USE OF PD-1 AND TIM-3 AS A MEASURE FOR CD8+ CELLS IN PREDICTING AND TREATING RENAL CELL CARCINOMA

Abstract

The present invention relates to a method for predicting the survival time of a subject suffering from renal cell carcinoma comprising the steps of: i) quantifying the percent of CD8+ T cells co-expressing PD-1 and Tim-3 in a tumor tissue sample obtained from the subject, ii) comparing the percent quantified at step i), with its corresponding predetermined reference value and iii) concluding that the subject will have a short survival time when the percent of CD8+ T cells co-expressing PD-1 and Tim-3 is higher than its corresponding predetermined reference value or concluding that the subject will have a long survival time when the percent of CD8+ T cells co-expressing PD-1 and Tim-3 is lower than its corresponding predetermined reference value.

Claims

1. A method for predicting the survival time of and treating a subject suffering from renal cell carcinoma comprising the steps of: i) quantifying the percent of CD8+ T cells co-expressing PD-1 and Tim-3 in a tumor tissue sample obtained from the subject, ii) comparing the percent quantified at step i) with its corresponding predetermined reference value iii) concluding that the subject will have a short survival time when the percent of CD8+ T cells co-expressing PD-1 and Tim-3 is higher than its corresponding predetermined reference value or concluding that the subject will have a long survival time when the percent of CD8+ T cells co-expressing PD-1 and Tim-3 is lower than its corresponding predetermined reference value; and iv) administering to a subject with a short survival time a therapeutically effective amount of a treatment that prolongs the survival time of the subject beyond that expected in the absence of such treatment.

2. The method of claim 1 wherein, the quantification of percent of CD8+ T cells co-expressing PD-1 and Tim-3 is determined by Immunohistochemistry (IHC).

3. The method of claim 1 wherein, the quantification of percent of CD8+ T cells co-expressing PD-1 and Tim-3 is determined by an automatized microscope.

4. A method for determining whether a subject suffering from a renal cell carcinoma will achieve a response with an immune-checkpoint inhibitor and treatment of the subject comprising the steps of i) quantifying the percent of CD8+ T cells co-expressing PD-1 and Tim-3 in a tumor tissue sample obtained from the subject treated with an immune-checkpoint inhibitor, ii) comparing the percent CD8+ T cells co-expressing PD-1 and Tim-3 quantified at step i) with its corresponding predetermined reference values and iii) concluding that the subject will not respond to the treatment when the percent of CD8+ T cells co-expressing PD-1 and Tim-3 is higher than its corresponding predetermined reference value or concluding that the subject will respond to the treatment when the percent of CD8+ T cells co-expressing PD-1 and Tim-3 is lower than its corresponding predetermined reference value, and, iii) administrating a therapeutically effective amount of a combination of immune checkpoint inhibitors if the subject is identified as a non-responder.

5. The method of claim 4, wherein the immune checkpoint inhibitor is an antibody.

6. The method of claim 4, wherein the immune checkpoint inhibitor is a monoclonal antibody.

7. The method of claim 4, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

8. The method of claim 4, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

9. The method of claim 4, wherein the immune checkpoint inhibitor is an anti-PD-L2 antibody.

10. The method of claim 4, wherein the immune checkpoint inhibitor is an anti-Tim-3 antibody.

11. The method of claim 4, wherein the immune checkpoint inhibitor is a small organic molecule.

12. The method of claim 4, wherein the immune checkpoint inhibitor is an aptamer.

13. (canceled)

Description

FIGURES

[0078] FIG. 1: Relationships between the co-expression of PD-1 and Tim-3 on CD8+T cells and clinical outcome. A: RCC patients were divided into two groups depending on their percent of co-expression of PD-1 and Tim-3 on CD8+T cells above or below the median (34.7). Kaplan Meier curves for the progression free survival for the two groups of patients are shown. B: The relationship between the percent of co-expression of PD-1 and Tim-3 on CD8+T cells selected as a quantitative variable and the 36-month survival is shown. The blue line corresponds to this relationship, whereas the red line represents the upper or lower limits of the 95% CI. Blue squares on the top indicate that corresponding patients are alive, while blue squares on the bottom correspond to patient death.

[0079] FIG. 2: Co-expression of PD-1+Tim-3+ on CD8+T cells correlates with high levels of PD-1 A: Mean fluorescence intensity (MFI) of PD-1 measured by cytometry on PD-1+Tim-3+ and PD-1+Tim-3 cells gated on CD8+T cells for one representative RCC patient. The same analysis is shown for 16 patients selected as they coexpress PD-1 and Tim-3 at least in 10% of the total CD8+T cell population. B: Example of the intensity of PD-1 detected at the cellular levels by Immunofluorescence in situ analysis on PD-1+Tim-3+ and PD-1+Tim-3neg CD8+T cells (left) and at individual levels after integrating the various cell signal on a tissue section. Comparative analysis of the mean PD-1 intensity measure by in situ Immunofluorescence on the 2 CD8+T cell subsets (PD-1+Tim-3 and PD-1+Tim-3+) in a series of patients for whom both tissue sections and TIL were available.

