METHODS AND KITS FOR LABELING, DETECTION AND ISOLATION OF FOXP3+ REGULATORY T CELLS, ISOLATED POPULATION OF FOXP3+ REGULATORY T CELLS THUS OBTAINED AND USES THEREOF

Abstract

Disclosed is a method for labeling, detecting and/or isolating Foxp3+ Treg cells from a biological sample containing peripheral blood mononuclear cells (PBMC) or lymphocytes including the following steps of: (i) coupling the surface of PBMC or lymphocytes to a capture moiety which binds to the cell through a cell surface molecule and to interleukin-34 (IL34), (ii) culturing the lymphocytes under conditions wherein IL34 is secreted, released and specifically captured by the capture moiety, (iii) labeling the IL34 expressing lymphocytes with a label moiety, and (iv) optionally detecting and/or isolating the IL34 expressing lymphocytes which are Foxp3+ Treg cells. Also disclosed is an isolated population of Foxp3+ Treg cells obtainable by the method and uses thereof.

Claims

1-18. (canceled)

19. A method for labeling, detecting and/or isolating forkhead box P3 (Foxp3+) regulatory T cells (Foxp3+ Treg cells) from a biological sample containing peripheral blood mononuclear cells (PBMC) or lymphocytes comprising the following steps of: (i) coupling the surface of PBMC or lymphocytes to a capture moiety which binds to the cell through a cell surface molecule and to Interleukin-34 (IL34), (ii) culturing the lymphocytes under conditions wherein IL34 is secreted, released and specifically captured by the capture moiety, and (iii) labeling the IL34 expressing lymphocytes with a label moiety.

20. The method according to claim 19, wherein the cell surface molecule is selected from the group consisting of CD3, CD4, CD8, CD45, TCR, chimeric antigen receptor, IFN?, IL10 and MCSF.

21. The method according to claim 19, wherein the capture moiety is selected from the group consisting of a bispecific antibody which binds to CD3 and IL34, a bispecific antibody which binds to CD4 and IL34, a bispecific antibody which binds to CD8 and IL34, a bispecific antibody which binds to CD45 and IL34, a bispecific antibody which binds to a TCR and IL34, and a bispecific antibody which binds to a chimeric antigen receptor and IL34.

22. The method according to claim 19, wherein the capture moiety is selected from the group consisting of a trispecific antibody which binds to IL34, IFN? and CD3, a trispecific antibody which binds to IL34, IL10 and CD3, a trispecific antibody which binds to IL34, MCSF and CD3, and a trispecific antibody which binds to IL34, CD3 and an antigen.

23. The method according to claim 19, wherein the label moiety is a labeled anti-IL34 antibody.

24. The method according to claim 19, wherein the biological sample is a blood sample obtained from a transplanted patient or a patient suffering from an autoimmune disease, allergy or cancer.

25. The method according to claim 19, wherein said method is for obtaining a population of antigen-specific Foxp3+ Treg cells, and wherein said method comprises a step of culturing the population of Foxp3+ Treg cells with a culture medium comprising an amount of antigen and a step of isolating the population of antigen-specific Foxp3+ Treg cells.

26. The method according to claim 19, wherein said method is an in vitro method for determining whether a patient is at risk of transplant rejection, autoimmune diseases, unwanted immune response against therapeutic proteins or allergies, wherein said method comprises a step of determining the presence Foxp3+ Treg cells in a biological sample obtained from said patient, and wherein the presence of Foxp3+ Treg cells is indicative of a reduced risk of transplant rejection, autoimmune diseases, unwanted immune response against therapeutic proteins or allergies.

27. The method according to claim 19, wherein said method is an in vitro method for predicting the survival time of a subject suffering from a cancer, wherein said method comprises the steps of determining the presence Foxp3+ Treg cells in a biological sample obtained from said subject, and concluding that the presence of Foxp3+ Treg cells is indicative of a poor prognosis.

28. A kit comprising at least one capture moiety which binds to the cell through a cell surface molecule and to IL34.

29. The kit according to claim 28, further comprising a labeled moiety.

30. The kit according to claim 28, wherein the at least one capture moiety which binds to the cell through a cell surface molecule and to IL34 is selected from the group consisting of a bispecific antibody which binds to CD3 and IL34, a bispecific antibody which binds to CD4 and IL34, a bispecific antibody which binds to CD8 and IL34, a bispecific antibody which binds to CD45 and IL34, a bispecific antibody which binds to a TCR and IL34, and a bispecific antibody which binds to a chimeric antigen receptor and IL34.

