METHODS AND KITS FOR IDENTIFYING EFFECTOR TREG CELLS

Abstract

The present invention relates to methods and kits for identifying effector regulatory T cells. In particular the present invention relates to use of CD15s as a biomarker for eTreg cells. The present invention also relates to a method for identifying effector Treg cells (eTreg) in a fluid sample comprising the steps of i) detecting the cell surface expression of CD4, CD25, CD127 and CD15s markers on the cell population contained in the fluid sample and ii) concluding that the cells expressing CD4, CD25, CD127 at low levels and CD15s are the effector Treg cells. The present invention also relates to a method for identifying effector Treg cells (eTreg) in a tissue sample comprising the steps of i) detecting the cell expression of CD4, CD25, Foxp3 and CD15s markers and ii) concluding that the cells expressing CD4, CD25, Foxp3 and CD15s are the effector Treg cells.

Claims

1. A method for identifying effector Treg (eTreg) cells in a sample comprising the steps of i) identifying a population of Treg cells; ii) detecting cell surface expression of CD15s in the population of Treg cells; and iii) concluding that Treg cells expressing CD15s are the eTreg cells.

2. The method of claim 1, wherein the sample is a fluid sample, and wherein the method of identifying a population of Treg cells comprises iv) detecting cell surface expression of CD127, CD4 and CD25 in the population of Treg cells; and v) concluding that cells expressing CD127 at low levels, and expressing CD4 and CD25, are the Treg cells.

3. The method according to claim 2 wherein the fluid sample is selected from the group consisting of blood samples, PBMC samples and samples of Treg cells in suspension.

4. The method of claim 2 wherein the step of detecting is performed with a set of antibodies specific for CD4, CD25 and CD127.

5. The method of claim 2 wherein the step of detecting comprises performing flow cytometry.

6. The method of claim 1, wherein the sample is a tissue sample, and wherein the method further comprises iv) detecting cell surface expression of CD4, CD25 and Foxp3 in the population of Treg cells; and v) concluding that Treg cells expressing CD4, CD25 and Foxp3 are the eTreg cells.

7. The method of claim 6 wherein the step of detecting comprises a step of staining the tissue sample with a set of antibodies specific for CD4, CD25 and Foxp3.

8. The method of claim 6 wherein the step of detecting is performed by immunochemistry.

9. The method of claim 6 wherein the tissue sample is selected from the group consisting of tissue sections of brain, adrenal glands, colon, small intestines, stomach, heart, liver, skin, kidney, lung, pancreas, testis, ovary, prostate, uterus, thyroid and spleen.

10. The method of claim 2 which further comprises a step of detecting cell surface expression of CD45RA.

11. The method of claim 2 which further comprises a step of determining the level of eTreg cells present in the sample.

12. The method of claim 2 which further comprises a step of isolating eTreg cells from the sample.

13. An isolated eTreg obtained by the method of claim 12.

14. A pharmaceutical composition comprising a population of isolated eTreg cells identified by the method of claim 1.

15. A method of treating a disease selected from the group consisting of autoimmune diseases, inflammatory disease, allergic disease and graft rejection in a subject in need thereof, comprising administering to said subject an efficient amount of isolated effector Treg (eTreg) cells identified by the method of claim 1.

16. An in vitro method for monitoring the treatment of a disease selected from the group consisting of autoimmune diseases, inflammatory diseases and allergic diseases comprising the steps of i) determining the level of a population of eTreg cells in a sample obtained from the subject before the treatment by performing the method of claim 11, ii) determining the level of a population of eTreg cells in a sample obtained from the subject after the treatment by performing the method of claim 11, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the treatment is efficient when the level determined at step ii) is higher than the level determined at step i).

17. An in vitro method for predicting the survival of a patient suffering from a cancer comprising the steps of i) determining the level of a population of eTreg cells in a sample obtained from the subject by performing the method of claim 11, ii) comparing the level determined at step i) with a reference value and iii) concluding that the patient has a poor prognosis when the level determined at step i) is higher than the reference value.

18. An in vitro method for determining whether a patient suffering from a cancer will respond to a treatment comprising the steps of i) determining the level of a population of eTreg cells in a sample obtained from the subject by performing the method of claim 11, ii) comparing the level determined at step i) with a reference value and iii) concluding that the patient will significantly respond to the treatment when the level determined at step i) is lower than the reference value.

