-2 for Tregs34, blockade of IL-2 blunted the homeostatic rebound following 50% Treg depletion, playing an essential role in the transient decrease in Treg apoptosis and a significant role in the boost to proliferation. The reliance of the Treg homeostatic feedback circuit on an inducible cytokine lies in stark contrast to the homeostatic feedback loops of B cells and non-regulatory T cells, which rely on the cytokines, BAFF ⁴⁰ and IL-7⁴¹ respectively, which are not made by activated T cells but constitutively produced by stromal cells^{42, 43}. Notably, a feature of static consumption-based homeostatic systems, such as those driven by BAFF and IL-7, is that only numerical, rather than functional, sufficiency is selected for. In the Treg homeostatic system described here, by contrast, the dynamic production of IL-2 in response to Foxp3+ regulatory T cell numbers makes the niche dependent on regulatory T cell function (i.e. restraining improper T cell activation) as opposed to merely numerical sufficiency. It is predicted that such a homeostatic model will prove to show increased robustness when challenged by variation in the efficiency of Treg suppression, as demonstrated by the expansion in Treg numbers in several models of impaired function^{44, 4S}.

A key feature of the Treg homeostatic feedback loops described here, and previously unappreciated, is the central role for apoptosis in regulating Treg numbers. Expansion of Tregs during numerical deficit was accompanied by an IL-2-dependent suspension of apoptosis, while contraction during surplus involved apoptotic processes, with Treg-specific deletion of the intrinsic apoptosis pathway leading to the accumulation of surplus Tregs. Our approach of systematic assessing pro-survival members of the intrinsic apoptosis pathway revealed that Bcl-2 and Bcl-xL were redundant, in contrast to prior supposition^{17 19"21}, while Mcl-1 was essential for Treg survival. Furthermore, Mcl-1 appears to represent a rheostat for controlling the Treg homeostatic niche, with positive regulation via IL-2 and antagonism by Bim during homeostatic perturbation (Figure 15). This role of Mcl-1 in driving the return of Tregs to homeostatic levels represents a potential intervention point for therapeutic manipulation.

Example 5. Therapeutic intervention to increase Treg number in mice

The following lentiviral vectors allowing specific expression of any gene/protein specifically in regulatory T cells were generated:

1) a lentiviral vector containing the basic Foxp3 promoter + the first exon of Foxp3 (which is located from the Foxp3 transcriptional start site at location -639 to +174). In addition, Treg specific enhancer regions (here called "conserved non-coding-sequence" or CNS) were added to increase specificity of expression to the Treg lineage. CNS1 (position 2003 to 2707) and CNS2 (4262 to 4787) are in the first intron and CNS3 (6909 to 7103) is in the intron after the translational start site. The three CNS were cloned together as a complex of 1510 bp (hereafter called CNSl+2+3).

2) A lentiviral vector containing the basic Foxp3 promoter (without exon) with the CNSl+2+3 enhancer.

The CNSl+2+3 enhancer sequences were cloned either at the front of the construct, or at the end.

In first instance, eGFP was cloned in the plasmid to check whether the construct is really only active in

Regulatory T cells. a) treatment of IPEX using transduced stem cells

In the best working plasmids, the eGFP will be exchanged with the coding sequence of the Foxp3 protein, and optionally with another reporter that is nontoxic and tested in human/mouse (e.g. tCD34 linked via T2A site). Additionally, a construct will be made in which both the coding sequences of the Foxp3 protein and the Mcl-1 protein are both operably linked to the Foxp3 promoter. These vectors will be used to transduce hematopoietic stem cells. The transduced cells will be introduced in the scurfy mouse (a mouse model of IPEX). In other words, mice with depleted Treg number will be treated as follows: stem cells will be isolated from their bone marrow, these stem cells will be transfected or transduced with a lentiviral vector expressing Foxp3 and Mcl-l (e.g. a construct consisting of the Foxp3 promoter driving expression of both Foxp3 and Mcll). The stem cells will be introduced in an identical mouse, and at least part of the stem cells will develop into Treg cells. This because the transcription factor Foxp3 would turn on Treg development, Mcl-l would ensure survival of the Tregs. Thus, even when the stem cells will only produce a fraction of the T cells, those that expressed Foxp3 and Mcl-l would get a big survival boost and proliferate up, thereby rescuing the Treg deficiency in the mouse. b) treatment of GVHD using transduced T cells

