REGULATORY T CELLS TARGETED BY LYMPHOTOXIN ALPHA BLOCKING AGENT AND USES THEREOF

Abstract

The present invention relates to regulatory T cell and uses thereof. By their immunosuppressive and anti-inflammatory activities, regulatory T cells play a central role in peripheral tolerance and thus critically prevent the development of autoimmune and inflammatory disorders. The inventors showed that Foxp3+CD4+ Tregs express high levels of LTα, which negatively regulates their immunosuppressive signature. The inventors have demonstrated that the adoptive transfer of Tregs previously incubated with soluble lymphotoxin-β receptor in mice protects from dextran sodium sulfate (DSS)-induced colitis. Thus, the number of cells to be injected in adoptive transfer may be reduced and a transfection or transduction step avoided, which represents a technical facilitation. In particular, the present invention relates to a method of treating or preventing autoimmune disorders and inflammatory-associated cancers in a subject in need thereof comprising the step of administrating to the subject a therapeutically effective amount of regulatory T cells which have been previously incubated with effective amount of soluble lymphotoxin-β receptor.

Claims

1. A regulatory T cell which has been previously incubated with an amount of a lymphotoxin alpha blocking agent sufficient to increase the suppressive activity of the regulatory T cell.

2. The regulatory T cell of claim 1, wherein the lymphotoxin alpha blocking agent is a soluble lymphotoxin-β receptor (LTβR).

3. A population of regulatory T cells according to claim 1.

4. A The regulatory T cell of claim 1 wherein the regulatory T cell expresses a TCR or a chimeric antigen receptor which recognizes/binds to an autoantigen.

5. A population of regulatory T cells according to claim 4.

6. A method of producing the regulatory T cell according to claim 4, comprising, transfecting or transducing a regulatory T cell in vitro or ex vivo with a vector encoding for a chimeric antigen receptor or a TCR.

7. A method of conducting adoptive cell therapy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the population of regulatory T cells according to claim 3.

8. A pharmaceutical composition comprising the population of regulatory T cells according to claim 3.

9. An ex vivo method for stimulating regulatory T cells immunosuppressive activity, said method comprising: i. obtaining a biological sample from a subject; ii. isolating regulatory T cells from said sample; iii. expanding the regulatory T cells in vitro; iv. incubating the regulatory T cells with an amount of lymphotoxin alpha blocking agent sufficient to block LTα1β2/LTβR or LTα2β1/LTβR interactions.

10. The method according to claim 9 wherein the biological sample is a blood sample.

11. A method of treating or preventing an autoimmune disorder& in a subject in need thereof comprising the step of administering to the subject a therapeutically effective amount of the population of regulatory T cells according to claim 3.

12. The method according to claim 11, wherein the autoimmune disorder is inflammatory bowel disease.

13. A method of treating or preventing inflammation-associated cancer or allergy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of regulatory T cells according to claim 3.

14. The method according to claim 13, wherein the inflammation-associated cancer is colitis-associated cancer.

15. A method of treating or preventing immune reactions against molecules that are exogenously administered or immune reactions against a grafted tissue or grafted cells in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of regulatory T cells according to claim 3.

Description

FIGURES

[0138] FIG. 1. The adoptive transfer of LTα−/− Tregs protects from the severity of DSS-induced colitis. (A) Representative flow cytometry profiles of Foxp3 expression in purified splenic CD4+CD25+ cells from WT and LTα−/− mice. (B) Experimental setup: colitis was induced by the administration of 2% DSS in drinking water for 7 days followed by water only until day 11 in WT mice injected 2 days before with 2×10.sup.5 WT or LTα−/− Tregs. Colon inflammation and CD4.sup.+ T cell priming in mesenteric lymph nodes were analyzed at day 11 and day 4 of the protocol, respectively. (C) Body weight loss relative to the initial weight on day 0 of WT mice injected with 2×10.sup.5 WT or LTα−/− Tregs. Data are derived from 3 independent experiment with 4 mice per group. (D) Disease activity index (DAI) was monitored during the course of DSS-induced colitis. (E) The histogram shows the histological score of the colon in both groups of mice.