19. The method of claim 18 wherein the treatment is done with a immunotherapeutic agent.

20. A kit comprising means for detecting, on a cell population, the cell surface expression of CD4, CD25, CD127 and CD15s markers; or CD4, CD25, Foxp3 and CD15s markers.

21. The kit of claim 20 wherein said means are antibodies.

Description

FIGURES

[0127] FIG. 1A-B Cell surface markers for FoxP3, Helios and Ki-67 expressing CD4.sup.+ T cell subsets.

[0128] (A) 18 surface markers upregulated in FoxP3.sup.high effector Treg cells among more than 340 analyzed. Expression of intracellular FoxP3 and each indicated surface markers by flow cytometry of PBMCs gated on CD4.sup.+ T cells. Data are representative of 6 healthy donors.

[0129] (B) To assess which of the makers shown in FIG. 1A are preferentially expressed on FoxP3.sup.+CD4.sup.+ cells, we calculated the ratio of the proportion of positive cells among FoxP3.sup.+ cells (population A, shown in the lower cytometry scheme representing CD4+T subsets defined with FoxP3 and one the aforementioned surface marker expression) to the proportion of positive cells among FoxP3.sup. cells among CD4.sup.+ T cells (population B) for each marker (ratio I). We defined that the markers that were the most specific for FoxP3.sup.+ cells were those with the highest ratio I (upper histogram).

[0130] We also verified whether such markers were preferentially expressed on eTreg cells (shown in upper cytometry scheme defined by the CD45RA.sup.FoxP3.sup.high phenotype) or were also expressed on other CD45RA.sup.FoxP3.sup.low non Treg cells by calculating the ratio of the proportion of CD45RA.sup.FoxP3+ cells expressing the marker (population A, lower cytometry scheme) devided by the proportion of eTreg cells defined by the CD45RA.sup.FoxP3.sup.high phenotype among CD4.sup.+ T cells (ratio II). We considered that markers with ratio II close to 1 are more specific for eTreg cells as they are not expressed by FoxP3.sup.low cells (lower histogram). Data are obtained from 6 healthy donors.

[0131] FIG. 2A-D CD15s is a marker for functional FoxP3.sup.high Treg cells

[0132] (A) CD15s is expressed on eTreg cells but not non sialylated CD15. Expression of intracellular FoxP3 and indicated surface markers by flow cytometry of PBMCs gated on CD4.sup.+ T cells. Data representative of 6 independent experiments.

[0133] (B) CD15s.sup.+FoxP3.sup.+ are suppressive while CD15s.sup.FoxP3.sup.+ cells are not. CFSE dilution by 10.sup.4 labeled CD25.sup.CD45RA+CD4.sup.+ responder T cells assessed after 84-90 hours of coculture with indicated CD4.sup.+ T cell subset at a 1 to 1 ratio with TCR stimulation. CFSE dilution by nTregs or eTregs isolated using the protocole previously described in ref 8 is used as positive controls for complete suppression. As shown by the arrows, complete suppression is characterized by fewer proliferation cycles (2 distinguishable peaks vs 4 in expanding effector cells) and decreasing amplitudes in consecutive cycle peaks while ongoing proliferation is characterized by increasing amplitudes in consecutive cycle peaks. Data are representative of 3 independent experiments.

[0134] (C) Flow cytometry of the expression of CD45RA and intranuclear FoxP3 (top), nuclear Ki-67 and FoxP3 (middle) and surface CD15s and intranuclear FoxP3 (bottom) in CD4.sup.+ T cells. Red line separates Ki-67.sup.+/FoxP3.sup.high from Ki-67.sup.FoxP3.sup.low cells and CD45RA.sup.+/ FoxP3.sup.low from CD45RA.sup.FoxP3.sup.high cells while black lines in bottom panels separated CD15s.sup.+FoxP3.sup.+ cells from CD15s.sup.FoxP3.sup.+ cells. The percentages of CD45RA.sup.FoxP3.sup.high, Ki-67.sup.+ FoxP3.sup.high cells and CD15s.sup.+FoxP3.sup.high cells among CD4.sup.+ cells are indicated. Data are representative of 8 independent experiments.