In the best working plasmids, the eGFP will be exchanged with the coding sequence of the Mcl-l protein, and optionally with another reporter that is nontoxic and tested in human/mouse (e.g. tCD34 linked via T2A site).

For treatment of Graft-versus-host disease (GvHD), peripheral regulatory T cells can be isolated from the blood of a donor, transduced with the plasmid and the more stable Tregs (i.e., with a higher resistance to apoptosis) can be introduced into the recipient. As it is difficult to just isolate pure Treg from the donor, the use of the Foxp3 promoter/CNSl+2+3 plasmid allows to express the Mcll protein specifically in Treg.

Materials and Methods

Mice

Bak-/- ^1S, Baxfl/fl ⁵³, Bcl-x(Bcl2ll)fl/fl ^2S, CDllc-Cre-Tg ⁵⁴, CD127-Cre-Tg ²⁷ , Foxp3GFP ^5S, Foxp3YFPCre ²⁴, Foxp3Thyl.l ⁵⁶, Mcllfl/fl ²⁶, Rag2-/- ⁵⁷ , ag2GFP-Tg ^5S, Rosa26-flstopfl-YFP ⁵⁹, Rosa26-flstopfl-DTR ⁶⁰ and Rosa26Cre-ERT2 ⁶¹ mice were all generated on, or backcrossed to, the C57BL/6 background. Foxp3DTR mice ⁶² were backcrossed to the C57BL/6.Ly5.1 background. Experimental mice were housed under specific pathogen-free conditions. Disease development was monitored by frequent observation and post-mortem analysis. Cohorts of mice for the survival test were removed from the study at death or when veterinary advice indicated likely death within 48 hours. All experiments were approved by the University of Leuven animal ethics committee or the WEHI animal ethics committee. Histological examination was performed by Histology Consultation Services and pathology reports were generated by BioGenetics. In vivo treatments

Heterozygous females were injected i.p. with a dose of 5C^g/kg of Diphtheria Toxin (DT) (Sigma- Aldrich) diluted in saline on days 0 and 1 (For CDllc-depletion on days 0,1,2 and 3) of the experiment. A daily dose of 50μg of IL-2 neutralizing antibody (S4B6) or lgG2a isotype-matched control antibody was administered i.p. starting on the day of the first DT injection. CTLA4-lg (abatacept, Bristol-Myers Squibb) was injected i.p. (25mg/kg) on days 0, 2 and 4. Recipient C57BL/6 Rag2-/- mice were sublethal^ irradiated with 9.5Gy and reconstituted within 24 hours using an intravenous injection with a total of 2 x 10^s hematopoietic cells from bone marrow donors. Chimeric mice were analyzed at or after 6 weeks after reconstitution. For inducible deletion of Bim(Bcl2ll) or Mcll, mice were given 2 doses of 200mg/kg of tamoxifen (Sigma, T5648) via oral gavage on days 0 and 1. BrdU exposure was initiated via an i.p. injection of 100μg/100μL BrdU (Sigma). A subset of mice were additionally given continuous exposure to 8mg/mL BrdU in the drinking water, changed daily.