[0139] FIG. 2. The adoptive transfer of WT Tregs pre-incubated with the soluble LTβR□Fc fusion protein attenuates DSS-induced colitis. (A) Experimental setup: colitis was induced by the administration of 2% DSS in drinking water for 7 days followed by water only until day 11 in WT mice injected 2 days before with either 2×105 WT Tregs pretreated or not with LTβR-Fc protein or LTα−/− Tregs. Body weight was monitored every day and colon length was measured at day 11 of the protocol. (B) Body weight loss relative to the initial weight on day 0 of WT mice injected with either WT Tregs pre-incubated or not with the soluble LTβR-Fc fusion protein or LTα−/− Tregs. Data are derived from 1 independent experiment with 3-9 mice per group. (C) The histogram shows the colon length observed at the end of the experimental protocol.

[0140] FIG. 3. The suppressive signature of Treg cells is controlled by the LTα1β2/LTβR axis. (A) The expression level of Il10, Ebi3, Tgfb1, Ifn-γ, Gzmb and Fasl mRNAs was measured by qPCR in purified CD45.2 WT and LTα−/− Tregs from donor groups. (B) Purified WT Foxp3+CD4+ Tregs pre-incubated or not with a soluble LTβR-Fc fusion protein and co-cultured during 24 h with purified CD11c+ dendritic cells were analyzed for the expression level of Klrg1, Il10, Ebi3, Tgfb1, Gzmb and Fasl by qPCR.

EXAMPLE

[0141] Material & Methods

[0142] Mice

[0143] All mice—CD45.1 WT, CD45.1×CD45.2 WT, CD45.2 WT and CD45.2 LTα−/− mice—were on a pure C57BL/6 background and maintained under specific pathogen free conditions at the CIML (France). Standard food and water were given ad libitum. Males and females were used at the age of 6-8 weeks. All procedures involving animals have been performed in accordance with the institutional and ethical guidelines.

[0144] Treg Cell Isolation

[0145] Splenic Treg cells were isolated by scratching the spleen through a 70 μm mesh. Splenic red blood cells were lysed with lysis buffer (eBioscience). Before cell-sorting, CD4+ T cells were pre-enriched by depletion of CD8+ and CD19+ cells using anti-CD8a (clone 53.6.7) and anti-CD19 (clone 1D3) biotinylated antibodies with anti-biotin microbeads by AutoMACS (Miltenyi Biotech) via the deplete program. CD4+CD25+ Tregs were sorted using a FACSAriaIII cell sorter (BD).

[0146] DSS-Induced Colitis Experiments

[0147] Two days before the induction of colitis, WT recipient mice were injected i.v. with 2.Math.10.sup.5 CD4+CD25+ splenic Tregs sorted from WT or LTα−/− mice. The induction of colitis was assessed by given 2% DSS (Alfa Aesar) in drinking water for 7 days, followed by only water until sacrifice at dl 1. Body weight, rectal bleeding and stool consistency were monitored every day after DSS administration and used to determine the DAI.

[0148] In Vitro Co-Culture Assays

[0149] For co-culture assays, 2.Math.10.sup.3 cell-sorted total CD11c.sup.hi dendritic cells were co-cultured during 24 h at 37° C. with 10.sup.4 purified CD4.sup.+CD25.sup.+ Tregs that were or not pre-incubated during 1 h with a soluble LTβR-Fc recombinant protein (2 μg/ml; R&D systems).