[0080] FIG. 3: Functional analysis of TIL depending on their expression of PD-1 alone or combined with Tim-3. Cells collected after sorting were activated or not by anti-CD3 and anti-CD28 for 24 hours and then IFN was measured by Elisa in the supernatant. *p<0.01

[0081] FIG. 4: Co-expression of PD-1 and Tim-3 on CD8+T cells correlate with clinical parameters of RCC aggressivity The percent of PD-1+Tim-3+ on CD8+T cells selected as a continuous variable and measured by in situ imunofluorescence technique was plotted against various clinical parameters defined as a binary (TNM, Fuhrman grade, UISS score) or a continuous variable (tumor size). TNM was divided in two groups : localized disease (pT1 and pT2) and advanced disease (pT3, pT4, N+or M+). The Fuhrman grade was defined as low (grade I or II) and high (grade III or IV) and the UISS score into 3 classes (0, 1, 2).

[0082] FIG. 5: Patients who co-express PD-1 and Tim-3 on CD8+T cells more often exhibited a clear cell RCC histology The percent of PD-1+Tim-3 and PD-1+Tim-3+ on CD8+T cells selected as a continuous variable and measured by cytometry were plotted against the histological group of RCC patients divided into two classes: Clear cell histology or non-clear cell histology (papillary, chormophobe, medullary carcinoma, oncocytoma). Box and whisker plot analysis is shown.

[0083] FIG. 6: Expression of Tim-3 is decreased on PD-1+CD8+T cells after treatment with collagenase A: The percentage of PD-1+Tim-3+/CD8+T cells was determined by in situ immunofluorescence and cytometry: in a serie of 10 RCC patients for whom both frozen sections and fresh TIL were available. B: MFI of Tim-3 on PD-1+CD8+T cells measured by cytometry for one RCC patient whose TIL were pretreated with collagenase or cell recovery solution. Integrated data for 4 TIL derived from RCC patients. C: Four PBMC were activated by anti-CD3 and anti-CD28 for 48 hours. Then, the PBMC were treated or not with collagenase or a cell recovery solution and the MFI of Tim-3 on PD-1+CD8+T cells were measured by cytometry.

EXAMPLE

Introduction

[0084] Immunotherapy based on the inhibition of checkpoint inhibitors (CTLA-4, PD-1) expressed on T cells has demonstrated their clinical efficacy in various phase 3 clinical trials in metastatic melanoma, non small cell lung carcinoma (NSCLC) and renal cell carcinoma (RCC) (1). Overall, this novel therapeutic approach leads to about 30% clinical responses in cancer patients (2). Pre-existing anti-tumor CD8+T cells seems to be required for the success of PD-1-PD-L1/2 blockade in cancer patients (3). Various arguments suggest that co-expression of inhibitory receptors (PD-1, CTLA-4, Tim-3, Lag3 . . . ) on CD8+T cells may represent a clue to explain resistance mechanisms to checkpoint inhibitors blockade. Indeed co-expression of distinct inhibitory receptors was associated with greater T cell exhaustion and resistance to the ability of anti-PD-1/PD-L1 antibodies to revigorate these dysfunctional T cells in both infections and cancer (4-7).

[0085] Up until now the co-expression of inhibitory receptors has mainly been performed on fresh tumor cells by multiparametric cytometric analysis which precludes the determination of the prognostic significance of this parameter on large cohort of patients or in retrospective studies. To overcome this hurdle, we have developed multiparametric in situ immunofluorescence analysis with multispectral imaging. The use of software for computing the pure spectrum of a fluorophore from a mixed emission signals combined with automated image analysis avoids the usual risk of overlapping signals from various fluorophores and the variation of manual counting between different operators. In human, RCC represent a good model to analyze the clinical significance of this coexpression, as some previous studies already detected inhibitory receptors (PD-1, Lag3, PDL1, PD-L2) endowed with bad prognostic value in this cancer (8, 9). We focus on the expression of PD-1 and Tim-3 on CD8+T cells, as in murine acute myelogenous leukemia, human melanoma and NSCLC the co-expression of PD-1 with Tim-3 has been shown to correlate with T cell dysfunction (4, 10, 11). Secondary to VHL gene inactivation in most RCC with clear cell histology, there is an overexpression of VEGF explaining that RCC is a highly vascular cancer. We recently showed that VEGF induced the expression of PD-1 and Tim-3 on CD8+T cells (12). Lastly, TCGA data base reported a high expression of Tim-3 in kidney renal clear cell carcinoma (13), but since Tim-3 could also be expressed by many cells including tumor, myeloid and endothelial cells, only multiparametric in situ immunofluorescence analysis will permit to define its role and clinical significance, when expressed on CD8+T cells. The aim of this study was to evaluate the biological significance and clinical impact of the co-expression of Tim-3 on intratumoral PD-1+CD8+T cells in RCC patients.