Flow Cytometry

Leukocytes from peripheral blood, thymus, spleen or lymph nodes were analyzed using the following antibodies: anti-BrdU-APC (BD), anti-CD25-PEcy7, anti-CD25-PE (BD), anti-CD4-APC-H7 (BD), anti-CD4- PerCP (BioLegend), anti-huCD4-PE, anti-CD4-PE, anti-CD4-FITC, anti-huCD4-PE-Cy7, anti-huCD4-PE, anti-TCR-beta-PE-Cy7 (BioLegend), anti-CD44-PerCP-Cy5.5, CD62L-PE-Cy7, anti-CD8- PerCP-Cy5.5, anti- CD8-PerCP, anti-CD8-APC-eFluor780, anti-CD8-Qdot-655, anti-Foxp3-APC, anti-Foxp3-FITC, anti-GFP- Alexa488 (Invitrogen), ΚΪ67-ΡΕ (BD), Ki67-FITC (BD) and anti-Thyl.l-PerCP-Cy5.5, IFN-y-PerCP-cy5.5, IL- 17A-APC, IL-4-PEcy7 (all eBioscience unless indicated otherwise). Intracellular staining for Ki67 and Foxp3 was performed following fixation and permeabilization using the reagents from the eBiosciences Foxp3 staining kit. Intracellular staining for BrdU was performed following Foxp3 staining, using the BrdU staining kit (BD). For Intracellular cytokine staining, cells were stimulated for 4 hours in complete RPMI in presence of Phorbol myristate acetate (50ng/mL; Sigma-Aldrich), ionomycin (500ng/mL; Sigma-Aldrich), and monensin (1/1000; BD), reagents from the BD cytofix/cytoperm kit were used. Apoptosis was assessed using the Abeam active Caspase-3 FITC Staining Kit. Biochemical Analyses

Anti-dsDNA titers in individual plasma samples were determined by enzyme-linked immunosorbent assay (ELISA). IgE levels were measured with a mouse IgE Ready-SET-Go! ^® ELISA assay (eBioscience). IL-2 concentrations were determined with a mouse IL-2 High Sensitivity ELISA (eBioscience). For in vitro stimulation of Tregs with IL-2, pooled splenic and lymph node cells from Foxp3Cre mice were labeled with anti-CD4 microbeads and the CD4+ T cells enriched on an AutoMACS separator (Miltenyi Biotec). Enriched CD4+ cells were stained with anti-CD4-PerCP-Cy5.5 and anti-CD25-PE and YFP+ Treg or YFP- conventional T cells were purified by cell sorting on a MoFlo FACS machine (Cytomation). Tregs were plated at 10^s cells per well in Complete medium with 200U/well IL-2 (Peprotech). The pan-caspase inhibitor QVD-OPH was added where indicated to prevent Treg apoptosis in the absence of IL-2. Following 24 hours of culture, Tregs were recovered in lysis buffer and lysates run on SDS-PAGE, then transferred to PVDF membrane for probing with rabbit anti-Mcl-1 (Rockland Immunochemicals), HRPO conjugated anti-rabbit Ig (Southern Biotech) and development with ECL reagents (GE Healthcare). Statistical Analyses

Differences in animal survival rates were analyzed using a log rank test (Prism). All other statistical analyses were performed through an ANOVA with Tukey's post-test, followed by individual t-test comparisons, with p<0.05 used as the threshold for statistical significance.

References

1. Gavin, M.A. et al. Foxp3-dependent programme of regulatory T-cell differentiation. Nature 445,

771-775 (2007).

2. Sakaguchi, S., Miyara, M., Costantino, CM. & Hafler, D.A. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol 10, 490-500 (2010).

3. Fontenot, J.D., Rasmussen, J. P., Gavin, M.A. & Rudensky, A.Y. A function for interleukin 2 in Foxp3- expressing regulatory T cells. Nat Immunol 6, 1142-1151 (2005).

4. Chougnet, C.A. et al. A major role for Bim in regulatory T cell homeostasis. J Immunol 186, 156-163 (2011).

5. Siggs, O.M. et al. Opposing functions of the T cell receptor kinase ZAP-70 in immunity and tolerance differentially titrate in response to nucleotide substitutions. Immunity 27, 912-926 (2007).