[0150] Treg Cell Incubation with Soluble LTβR-Fc Protein in the DSS-Induced Colitis Model

[0151] 2.105 CD4+CD25+ splenic Tregs purified from CD45.2 WT mice were pre-incubated or not with the soluble LTβR-Fc fusion protein (50 μg/ml; R&D Systems) for 30 min in culture medium (RPMI ThermoFisher with 10% FBS (Sigma Aldrich), 2 mM L-glutamine (ThermoFisher), 1 mM sodium pyruvate (ThermoFisher) and 2×10-5 M 2-mercaptoethanol (ThermoFisher)). Tregs were then adoptively transferred intravenously into CD45.2 WT recipients. 2.105 CD4+CD25+ splenic Tregs from LTα−/− mice were used as a control. Two days later, colitis was induced by given 2% DSS (Alfa Aesar) in drinking water for 7 days, followed by only water until sacrifice at dl 1. Body weight was monitored every day after DSS administration.

[0152] Flow Cytometry

[0153] Anti-CD4 (RM4.5) antibody was from BD. For intracellular staining with Foxp3 antibody, cells were fixed, permeabilized and stained with the Foxp3 staining kit according to the manufacturer's instructions (eBioscience). Stained cells were analyzed with FACSCanto II (BD) and data were analyzed using FlowJo software.

[0154] Quantitative RT-PCR

[0155] Total RNA was isolated with TRIzol (Invitrogen) and cDNA was synthesized with random oligo dT primers and Superscript II reverse transcriptase (Invitrogen). qPCR was performed with SYBR Premix Ex Taq master mix (Takara) on a ABI 7500 fast real-time PCR system (Applied Biosystem). Results were normalized to actin mRNA.

[0156] Statistical Analysis

[0157] Statistical significance was assessed with GraphPad Prism 6 software using unpaired Student's t test or Mann-Whitney test. The two-way Anova test with Bonferroni correction was used for the analysis of tumor growth, the loss of weight and DAI. *, P<0.05; **, P<0.01; ***, P<0.001, ****, P<0.0001. Normal distribution of the data was assessed using d'Agostino-Pearson omnibus normality test. Error bars represent mean±SEM.

[0158] Results

[0159] The Adoptive Transfer of LTα.sup.−/− Tregs Protects from Ulcerative Colitis

[0160] Given that LTα.sup.−/− Tregs highly express several genes implicated in Treg suppressive functions (data not shown), we evaluated whether the adoptive transfer of LTα.sup.−/− Tregs shows therapeutic benefits to protect from dextran sodium sulfate (DSS)-induced colitis. 2.Math.10.sup.5 CD4.sup.+CD25.sup.+ cells that predominantly contain Foxp3.sup.+ Tregs (FIG. 1A) purified from WT or LTα.sup.−/− mice were injected into WT recipient mice two days before the induction of colitis with 2% DSS (FIG. 1B). We observed that mice injected with LTα.sup.−/− Tregs lost significantly less weight than those injected with WT Tregs (FIG. 1C). Moreover, the disease activity index (DAI), which combines stool consistency, rectal bleeding and weight loss was substantially less important in these mice (FIG. 1D). In accordance with the weight loss and DAI, these mice displayed less damages of the colonic epithelium (data not shown) and a reduced colitis histological score at the end of the experiment (FIG. 1E). We also observed a reduced expression of pro-inflammatory cytokines such as Il6, Ifnγ, Tnf-α, Il17A, Il1α and Il33 as well as of chemokines implicated in the recruitment of immune cells such as Ccl2 and Cxcl12 in colons of mice transferred with LTα.sup.−/− Tregs (data not shown). We further analysed the nature of colon-infiltrating immune cells by flow cytometry. Numbers of neutrophils, macrophages, dendritic cells, B cells and CD4.sup.+ T cells were drastically reduced in mice transferred with LTα.sup.−/− Tregs compared to those transferred with WT Tregs (data not shown). A reduced infiltration of CD3.sup.+ and B220.sup.+ cells was confirmed on histological colon sections (data not shown). Numbers of Th1 and Th17 effector CD4.sup.+ T cells were also reduced (data not shown). Consequently, Treg/Th1 and Treg/Th17 ratios were increased in the colon of mice transferred with LTα.sup.−/− Tregs (data not shown). We then assessed the potential of Lta.sup.−/− Tregs to protect against colitis by reducing the number of adoptively transferred cells from 2.Math.10.sup.5 to 1.Math.10.sup.5 and then to 0.5.Math.10.sup.5 cells. We observed that 1.Math.10.sup.5 Lta.sup.−/− Tregs still shows a better protection than 2.Math.10.sup.5 WT Tregs characterized by reduced weight loss (data not shown). Interestingly, 0.5.Math.10.sup.5 Lta.sup.−/− Tregs show the same protective effect than 2.Math.10.sup.5 WT Tregs, indicating that Lta.sup.−/− Tregs are ˜4 times more suppressive in vivo than their WT counterparts.