[0135] (D) Expanding nave Treg cells upregulate CD15s in vitro. CD25.sup.CD45RA.sup.+CD4.sup.+ conventional T cells and nTreg cells were flow isolated and cultured for 14 days in the presence of anti-CD3/CD28 beads, IL-2 and rapamycin analyzed for CD15s and FoxP3 expression. % of CD15s.sup.+ and CD15s.sup. FoxP3.sup.+ cells are indicated in corresponding quandrants. Data representative of 3 independent experiments.

[0136] FIG. 3A-B Expression of CD15s by FoxP3 expressing developping Treg cells in the thymus

[0137] (A) Flow cytometry of the expression of intranuclear FoxP3 and indicated markers by double negative (DN), double positive (DP) and single positive (SP) CD4.sup.+ Thymocytes. Data are representative of 3 independent experiments.

[0138] (B) Modification of surface and intracellular markers by developing Treg cells in the thymus. Changes in markers are indicated in bold.

[0139] FIG. 4A-B CD15s expression in CD4.sup.+ T cell subsets in canonical diseases with FoxP3 expressing cell subset abnormalities

[0140] (A) Flow cytometry of PBMCs gated on CD4.sup.+ T cells of a representative healthy donor, a subject with active sarcoidosis and active SLE. Expression of CD45RA and FoxP3 (up) and of CD15s and FoxP3 (bottom). Numbers indicate % of eTreg cells defined by CD45RA.sup.FoxP3.sup.high (up) and by CD15s.sup.+FoxP3.sup.high (bottom) phenotypes. Doted vertical line represents the threshold for FoxP3 expression for the delineation between FoxP3.sup.low and FoxP3.sup.high defined by CD45RA expression. On the right panels, the thin vertical line represents the limit between FoxP3.sup.low and FoxP3.sup.high cells defined by the expression of CD15s.

[0141] (B) Comparisons of the % of eTreg cells defined by the expression of CD45RA and FoxP3 expression and by the expression of CD15s and FoxP3 in 8 healthy donors (top), 8 subjects with active sarcoidosis (middle) and 8 subjects with active SLE (bottom). Red lines and numbers represent mean %. Statistical comparisons were performed using a Student t-test. P<0.05 is considered significant.

[0142] Flow cytometry of PBMCs gated on CD4.sup.+ T cells of a subject with untreated mycosis fungoides analyzing the parameters described in (A).

[0143] FIG. 5 The absolute count of CD15s.sup.+ effector Treg cells is significantly decreased in active SLE when compared to healthy donors

[0144] Proportions among CD4.sup.+T cells (A) and absolute counts (B) of CD15s.sup.+effector Treg cells of the 8 healthy donors and the 8 active SLE subjects shown in FIG. 4 are compared using a non parametric Mann-Whitney U test. Mean values with standard errors are shown in red.

EXAMPLE

[0145] Material & Methods

[0146] Surface Marker Analysis of FoxP3, Ki-67 and Helios Expressing CD4.sup.+ T Cells and Flow Cytometry

[0147] Blood samples were obtained from young healthy adult volunteers and from active sarcoidosis or SLE. Diagnosis of SLE and sarcoidosis were made according to previously described criteria.sup.1, 2. The study was done according to the Helsinki declaration with the approval from the local human ethics committee (Comit Consultatif de Protection des Personnes dans la Recherche Biomdicale of Piti-Salptrire Hospital, Paris). For the analysis of thymocytes the approval by the Biomedecine agency (no PFS13-007) was obtained.

[0148] Human peripheral blood PBMC and human thymocytes were prepared by Ficoll gradient centrifugation and stained with anti-CD3, anti-CD8 anti-hCD4-PerCP-Cy5.5 or -APC, anti-hCD25-PE, anti-hCD45RA-PE-Cy7, anti-ICOS-, anti-HLA-DR (-PE from BD biosciences), anti-CD31 (-APC from e-bioscience), anti-hCD127 (-Pacific blue). Intracellular detection of FoxP3 with anti-hFoxP3 (PE or Alexa Fluor 647, clone 259D/A7, BD biosciences) and of Ki-67 antigen with Ki-67 antibody (FITC or PE from BD) was performed on fixed and permeabilized cells using Cytofix/Cytoperm (e-Bioscience). Most mAbs used for the study were derived from the Lyoplate system (BD). All mAbs for the cell surface marker screening were unconjugated and secondary stained.