6. Tian, L. et al. Foxp3+ regulatory T cells exert asymmetric control over murine helper responses by inducing Th2 cell apoptosis. Blood 118, 1845-1853 (2011).

7. Miyara, M. et al. Human FoxP3+ regulatory T cells in systemic autoimmune diseases. Autoimmun Rev 10, 744-755 (2011).

8. Lages, C.S. et al. Functional regulatory T cells accumulate in aged hosts and promote chronic infectious disease reactivation. J Immunol 181, 1835-1848 (2008).

9. Raynor, J., Lages, C.S., Shehata, H., Hildeman, D.A. & Chougnet, C.A. Homeostasis and function of regulatory T cells in aging. Curr Opin Immunol 24, 482-487 (2012).

10. Shimizu, J., Yamazaki, S., Takahashi, T., Ishida, Y. & Sakaguchi, S. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological selftolerance. Nat Immunol 3, 135-142 (2002).

11. Baba, J. et al. Depletion of radio-resistant regulatory T cells enhances antitumor immunity during recovery from lymphopenia. Blood 120, 2417-2427 (2012).

12. Youle, R.J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9, 47-59 (2008).

13. Strasser, A. The role of BH3-only proteins in the immune system. Nat Rev Immunol 5, 189-200 (2005).

14. Huang, D.C. & Strasser, A. BH3-Only proteins-essential initiators of apoptotic cell death. Cell 103,

839-842 (2000).

15. Zheng, L., Sharma, R., Gaskin, F., Fu, S.M. & Ju, ST. A novel role of IL-2 in organspecific autoimmune inflammation beyond regulatory T cell checkpoint: both IL-2 knockout and Fas mutation prolong lifespan of Scurfy mice but by different mechanisms. J Immunol 179, 8035-8041 (2007).

16. Ikeda, T. et al. Dual effects of TRAI L in suppression of autoimmunity: the inhibition of Th l cells and the promotion of regulatory T cells. J Immunol 185, 5259-5267 (2010).

17. Zhan, Y. et al. Defects in the Bcl-2-regulated apoptotic pathway lead to preferential increase of CD25 low Foxp3+ anergic CD4+ T cells. J Immunol 187, 1566-1577 (2011).

18. Lindsten, T. et al. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 6, 1389-1399 (2000).

19. Wang, X. et al. Preferential control of induced regulatory T cell homeostasis via a Bim/Bcl-2 axis.

Cell Death Dis 3, e270 (2012).

20. Liu, N., Zheng, Y., Zhu, Y., Xiong, S. & Chu, Y. Selective impairment of CD4+CD25+Foxp3+ regulatory T cells by paclitaxel is explained by Bcl-2/Bax mediated apoptosis. Int Immunopharmacol 11, 212- 219 (2011).

21. Tischner, D. et al. Defective cell death signalling along the Bcl-2 regulated apoptosis pathway compromises Treg cell development and limits their functionality in mice. J Autoimmun 38, 59-69

(2012).

22. Motoyama, N. et al. Massive cell death of immature hematopoietic cells and neurons in Bcl-x- deficient mice. Science 267, 1506-1510 (1995).

23. Ma, A. et al. Bclx regulates the survival of dou ble-positive thymocytes. Proc Natl Acad Sci U S A 92, 4763-4767 (1995).

24. Rubtsov, Y.P. et al. I L-10 produced by regulatory T cells contributes to their suppressor function by limiting inflammation at environmental interfaces. Immunity 28, 546-558 (2008).

25. Wagner, K. U. et al. Conditional deletion of the Bcl-x gene from erythroid cells results in hemolytic anemia and profound splenomegaly. Development 127, 4949-4958 (2000).

26. Vikstrom, I. et al. Mcl-1 is essential for germinal center formation and B cell memory. Science 330, 1095-1099 (2010).