[0161] We next further determined whether the adoptive transfer of LTα.sup.−/− Tregs inhibits CD4.sup.+ T cell priming in mesenteric lymph nodes five days after the administration of DSS. Of note, we found that mice injected with LTα.sup.−/− Tregs already showed longer colon length and reduced colonic weight/length ratio at this time point, indicative of attenuated colon inflammation (data not shown). Strikingly, numbers of Th1 and Th17 effector CD4.sup.+ T cells were substantially reduced in mesenteric lymph nodes of these mice (data not shown), indicating that LTα.sup.−/− Tregs inhibit the conversion of naïve CD4.sup.+ T cells into effectors. Altogether, these data show that the adoptive transfer of LTα.sup.−/− Tregs protects from the development of ulcerative colitis by dampening colon inflammation and the priming of pathogenic CD4.sup.+ T cells in mesenteric lymph nodes.

[0162] The Adoptive Transfer of WT Tregs Pre-Incubated with the Soluble LTβR□Fc Fusion Protein Attenuates DSS-Induced Colitis.

[0163] We next evaluated whether the adoptive transfer of Tregs pre-incubated with the soluble LTβR-Fc fusion protein shows therapeutic benefits to protect from dextran sodium sulfate (DSS)-induced colitis. 2.Math.10.sup.5 CD4.sup.+CD25.sup.+ cells that predominantly contain Foxp3.sup.+ Tregs purified from LTα.sup.−/− or WT mice (FIG. 1A) were pre-incubated or not with the soluble LTβR-Fc fusion protein for 30 min in culture medium. Tregs were injected into WT recipient mice two days before the induction of colitis with 2% DSS (FIG. 2A). We observed that mice injected with Tregs pre-incubated with LTβR-Fc lost significantly less weight than those injected with WT Tregs and similarly to those injected with LTα.sup.−/− (FIG. 2B). Moreover, the colon of mice injected with Tregs pre-incubated with soluble LTβR-Fc is significantly longer than those injected with WT Tregs. However it is similar to the one of mice injected with LTα.sup.−/− (FIG. 2C).

[0164] Altogether, these data show that the adoptive transfer of WT Tregs pre-incubated with soluble LTβR-Fc protects from the development of ulcerative colitis as effectively as LTα.sup.−/− Tregs.

[0165] LTα Expression in Hematopoietic Cells and LTα1β2/LTβR Axis Negatively Control the Suppressive Signature of Treg Cells

[0166] Because LTα.sup.−/− mice show a disorganized thymic and splenic microenvironment, we analysed the contribution of non-hematopoietic stromal cells in the highly immunosuppressive phenotype of LTα.sup.−/− Tregs. For this, we generated bone marrow (BM) chimeras in which lethally irradiated CD45.2 WT or LTα.sup.−/− recipient mice were reconstituted with WT BM cells from CD45.1 congenic mice (WT CD45.1: WT and WT CD45.1: LTα.sup.−/− mice, respectively). Six weeks after BM transplantation, CD4.sup.+CD25.sup.+ Treg cells of CD45.1 donor origin were cell-sorted from the spleen and analysed for the expression of several genes associated with Treg effector function (data not shown). Similar frequencies and numbers of Foxp3.sup.+ Tregs were observed in both groups of mice (data not shown). Furthermore, the expression of several genes such as Klrg1, Tgfb, Gzmb and Fasl was similar in both groups of mice, indicating that non-hematopoietic cells are not implicated in the highly suppressive signature of Tregs observed in LTα.sup.−/− mice (data not shown).