[0149] Data were acquired by LSR-Fortessa or FACSCanto-II were analyzed with FloJo software.

[0150] Suppression Assay

[0151] PBMCs were isolated through Ficoll gradient separation from freshly drawn blood. CD4.sup.+T cells were first magnetically isolated using a CD4 T cell separation kit (Miltenyi) and subsequently surface stained using a combination of flurochrome-conjugated mAbs: anti-CD4-PErCP 5.5, anti-CD25-PE, anti-CD127-Pacific blue, anti-CD45RA-PECy7 and anti-CD15s-AF647 obtained from BD bioscience. CD127.sup.+CD25.sup.CD45RA.sup.+CD4.sup.+, nave FoxP3.sup.low CD45RA.sup.+, effector FoxP3.sup.highCD45RA.sup. Treg cells, CD127.sup.lowCD25.sup.+CD45RA.sup. were flow isolated from PBMCs following flow isolation according to the gating strategy we validated previously using a FACSAria (BD bioscience).sup.10 and CD15s.sup.FoxP3.sup.low CD4.sup.+ T cells and CD127.sup.lowCD25.sup.+CD127.sup.lowCD15s.sup.+CD4.sup.+T cells according to the gating strategy.

[0152] 110.sup.4 CFSE (1 M Invitrogen)-labeled responder CD25.sup.CD45RA.sup.+CD4.sup.+ T cells were cocultured with 110.sup.4 unlabeled cells assessed for their suppressive capacity and 110.sup.5 irradiated autologous accessory cells containing B cells and monocytes. Cells were stimulated with 0.5 g/mL plate-bound anti-CD3 (OKT3 mAb) in 96-well round-bottom plate in RPMI medium supplemented with 10% fetal bovin serum (Bio West), 2 mM L-glutamin, 1 mM sodium pyruvate, 1% non essential amino acid MEM, 100 U/mL penicillin, 100 g/ml streptomycin and amphotericin B (all from Gibco). Proliferation of CFSE-labeled cells was assessed by flow cytometry after 84-90 hr of culture.

[0153] nTreg Cells Expansion

[0154] Regarding nTreg cells expansion, 30*10{circumflex over ()}3 isolated nTreg were immediately distributed into U bottom well for culture. Cells were cultured in X-vivo 15 media, (Lonza) with 5% AB serum (Invitrogen Lifetech) and supplemented with 2 mM L-glutamin, 1 mM sodium pyruvate, 1% non essential amino acid MEM, 100 U/mL penicillin, 100 g/ml streptomycin and amphotericin B (all from Gibco), anti-CD3/anti-CD28 coated Treg expander beads (Invitrogen Lifetech), in the presence of 300 IU/mL IL-2 (Miltenyi Biotec) in culture media alone or in the presence of Rapamycin (Sigma-Aldrich) diluted in culture medium (1 microg/mL). 300 to 1000 IU/mL IL-2 was added every 3-4 days.

[0155] Statistical Analysis

[0156] To compare the % of eTreg with CD15s.sup.+FoxP3.sup.+ cells, paired t-test was performed. To compare the % of CD15s.sup.+ in healthy donors versus SLE subjects, non parametric U-Mann Whitney test was performed

[0157] Results

[0158] Cell Surface Markers for FoxP3, Helios and Ki-67 Expressing CD4.sup.+ T Cell Subsets

[0159] To determine specific surface markers that differentiate subsets within the FoxP3 expressing CD4.sup.+ T cells, we conducted a multiparameter cytofluorometric analysis of human CD4.sup.+ T cells by analyzing CD25, CD45RA, ICOS, HLA-DR, Ki-67, Helios and FoxP3 expression together with cell surface markers.

[0160] Extensive analysis of more than 340 cell surface markers indicated that 18 surface markers were highly prevalent on eTreg cells among which 6 have been already described (CD15s, CLA, HLA-DQ, CD30, CD66B, CD101, CD275 i.e. ICOS-L, CCR5, CCR6, CCR4, CD137, CD71 and already described CD25.sup.4, HLA-DR.sup.15, CD39.sup.17, 18, CD95.sup.19, CD147.sup.20, CD278 i.e. ICOS.sup.14; FIG. 1).