31. The kit according to claim 29, wherein the label moiety is a labeled anti-IL34 antibody.

32. The kit according to claim 28, wherein the at least one capture moiety which binds to the cell through a cell surface molecule and to IL34 is selected from the group consisting of a trispecific antibody selected from the group consisting of a trispecific antibody which binds to IL34, IFN? and CD3, a trispecific antibody which binds to IL34, IL10 and CD3, a trispecific antibody which binds to IL34, MCSF and CD3, and a trispecific antibody which binds to IL34, CD3 and an antigen.

33. An isolated population of Foxp3+ Treg cells obtainable by a method according to claim 19.

34. The isolated population of Foxp3+ Treg cells according to claim 33, wherein said Foxp3+ Treg cells are CD4+ Foxp3+ Treg cells and/or CD8+ Foxp3+ Treg cells.

35. The isolated population of Foxp3+ Treg cells according to claim 33, wherein said Foxp3+ Treg cells are antigen-specific Foxp3+ Treg cells.

36. A method for inducing immune tolerance and/or for suppressing and/or inhibiting immune responses in a subject, comprising administering to the subject an isolated population of Foxp3+ cells according to claim 33.

37. The method according to claim 36, for treating an autoimmune disease, graft rejection, any unwanted immune reaction against a therapeutic protein, or allergy.

38. The method of claim 19, further comprising: (iv) detecting and/or isolating said IL34 expressing lymphocytes which are Foxp3+ Treg cells.

Description

FIGURES

[0150] FIG. 1: IL34 is a human Foxp3.sup.+Treg-specific cytokine that can inhibit anti-donor immune responses. The percentage of IL34 positive cells was evaluated in healthy individuals among CD4.sup.+ or CD8.sup.+ CD45RC.sup.low or CD45RC.sup.high T cells (A) or among Foxp3.sup.+ vs Foxp3.sup.? CD45RC.sup.low CD8.sup.+ or CD4.sup.+ T cells (B). The mean +/? SEM of 27 healthy individuals was represented (A). A representative plot of 10 healthy individuals was shown (B). (C) The panel show results for the mean +/? SEM of percentage of expression of IL34 in Foxp3.sup.+CD45RC.sup.lowCD4.sup.+ or CD8.sup.+ T cells in healthy individuals.

EXAMPLE

Interleukin-34, a New Role in Regulatory T Cell Immunoregulation and Transplant Tolerance

[0151] Material & Methods

[0152] Healthy volunteers blood collection and PBMC separation. Blood were collected from healthy donors, after informed consent was given, at the Etablissement Fran?ais du Sang (Nantes, France). Blood was diluted 2-fold with PBS before PBMC were isolated by Ficoll-Paque density-gradient centrifugation (Eurobio, Courtaboeuf, France) at 2000 rpm for 20 at room temperature without braking. Collected PBMC were washed in 50 mL PBS at 1800 rpm for 10 min and remaining red cells and platelets are eliminated after incubation 5 min in an hypotonic solution and centrifugation at 1000 rpm for 10 min. Total PBMC were then stimulated with PMA (50 ng/ml) and ionomycin (1 ?g/ml) for 7 h in presence of brefeldin A (10 ?g/ml) during the last 4 h for FACS analysis of IL34 expression in Tregs. For culture assay, CD3.sup.+CD4.sup.+CD25.sup.? cells were sorted by FACS Aria after enrichment in CD14.sup.? CD16.sup.? and CD19.sup.? cells by negative selection. Allogeneic APCs used to stimulate effector cells in MLR were obtained by CD3.sup.+ cells depletion and 35 Gray irradiation.

[0153] Monoclonal antibodies and flow cytometry. Human antibodies against CD3-PeCy7 (SKY7), CD4-PercPCy5.5 (L200), CD25-APCCy7 (M-A251), CD45RC-FITC (MT2), Foxp3-APC (236A/E7) and IL34-PE (578416, R&D) were used to characterize cell's phenotype. Fluorescence was measured with a Canto II cytometer (BD Biosciences, Mountain View, Calif.), and the FLOWJO software (Tree Star, Inc. USA) was used to analyze data. Cells were first gated by their morphology excluding dead cells by selecting DAPI viable cells.

[0154] Statistical analysis. Mann Whitney test was used for PBMC phenotype analysis.