Material & methods

[0086] Patient Cohorts

[0087] Two independent cohorts of RCC patients who underwent a nephrectomy at the Urology department of European Georges Pompidou Hospital were included in this study. One of them was a prospective cohort of 42 patients enrolled between February 2012 and November 2015. All histological cancer types were included in this cohort except kystic lesions.

[0088] A second independent cohort of 87 patients who underwent surgery for a renal carcinoma between April 1999 and June 2005 was retrospectively selected from the biobank of Necker hospital for multiparametric immunofluorescence in situ analysis. These samples were directly frozen after nephrectomy and histologic assessment and only included clear cell carcinoma. Only non-treated patients harboring a clear renal cell carcinoma of were included in the two cohorts. Patient characteristics of the two cohorts are reported in Table S1 and S2. This research was conductd protocol was approved by the local ethics committee (CPP Ile de France no 2012-05-04)

[0089] Immunophenotyping by Cytometry Analysis

[0090] Immunofluorescence staining and flow cytometry analysis of TILs were conducted as previously described (14). Briefly, after dissociation of biopsies by DNAse I (30 IU/mL, Roche) and Collagenase D (1 mg/mL, Roche) for 60 min, cells were stained with a fixable viability Dye FVS 520 (eBioscience, Paris 75006, France), BV510 labeled anti-CD3 (BD Biosciences, Pont de Claix 38801, France), PE labeled anti-CD8 (BD Biosciences), BV421 labeled anti-PD-1 (BD Biosciences) and APC labeled anti-Tim-3 (Biolegend/Ozyme Saint Quentin Yvelines 78053 France). For the analysis, cells were gated on viable singulet positive CD3+T cells. Isotype control antibodies were included in each experiment. Detailed description of the antibodies used for the cytometry analysis are described in table S3.

[0091] In Situ TILs Immunofluorescence Staining

[0092] Tissue samples obtained at the day of nephrectomy were frozen and stored at 80 C. The quality of the sample was checked by an H&E stained section. Frozen specimens were sectioned at 4 to 6 m with a cryostat, placed on slids, air dried and fixed for 5 minutes with 100% acetone. Before incubation with antibodies, the slides were pretreated with avidin/biotin blocker (DAKO) for 10 minutes and Fc receptors were blocked with Donkey serum (DAKO) 5% in TBS for 30 minutes. Staining for CD8, PD-1 and Tim3 was performed using non labeled primary antibodies followed by fluorophore labeled secondary antibodies. The antibodies used for the various immunofluorescence stainings are described in the table S3. Isotype matched antibodies were used as negative controls. In each case, we checked that the secondary antibodies did not cross react with an unrelated primary antibodies used in the combination. Nuclei were highlighted using DAPI mounting medium.

[0093] Fluorescence Analysis and Automatized Cell Count

[0094] Slides of stained renal sections were read with an automatized microscope VectraR. This Perkin ElmerR technology allows measurement of morphometric and fluorescence characteristics in the different cell compartments (membrane/cytoplasm/nuclei). Coupled with an Inform software the system allows multiplex staining protocol. As recommended for the multiplex analysis, single-stained (Cyanine 5 or Cyanine 3 or Alexa Fluor 488) and non-stained slides were analyzed in Inform in order to integrate the corresponding spectrums in a fluo library.

[0095] For each slide (1 patient), image acquisition and subsequent count were done on at least 5 fields. Stained slides were visually examined by a pathologist before the analysis. The analysis of immunofluorescence labelling of the cells was performed using the Inform software. Briefly, for cell recognition, a cell segmentation was done based on the DAPI staining and the size. Then a sampling of 1 (under 5) image per patient was carefully examined for the phenotyping step (refer to FIG. 1). Cells mono-stained for CD8 (blue dot) or co-stained for PD1 and CD8 (red dot) or PD-1, Tim-3 and CD8 (green dot) were manually identified until the automatized recognition by the Inform software was concordant with visual count (error <5%). Each images of phenotyping were checked after the software analysis.