[0161] To assess which of those makers were preferentially expressed on FoxP3.sup.+CD4.sup.+ cells, we calculated the ratio of the proportion of positive cells among FoxP3.sup.+ cells (population A in FIG. 1b) to the proportion of positive cells among FoxP3.sup. cells among CD4.sup.+ T cells (population B in FIG. 1b) for each marker (ratio I). We defined that the markers that were the most specific for FoxP3.sup.+ cells were those with the highest ratio I.

[0162] We also verified whether such markers were preferentially expressed on eTreg cells or were also expressed on other CD45RA.sup.FoxP3.sup.low non Treg cells by calculating the ratio of the proportion of CD45RA.sup.FoxP3.sup.+ cells expressing the marker (population A in FIG. 1b) on the proportion of eTreg cells defined by the CD45RA.sup.FoxP3.sup.high phenotype among CD4.sup.+ T cells (ratio II). We considered that markers with ratio II superior to 1 were less specific for eTreg cells as they were also expressed by FoxP3.sup.low cells.

[0163] We observed that CD15s was the only marker with the highest ratio I. CD15s was also the only marker with a proportion of positive cells among FoxP3.sup.+ cells similar to eTreg cells proportions, i.e. ratio II close to 1 (FIG. 1b). These results indicate that CD15s is preferentially expressed on eTreg cells.

[0164] We also looked for markers that were downregulated only in eTreg cells. While 7 unknown markers were downregulated in eTregs (CD26, CD55, CD100, CD130, CD221, CD305, CD321), none of them was as discriminative as CD127.sup.21, 22.

[0165] Comparison of the expression of surface markers with the intracellular expression of proliferation marker Ki-67 and transcription factor Helios revealed that CD71 was highly correlated to the Ki-67 expression as well as ICOS and ICOS-L. This result indicates that ICOS is a good marker for proliferating eTreg cells. The only specific surface marker that corresponded to intracellular Helios expression was CD39 in some healthy donors but not all.

[0166] Because CD15s was highly expressed on FoxP3.sup.high eTreg cells but not on FoxP3.sup.low CD45RA.sup.FoxP3.sup.low cells and poorly expressed on FoxP3negative cells, we decided to focus our study on CD15s as a potential marker for eTreg cells.

[0167] CD15s is a Marker for Functional FoxP3.sup.high Treg Cells

[0168] We noticed that CD15S was highly expressed on eTreg cells while the non sialylated CD15 was not (FIG. 2A). CD15s is the result of the sialylation of CD15 through the fucosyltransferase 7 (alpha (1,3) fucosyltransferase). Our previous transcriptome.sup.10 analysis of CD4.sup.+T cells expressing FoxP3 indicates that this enzyme is specifically expressed on eTreg cells subsets compared to other CD4.sup.+ T cell subsets, indicating that sialylation of CD15 is specific for eTreg cells among CD4.sup.+ T cells (Microarray data available at the National Center for Biotechnology Information Gene Expression Omnibus (GEO) accession number 15659).

[0169] To verify that CD15s expression is indeed a marker that separates functional suppressive FoxP3 expressing CD45RA.sup. eTreg cells from non suppressive FoxP3 expressing CD45RA.sup. cells, we flow isolated CD15S.sup.+CD45RA.sup.CD127.sup.lowCD25.sup.++CD4.sup.+ T cells and CD15S.sup.CD45RA.sup.CD127.sup.lowCD25.sup.++CD4.sup.+ T cells. As shown in FIG. 2B, CD15s.sup.+FoxP3.sup.high cells were highly suppressive while CD15S.sup.FoxP3.sup.low cells did not suppress. This indicates that CD15s is a surface marker that enables the flow separation of eTreg cells from FoxP3.sup.low non Treg cells in human PBMCs.

[0170] We also verified that the inflammatory cytokine profiles of CD4+ T cells subsets defined according to CD15s expression was in agreement with cytokine profiles of corresponding subsets previously defined using FoxP3 and CD45RA markers.sup.8. As expected, CD45RAFoxP3+CD15s cells were among healthy but also SLE CD4+FoxP3+ cells the highest IFN- and IL-2 producers.