[0155] Results

[0156] IL34 is expressed by human T cells and possessed a strong suppressive potential. IL34 has never been demonstrated as being expressed by Tregs and inhibiting anti-donor immune responses in human, we wanted to assess the potential and applicability of our findings in human. It has been suggested in the literature that human CD45RC.sup.low T cells were associated to Tregs while CD45RC.sup.high T cells were associated to naive and effector T cells (4, 5). We first analyzed the IL34 protein expression on CD8.sup.+ and CD4.sup.+ CD45RC.sup.low vs CD45RC.sup.high T cells by multicolor flow cytometry (FIG. 1A). Interestingly, we observed that both human CD4.sup.+ and CD8.sup.+ T cells expressed significant amount of IL34 protein in contrast to CD45RC.sup.high T cells. Moreover, we observed that CD8.sup.+ T cells expressed slightlymore IL34 than CD4.sup.+ T cells. To discriminate IL34 expression by Tregs inside the CD45RC.sup.low cells, we analyzed IL34 expression within Foxp3.sup.+ Tregs (FIG. 1B). Strikingly, we observed that IL34 expression was specific of CD4.sup.+Foxp3.sup.+ and CD8.sup.+Foxp3.sup.+ Tregs versus CD4.sup.+Foxp3.sup.?, CD8.sup.+Foxp3.sup.?, CD4.sup.+CD45RC.sup.high and CD8.sup.+CD45RC.sup.high T cells subsets, indicating that IL34 is a Treg-specific cytokine that may play a crucial role in Treg-mediated tolerance. More precisely, we observed that about half of the Foxp3.sup.+ Tregs (CD4.sup.+ or CD8.sup.+) express IL34 (FIG. 1C).

[0157] As we suspected a strong suppressive potential of IL34 in human, we added different doses of soluble human IL34 to a MLR where CD4.sup.+CD25.sup.?CFSE-labelled effector T cells were in presence of T-depleted allogeneic APCs. We observed a significant inhibition of effector T cells proliferation in presence of 5 ?g/ml of IL34, thus confirming the dose-dependent suppressive potential of IL34 on anti-donor immune response.

[0158] Altogether, these data provided the proof of concept of IL34 as a Treg-specific protein and a potential therapeutic target in manipulating the anti-donor immune response.

[0159] Discussion:

[0160] The biological relevance of IL34 remains to date largely unknown and controversial. In our study, in an attempt to unravel the complex mechanisms of tolerance induction in transplantation, we provide evidences, for the first time, of the unexpected properties of IL34 as a master regulator of immune responses and tolerance. We also provide the first proof that IL34 can be expressed by CD8.sup.+CD45RC.sup.low Tregs, and most importantly can induce potent regulatory T cells.

[0161] We analyzed IL34 expression by human Foxp3.sup.+CD45RC.sup.low CD4.sup.+ and CD8.sup.+ Tregs. Strikingly, we observed a specific and unique high level expression of IL34 by Foxp3.sup.+CD45RC.sup.low CD4.sup.+ and CD8.sup.+ Tregs, while CD45RC.sup.high and Foxp3.sup.?CD45RC.sup.low CD4.sup.+ and CD8.sup.+ T cells did not, demonstrating that IL34 is a Treg-specific cytokine in human. In addition, we also demonstrated the ex vivo suppressive potential of IL34 by itself on the anti-donor immune response, suggesting its potential as a target therapeutic in transplantation, and by extension in autoimmune diseases.

[0162] In conclusion, we described here the role in transplantation tolerance of a new cytokine, IL34, and we revealed its potential as a therapy in transplantation and by extension in other diseases. We also demonstrated for the first time that this cytokine can be produced by Tregs, opening new possibilities in the generation of Tregs transferrable to the human setting.

REFERENCES

[0163] 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.

[0164] 1. Waldmann, H., Hilbrands, R., Howie, D., and Cobbold, S. 2014. Harnessing FOXP3+ regulatory T cells for transplantation tolerance. J Clin Invest 124:1439-1445.

[0165] 2. Robb, R. J., Lineburg, K. E., Kuns, R. D., Wilson, Y. A., Raffelt, N. C., Olver, S. D., Varelias, A., Alexander, K. A., Teal, B. E., Sparwasser, T., et al. 2012. Identification and expansion of highly suppressive CD8(+)FoxP3(+) regulatory T cells after experimental allogeneic bone marrow transplantation. Blood 119:5898-5908.

[0166] 3. Wei, S., Nandi, S., Chitu, V., Yeung, Y. G., Yu, W., Huang, M., Williams, L. T., Lin,

[0167] H., and Stanley, E. R. 2010. Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells. J Leukoc Biol 88:495-505.

[0168] 4. Ordonez, L., Bernard, I., L'Faqihi-Olive, F. E., Tervaert, J. W., Damoiseaux, J., and Saoudi, A. 2009. CD45RC isoform expression identifies functionally distinct T cell subsets differentially distributed between healthy individuals and AAV patients. PLoS One 4:e5287.

[0169] 5. Ordonez, L., Bernard, I., Chabod, M., Augusto, J. F., Lauwers-Cances, V., Cristini, C., Cuturi, M. C., Subra, J. F., and Saoudi, A. 2013. A higher risk of acute rejection of human kidney allografts can be predicted from the level of CD45RC expressed by the recipients' CD8 T cells. PLoS One 8:e69791.