[0167] We next determined the respective contribution of the hematopoietic compartment by generating mixed bone marrow chimaeras in which lethally irradiated CD45.1×CD45.2 WT recipient mice were reconstituted with BM cells (50:50) from WT CD45.1 and WT CD45.2 (WT donor group), or WT CD45.1 and LTα.sup.−/− CD45.2 (LTα.sup.−/− donor group) (data not shown). Six weeks later, we found increased frequencies and numbers of CD4.sup.+Foxp3.sup.+ Tregs derived from LTα.sup.−/− CD45.2 BM cells compared to those derived from WT CD45.2 BM cells in the spleen (data not shown). Strikingly, purified LTα.sup.−/− CD45.2 Tregs showed increased expression of Il10, Ebi3, Tgfb1, Ifng, Gzmb and Fasl genes compared to WT CD45.2 Tregs (FIG. 3A). These data indicate that the expression of LTα in hematopoietic cells negatively controls the immunosuppressive signature of Treg cells.

[0168] Since we observed that Tregs express LTα, as a membrane anchored LTα1β2 heterocomplex (data not shown), we assessed the contribution of LTα1β2/LTβR axis in controlling the suppressive signature of Tregs. In particular, we analyzed whether blocking LTα1β2/LTβR interactions between Tregs and dendritic cells impacts the suppressive signature of Treg cells. For this, purified WT CD4.sup.+CD25.sup.+ Tregs pre-incubated or not with a soluble LTβR-Fc fusion protein were co-cultured with purified CD11 dendritic cells. Interestingly, Tregs that were pre-incubated with LTβR-Fc upregulated the expression of several genes associated with Treg suppressive function such as Klrg1, Il10, Ebi3, Tgfb1, Gzmb and Fasl compared to un-pretreated Tregs (FIG. 3B). These data thus indicate that LTα1β2/LTβR interactions between Tregs and dendritic cells negatively regulate the suppressive signature of Tregs.

[0169] LTα Expression is Conserved in Human Tregs Derived from Peripheral Blood

[0170] We next assessed whether LTα expression is conserved in human Tregs derived from peripheral blood of female and male healthy donors. Foxp3.sup.+CD4.sup.+ Tregs were classically identified as CD4.sup.+CD25.sup.+CD127.sup.lo cells. Intracellular LTα protein (data not shown) and the cell-surface LTα1β2 heterocomplex (data not shown) were substantially detected by flow cytometry in Tregs of all donors analyzed, indicating that this expression is conserved in mice to human.

DISCUSSION

[0171] Several studies have identified numerous molecules implicated in the positive regulation of Treg cell development and function. In contrast, few reports have described signals that negatively regulate Treg function. Here, by analyzing distinct T cell populations endowed with regulatory properties, we found that Foxp3+ Tregs substantially express Lta, as a membrane anchored LTα1β2 heterocomplex. Similarly to LTβR.sup.−/− mice, LTα.sup.−/− mice do not show any obvious defect in CD4.sup.+Foxp3.sup.+ Treg cell development in the thymus. However, the signature of genes associated with suppressive functions was greatly enhanced both in LTα.sup.−/− Tregs, indicating that LTα, and more precisely the LTα1β2/LTβR interactions between Tregs and dendritic cells negatively regulates their immunosuppressive signature.