[0171] We had previously shown that the boundaries between FoxP3.sup.low cells (both nTreg cells and non Treg cells) and FoxP3.sup.high cells could be schematically defined by the vertical limit drawn by the expression of CD45RA and Ki-67 (red vertical line in FIG. 2C).sup.8. As shown in FIG. 2C, the boundaries in FoxP3 expression defined by the expression of CD15s are close to the ones defined by CD45RA and Ki-67, but at slightly lower levels. The use of CD15s as a marker for FoxP3.sup.high cells indicates the existence of a narrow zone of overlap in FoxP3 expression between CD15s.sup.+eTreg cells and CD15s.sup. non Treg cell (FIG. 2C).

[0172] Finally, because eTreg cells derive from nTreg cells in vivo, we also verified whether CD15s expression could be induced in expanding nave Treg cells in vitro in the presence of high dose IL-2 and rapamycin.sup.23, 24. While few expanding nTreg cells expressed CD15s after 7 days of culture, a significant proportion of expanding cells upregulated CD15s whereas CD15s was weakly expressed on expanding conventional CD4.sup.+ T cells (FIG. 2D). However a majority of expanding nTreg cells did not upregulate CD15s indicating that IL-2 alone and rapamycin may not be sufficient to convert all nTreg cells in efficient suppressive CD15s.sup.+ eTreg cells.

[0173] Expression of CD15s by FoxP3 Expressing Developing Treg Cells in the Thymus

[0174] Because nTreg cells that derive from the thymus do not express most markers present on eTreg cells i.e. CD25 at low levels and absence of ICOS while some developing thymic Treg cells have been reported to express these markers, we thought to determine how CD15s together with these aforementioned activation markers was expressed in the thymus by developing natural Treg cells.

[0175] As shown by others, thymic expression of FoxP3 begins at the double positive stage.sup.25. At this stage, developing double positive Treg cells display the FoxP3.sup.highCD25.sup.highICOS.sup.+CD15s.sup.+CD45RA.sup.CD31.sup. phenotype and start to proliferate as some of these cells are Ki-67.sup.+. At the single CD4 positive stage, we observe two subsets of FoxP3 expressing cells, one with high expression and another with low expression of FoxP3. FoxP3.sup.high CD4.sup.+ thymocytes are proliferating since they express Ki-67 and share similar surface markers with proliferating double positive FoxP3.sup.high cells as they also express ICOS, high levels of CD25 and CD15s.

[0176] FoxP3.sup.low single CD4.sup.+ thymocytes can be divided in CD45RA.sup.CD31.sup. cells, CD45RA.sup. CD31.sup.+ and CD45RA.sup.+CD31.sup.+ cells that do not express either Ki-67, ICOS or, CD15s and have low levels of CD25 (FIG. 3A). We can therefore postulate that activation markers present on eTreg cells in the periphery are transiently expressed in developing Treg cells in thymus at the double positive stage and when they differentiated into single positive CD4.sup.+ T cells. Such markers are downregulated in parallel with FoxP3 expression when the FoxP3.sup.+ thymocytes acquire the nave Treg phenotype and emigrates out of the thymus as CD45RA.sup.+CD31.sup.+FoxP3.sup.low CD4.sup.+ nave Treg cells (FIG. 3B).

[0177] Thus, the analysis of human thymocytes indicates that CD15s expression parallels the expression of other Treg related markers that are transiently expressed in the thymus and absent in nTreg emigrating from the thymus.

[0178] CD15s Expression in CD4.sup.+T Cell Subsets in Canonical Diseases with FoxP3 Expressing Cell Subset Abnormalities

[0179] Finally we studied the expression of CD15s in PBMCS of healthy donors (n=8) and of subjects with diseases known for the prevalence of abnormalities in FoxP3 expressing subsets i.e. sarcoidosis (n=8) and systemic lupus erythematosus (n=8).

[0180] In both diseases, eTreg cells could be identified using CD15s and separated from FoxP3.sup.low non Treg cells (FIG. 4A). Of note, we also observed, especially in SLE, that the zone of overlap in FoxP3 expression between CD15s.sup.+eTreg cells and CD15s.sup. non Treg cells was wider. The proportion of eTreg cells measured using CD45RA and/or Ki-67 was slightly but significantly underestimated while eTreg in sarcoidosis were, yet augmented, overestimated (FIG. 4B). While the proportion of CD15s.sup.+Treg cells was not significantly different in active SLE when compared to healthy donors, the absolute counts was significantly lower, which is consistent with our previous observations.sup.2, 10, 26 (FIG. 5). This further indicates the necessity to add CD15s in the analysis of FoxP3 expressing CD4.sup.+ subsets in order to distinguish eTreg cells from non Treg cells efficiently especially in diseases with reported increase in FoxP3 expressing CD4.sup.+ T cells such as cancer.