27. Schlenner, S. M. et al. Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus. Immunity 32, 426-436 (2010).

28. Dzhagalov, I., Dunkle, A. & He, Y.W. The anti-apoptotic Bcl-2 family member Mcl-1 promotes T lymphocyte survival at multiple stages. J Immunol 181, 521-528 (2008).

29. Opferman, J.T. et al. Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature 426, 671-676 (2003).

30. Strasser, A., Cory, S. & Adams, J.M. Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. Embo J 30, 3667-3683 (2011). 31. Bouillet, P. et al. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 286, 1735-1738 (1999).

32. Gray, D.H. et al. The BH3-only proteins Bim and Puma cooperate to impose deletional tolerance of organ-specific antigens. Immunity 37, 451-462 (2012).

33. Bautista, J.L et al. Intraclonal competition limits the fate determination of regulatory T cells in the thymus. Nat Immunol 10, 610-617 (2009).

34. Liston, A. & Rudensky, A.Y. Thymic development and peripheral homeostasis of regulatory T cells.

Curr Opin Immunol 19, 176-185 (2007).

35. Tai, X. et al. Foxp3 is pro-apoptotic and lethal to developing regulatory T cells unless counterbalanced by cytokine survival signals. . Immunity In press (2013).

36. Walker, L.S. CD4+ CD25+ Treg: divide and rule? Immunology 111, 129-137 (2004).

37. McNeill, A., Spittle, E. & Backstrom, B.T. Partial depletion of CD69low-expressing natural regulatory T cells with the anti-CD25 monoclonal antibody PC61. Scand J Immunol 65, 63-69 (2007).

38. Suffner, J. et al. Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice. J Immunol 184, 1810-1820 (2010).

39. Guy, C.S. et al. Distinct TCR signaling pathways drive proliferation and cytokine production in T cells. Nat Immunol 14, 262-270 (2013).

40. Khan, W.N. B cell receptor and BAFF receptor signaling regulation of B cell homeostasis. J Immunol

183, 3561-3567 (2009).

41. Carrette, F. & Surh, CD. IL-7 signaling and CD127 receptor regulation in the control of T cell homeostasis. Semin Immunol 24, 209-217 (2012).

42. Dummer, W., Ernst, B., LeRoy, E., Lee, D. & Surh, C. Autologous regulation of naive T cell homeostasis within the T cell compartment. J Immunol 166, 2460-2468 (2001).

43. Gorelik, L. et al. Normal B cell homeostasis requires B cell activation factor production by radiation- resistant cells. J Exp Med 198, 937-945 (2003).

44. Wing, K. et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science 322, 271-275 (2008).

45. Liston, A., Lu, L.F., O'Carroll, D., Tarakhovsky, A. & Rudensky, A.Y. Dicerdependent microRNA pathway safeguards regulatory T cell function. J Exp Med 205, 1993-2004 (2008).

46. d'Hennezel E, Bin Dhuban K, Torgerson T, Piccirillo CA. The immunogenetics of immune dysregulation, polyendocrinopathy, enteropathy, X linked (IPEX) syndrome. J Med Genet. 2012;

49(5):291-302.

47. Santagata S, Villa A, Sobacchi C, Cortes P, Vezzoni P (2000). "The genetic and biochemical basis of Omenn syndrome". Immunol Rev. 178: 64-74. 48. Koreth, J. et al. lnterleukin-2 and regulatory T cells in graft-versus-host disease. N. Engl. J. Med. 365, 2055-2066 (2011).

49. Saadoun, D. et al. Regulatory T-cell responses to lowdose interleukin-2 in HCV-induced vasculitis.

N. Engl. J. Med. 365, 2067-2077 (2011)

50. Gavin, M. A. et al. Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proc. Natl Acad. Sci. USA 103, 6659-6664 (2006).