[0172] Interestingly, we have previously demonstrated that the adoptive transfer of LTα.sup.−/− Tregs protects from DSS-induced colitis (FIG. 1) and treats from IBD more efficiently than WT Tregs. This was reflected by a reduced body weight loss, a higher colon length and a reduced histological score in mice transferred with LTα.sup.−/− Tregs compared to mice injected with WT Tregs. Furthermore, we observed that LTα.sup.−/− Tregs substantially reduce colon inflammation and the infiltration of inflammatory immune cells. In the DSS-induced colitis model, we found that the transfer of LTα.sup.−/− Tregs before the induction of colitis reduces the priming and/or expansion of Th1 and Th17 pathogenic cells in mesenteric lymph nodes. Importantly, the ratios Treg/Th1 and Treg/Th17 were increased in the colon in both the DSS-induced colitis and IBD models, suggesting that Tregs can also exert their suppressive effects locally in this tissue. By their ability to suppress colon inflammation, the adoptive transfer of LTα.sup.−/− Tregs also attenuates the development of CAC, which is known to be promoted by chronic inflammation. Altogether, these data indicated that compared to their WT counterparts, LTα.sup.−/− Tregs show a higher capacity to treat colitis and protect from both colitis and CAC development. We also revealed that LTα1β2/LTβR interactions between Tregs and dendritic cells, particularly Sirpα.sup.+ conventional dendritic cells and plasmacytoid dendritic cells, negatively control the suppressive signature of Treg cells, suggesting that a direct cell contact with antigen-present cells regulates Treg suppressive activity.

[0173] Herein, we show that the adoptive transfer of WT Tregs pre-incubated with soluble LTβR-Fc protects from the development of ulcerative colitis as effectively as LTα.sup.−/− Tregs (FIG. 2B-C). Furthermore, WT Tregs pre-incubated with soluble LTβR-Fc displays a similar suppressive signature than the LTα.sup.−/− Tregs (FIG. 3). Therefore, the adoptive transfer of WT Tregs pre-incubated with soluble LTβR-Fc is expected to not only protect from DSS-induced colitis but also treat from IBD and attenuate the development of CAC as effectively as the adoptive transfer of LTα.sup.−/− Tregs.

REFERENCES

[0174] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0175] 1. Sakaguchi, S., et al. (2008). Regulatory T cells and immune tolerance. Cell 133, 775-787. [0176] 2. Dhamne, C. et al. (2013). Peripheral and thymic foxp3(+) regulatory T cells in search of origin, distinction, and function. Front Immunol 4, 253. [0177] 3. Fontenot, J. D. et al. (2005). Developmental regulation of Foxp3 expression during ontogeny. The Journal of experimental medicine 202, 901-906. [0178] 4. Brunkow, M. E. et al. (2001). Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nature genetics 27, 68-73. [0179] 5. Bennett, C. L. et al. (2001). The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nature genetics 27, 20-21. [0180] 6. Maloy, K. J. et al. (2003). CD4+CD25+ T(R) cells suppress innate immune pathology through cytokine-dependent mechanisms. The Journal of experimental medicine 197, 111-119. [0181] 7. Szanya, V. et al. (2002). The subpopulation of CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7. Journal of immunology 169, 2461-2465. [0182] 8. McGeachy, M. J. et al. (2005). Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system. Journal of immunology 175, 3025-3032. [0183] 9. Presser, K. et al. (2008). Coexpression of TGF-beta1 and IL-10 enables regulatory T cells to completely suppress airway hyperreactivity. Journal of immunology 181, 7751-7758. [0184] 10. Waldner, M. J., and Neurath, M. F. (2009). Colitis-associated cancer: the role of T cells in tumor development. Semin Immunopathol 31, 249-256. [0185] 11. Riley, J. L. at al. (2009). Human T regulatory cell therapy: take a billion or so and call me in the morning. Immunity 30, 656-665. [0186] 12. Bluestone, J. A., et al. (2015). Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sci Transl Med 7. [0187] 13. Desreumaux, P. et al. (2012). Safety and efficacy of antigen-specific regulatory T-cell therapy for patients with refractory Crohn's disease. Gastroenterology 143. [0188] 14. Di Ianni, M. et al. (2011). Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation. Blood 117, 3921-3928.