[0181] We thus applied this strategy when analyzing the PBMCs of a subject with untreated mycosis fungoides which is known to display expanded circulating Treg cells.sup.27, 28. As shown in FIG. 4C, an increase in FoxP3 expressing CD4.sup.+ T cells with very few nTreg cells was observed. The use of CD15s as a marker for eTreg cells confirmed that eTreg cells were indeed highly increased and identifiable as a discreet population.

[0182] The combination of low expression of CD127 and high expression of CD25 in CD4+ T cells is well accepted as a surrogate to define FoxP3 expressing CD4+ T cells in humans.sup.19, 20. Of note, in SLE, Sjgren syndrome, systemic sclerosis, myasthenia gravis or sarcoidosis, the combination of CD127 and CD25 did not efficiently separate FoxP3+ from FoxP3 cells as a significant proportion of each FoxP3+ cells i.e. nave Treg cells, Foxp3low CD15s non Treg cells and CD15s+ eTreg cells resided is either CD127high or CD25 cells. This indicates that the combination of low expression of CD127 and high expression of CD25 may not be suited for the identification of FoxP3+ cell in autoimmune and/or inflammatory conditions in humans

[0183] Discussion:

[0184] Because human FoxP3 expressing CD4.sup.+ T cells are heterogeneous in function.sup.4, markers that enable their separation into suppressive Treg cells and effector T cells that express FoxP3, are required, especially in immune mediated diseases such as autoimmune and/or inflammatory/inflammatory diseases, transplantation and cancer. We had previously shown that FoxP3 expressing CD4.sup.+ T cells could be separated into 3 subsets based on their levels of intracellular FoxP3 and expression of CD45RA.sup.10. It is now well accepted that CD4.sup.+ T cells with low expression of FoxP3 bearing a nave phenotype are a distinct subset of Treg cells that derive from the thymus corresponding to murine natural thymic derived Treg cells. These cells can be simply flow isolated as CD45RA.sup.+CD25.sup.+CD4.sup.+ T cells.sup.10.

[0185] Regarding CD45RA.sup.FoxP3 expressing cells, FoxP3.sup.high cells are highly suppressive while CD4.sup.+ T cells with low expression of FoxP3 do not suppress. The latter probably correspond to conventional T cells with activation induced expression of FoxP3, although not high enough to acquire a suppressive function.sup.29, 30.

[0186] FoxP3.sup.high cells encompass Treg cells expressing HLA-DR and/or ICOS. Both markers have been shown to characterize subsets of Treg cells that are highly and rapidly suppressive.sup.15 and that produce IL-10 respectivelyl.sup.4. More recently, Shevach's group has proposed that transcription factor Helios was a marker for natural Treg cells.sup.16. However recent findings have demonstrated that CD4.sup.+ T cells with induced expression of FoxP3 and other activated CD4.sup.+ T cells could also express Helios.sup.31-33.

[0187] Because it was unclear whether the FoxP3.sup.high effector Treg cells could be defined as a discreet population with a single specific surface marker, we decided to analyze all known surface markers for which antibodies for flow cytometry were available.

[0188] The study of more than 340 surface markers allowed us to define 18 markers highly upregulated by eTreg cells among which 6 markers (CD25, HLA-DR, CD39, CD95, CD147, and ICOS).sup.4, 14, 15, 17-20 have been already described. 7 other surface molecules were downregulated but none of them was better than CD127 to define FoxP3.sup.+ cells.

[0189] ICOS was highly correlated with the expression of Ki-67, indicating that ICOS is rather a marker for proliferating eTreg cells than a marker for a particular population. Together with Ito's report, this result indicates that proliferating ICOS.sup.+Ki-67.sup.+ eTreg cells produce IL-10.sup.14. We failed to determine a specific surface marker for Helios except CD39 which was highly correlated with Helios in some donors but not all. This result indicates that Helios.sup.+ and CD39.sup.+Treg cells are probably the same subset within the eTreg cells.