51. Allan, S. E., Song-Zhao, G. X., Abraham, T., McMurchy, A. N. & Levings, M. K. Inducible reprogramming of human T cells into Treg cells by a conditionally active form of FOXP3. Eur. J. Immunol. 38, 3282-3289 (2008).

52. Hoffmann, P. et al. Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation. Eur. J. Immunol. 39, 1088-1097 (2009).

53. Takeuchi, O. et al. Essential role of ΒΑΧ,ΒΑΚ in B cell homeostasis and prevention of autoimmune disease. Proc Natl Acad Sci U S A 102, 11272-11277 (2005).

54. Caton, M. L., Smith-Raska, M. R. & Reizis, B. Notch-RBP-J signaling controls the homeostasis of CD8- dendritic cells in the spleen. J Exp Med 204, 1653-1664 (2007).

55. Fontenot, J. D. et al. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22, 329-341 (2005).

56. Liston, A. et al. Differentiation of regulatory Foxp3+ T cells in the thymic cortex. Proc Natl Acad Sci U S A 105, 11903-11908 (2008).

57. Mombaerts, P. et al. RAG-l-deficient mice have no mature B and T lymphocytes. Cell 68, 869-877 (1992).

58. Yu, W. et al. Continued RAG expression in late stages of B cell development and no apparent re- induction after immunization. Nature 400, 682-687 (1999).

59. Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1, 4 (2001).

60. Buch, T. et al. A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration. Nat Methods 2, 419-426 (2005).

61. Seibler, J. et al. Rapid generation of inducible mouse mutants. Nucleic Acids Res 31, el2 (2003). 62. Kim, J.M., Rasmussen, J. P. & Rudensky, A.Y. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat Immunol 8, 191-197 (2007).

Claims

1. A vector containing nucleic acid encoding Foxp3 and Mcl-1; or

nucleic acid encoding Foxp3 and/or Mcl-1 operably linked to a Foxp3 promoter.

2. The vector according to claim 1, which is a viral vector, particularly a lentiviral vector.

3. The vector according to claim 1 or 2, wherein Foxp3 and Mcl-1 are operably linked to a common promoter.

4. The vector according to claim 3, wherein the promoter is the Foxp3 promoter.

5. The vector according to any one of claims 1 to 4 for use as a medicament.

6. The vector according to any one of claims 1 to 4 for use in treatment of disease characterized by T_reg deficiency.

7. The vector for use according to claim 6, wherein the disease characterized by T_reg deficiency is selected from immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), Omenn syndrome and graft versus host disease (GVHD).

8. A method of treating a disease characterized by T_reg deficiency in a subject in need thereof, comprising:

introducing a vector containing nucleic acid encoding Foxp3, or encoding Foxp3 and Mcl-1, in stem cells, and;

introducing the stem cells in the subject.

9. The method according to claim 8, further comprising a step of allowing expression of Foxp3 and Mcl-1 in said stem cells.

10. The method according to claim 8, wherein the stem cells are stem cells from the subject.

11. The method according to claim 10, further comprising a step of isolating stem cells from the subject prior to the introduction step.

12. The method according to claim 11, wherein the stem cells are isolated from the blood of the subject.

13. The method according to any one of claims 8 to 12, wherein at least part of the reintroduced stem cells differentiates into T_reg cells, thereby rescuing the T_reg deficiency.

14. A method of treating a disease characterized by T_reg deficiency in a subject in need thereof, comprising:

introducing a vector containing nucleic acid encoding Mcl-1 operably linked to a Foxp3 promoter in T cells, and;

introducing the T cells in the subject.

15. The method according to claim 14, further comprising a step of allowing expression of Mcl-l in at least part of the T cells.

16. The method according to claim 14, wherein the T cells are T cells from the subject.

17. The method according to claim 16, further comprising a step of isolating T cells from the subject prior to the introduction step.

18. The method according to claim 16, wherein the T cells are T_reg cells.