[0190] We finally found that CD15s was the best candidate as a putative specific marker for eTregs as CD15s was highly expressed on FoxP3.sup.high cells but not on both nTreg cells and FoxP3.sup.lowCD45RA.sup.CD4.sup.+ T cells. We could confirm that CD15s.sup.+FoxP3.sup.+(high) cells were highly suppressive while CD15s.sup.FoxP3.sup.+(low) were not conferring to this marker a high discriminative power. We therefore describe here for the first time a marker that is highly specific for eTreg cells.

[0191] CD15s is the 2-3 sialosylated form of lacto-N-fucopentaose III (CD15), also known as sialyl Lewisx (sLEx) which is a tetrasaccharide carbohydrate.sup.34. The glucidic nature of this antigen explains why previous attempts to determine specific surface markers through transcriptome analysis failed to identify this molecule.sup.10. However, careful retrospective analysis of our previous transcriptome analysis indicated that fucosyltransferase 7 (alpha (1,3) fucosyltransferase), which is required for the synthesis of CD15s from CD15.sup.35, was indeed specifically upregulated in the eTreg subset.

[0192] We also analyzed the expression of CD15s together with other known activation markers born by peripheral eTreg cells in Treg cells developing in the thymus. We could establish a stepwise differentiation dynamic for thymic developing Treg cells by analyzing the levels of FoxP3, Ki-67, CD45RA and CD31 in double positive and single CD4.sup.+ positive FoxP3.sup.+ thymocytes. We could confirm that FoxP3 expression was initiated at a DP stage at high levels together with the expression of ICOS and CD15s.sup.14. Both markers were not present in SP CD4.sup.+ CD31.sup.+FoxP3.sup.low cells indicating that both markers are transiently expressed in the thymus and are not present in thymus emigrating cells.

[0193] Finally, we confirmed that CD15s was a relevant marker for eTreg cells in diseases. We observed that the prevalence of eTreg cells using the definition of Treg subsets based on the expression of FoxP3, CD45RA and Ki-67 was unmodified in healthy donors and slightly overestimated in sarcoidosis' and slightly underestimated in SLE.sup.2 when using CD15s as an additional marker for eTreg cells. Of note, the abnormalities observed in FoxP3 expressing CD4.sup.+ T cell subsets were still present and remarkable using this marker. Finally, we applied this new definition to the PBMCs of a subject with mild untreated mycosis fungoides.sup.27, 28 and could observe a clear distinct population of CD15s.sup.+FoxP3.sup.high eTreg cells.

[0194] We therefore propose that CD15s be added in any phenotypic flow analysis of FoxP3 expressing CD4.sup.+ T cells subsets in human studies in addition to, at least, CD45RA and to CD25, CD127 and CD45RA for flow separation of FoxP3 expressing subsets for functional analysis.

[0195] Because CD15s is upregulated on expanding nTreg cells, we also propose CD15s as a quality control marker to assess the purity of expanding Treg cells in vitro. Indeed, using IL-2 and rapamycin, CD15s was present in only 20-30% of proliferating nTreg cells indicating that IL-2 and ramycin alone may not be optimal to obtain highly pure FoxP3.sup.high eTreg cells upon culture.sup.36.

[0196] Because we flow isolated CD15s.sup.+ eTreg cells using antibodies that specifically recognize CD15s and not CD15, it is highly improbable that CD15s itself participates in the in vitro and the in vivo contact dependent Treg mediated suppression.sup.37. However, because CD15s is a ligand for selectins and is involved in the cellular interaction with endothelial cells in order to promote the migration from peripheral blood into the tissues.sup.38, CD15s is probably involved in the transmigration of eTreg cells in target tissues, thus participating in their suppressive function in vivo. CD15s is also highly expressed on monocytes and some myeloid precursors.sup.39. This may compromise the use of monoclonal antibodies aiming the neutralization of the transmigration of eTreg cells into tissues in order to promote effector immune responses. Better understanding of the molecular mechanisms of the upregulation of CD15s in eTreg may help provide new therapeutic targets in enhancing or neutralizing suppressing effects of eTreg cells.

[0197] In conclusion, we have demonstrated that CD15s was a marker for eTreg cells that confirms the relevance of the previously proposed classification for FoxP3 expressing CD4+T cell subsets. We therefore recommend the use of CD15s in addition to CD45RA for the phenotypic and functional analysis of human FoxP3 expressing CD4.sup.+ T cells.

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