[0096] Cell Sorting and T Cell Activation

[0097] Fresh tumor infiltrating lymphocytes obtained after DNAse/collagenase digestion were stained with anti-CD3, anti-CD8, anti-PD-1 and anti-Tim-3 and were sorted into three populations PD-1+Tim-3+CD8+, PD-1+Tim-3CD8+, PD1Tim-3CD8+ using a FACS-ARIA sorter (BD Biosciences). Recovered T cell were incubated for 24 hours with medium or stimulated with an anti-CD3-anti-CD28 T cell activation kit (Miltenyi). IFN was measured by Elisa (Diaclone) in the supernatants collected 24 hours after T cell activation or not.

TABLE-US-00001 TABLE 1 Correlation between the expression of PD - 1 alone or combined with Tim - 3 on CD8+T cells and clinical prognostic parameters of RCC patients. The percent of PD - 1+, PD - 1 + Tim - 3+ or PD - 1 + Tim - 3 on CD8+T cells selected as a continuous variable measured by either in situ imunofluorescence technique (IF) or cytometry (Cytm) was correlated against various clinical parameters defined as a binary (TNM, Fuhrman grade, UISS score) or a continuous variable (tumor size). TNM was divided in two groups: localized disease (pT1 and pT2) and advanced disease (pT3, pT4, N+ or M+). The Fuhrman grade was defined as low (grade I or II) and high (grade III or IV) and the UISS score into 3 classes (0, 1, 2). The p values for significant correlation are in bold. % PD-1/CD8 % PD-1/CD8 % PD1.sup.+Tim3.sup.+/CD8 % PD1.sup.+Tim-3.sup.+/CD8 % PD-1.sup.+Tim-3.sup./ % PD-1.sup.+Tim-3.sup./CD8 (IF) (Cytm) (IF) (Cytm) (IF) (Cytm) TNM 0.04 0.28 0.003 0.047 0.22 0.77 Furhman 0.01 0.25 0.004 0.58 0.74 0.33 Grade Tumor Size 0.08 0.22 0.01 0.02 0.37 0.39 (mm) UISS 0.01 0.01 0.01 0.049 0.63 0.2 Score

TABLE-US-00002 TABLE S1 Baseline clinical characteristics of primary clear cell RCC patient selected for multiparametric immunofluorescence insitu analysis. Characteristics Number of patients (%) Sex Male 60 (69%) Female 27 (31%) Median Age (Years) 76 (46-101) pTNM pT1-T2 (localized disease) pT1 60 (69%) pT2 3 (3%) pT3-T4-N+M + (advanced disease) pT3 24 (28%) pT4 0 N+ 0 M+ 0 Sarcomatoid component 11 (13%) Tumor Size (major Axis (mm)) Median 4, mean 4.7 (10-120) Fuhrman Grade Grade I and II (low) Grade I 0 (0%) Grade II 25 (29%) Grade III and IV (high) Grade III 41 (47%) Grade IV 21 (24%) UISS Score Low 22 (25.3%) Intermediate 62 (71.3%) High 3 (3.4%)

TABLE-US-00003 TABLE S2 Baseline clinical characteristics of primary RCC patient with analysis of fresh tumor TIL. The Fuhrman grade could only be assessed for clear cell and papillary histology type. Missing data for 1 patients for Fuhrman grade and 3 for UISS score. Characteristics Number of patients (%) Sex Male 27 (64.3) Female 15 (35.7) Median Age (Years) 56 (28-81) pTNM pT1-T2 (localized disease) 24 (57.15) pT1 17 (40.5) pT2 7 (16.65) pT3-T4-N.sup.+ M.sup.+ (advanced disease) 18 (42.85) pT3 18 pT4 0 N+ 2 (1) M+ 1 (0.05) Histology Type Clear Cell 27 (64.3) Papillary 7 (16.7) Chromophobe 6 (14.2) Others (medullary carcinoma 2 (4.8) and oncocytoma) Sarcomatoid component 1 (2.4) Tumor Size (major Axis (mm)) 60 (20-110) Fuhrman Grade Grade I and II (low) 11 (33.33) Grade I 0 Grade II 11 (33.33) Grade III and IV (high) 22 (66.66) Grade III 15 (45.45) Grade IV 7 (21.2) UISS Score Low 6 Intermediate 27 High 0

TABLE-US-00004 TABLE S3 List of antibodies used for in situ immunofluorescence staining and cytometry Primary antibodies Secondary antibodies Revelation Cytometry CD3-CD8-PD-1-Tim-3 BV510 conjugated anti-CD3 (Clone UCHT1)(BD Biosciences) PE conjugated Anti-CD8 (Clone RPA-T8) (BD Biosciences) BV421 conjugated anti-PD-1 Clone MIH4) (BD Biosciences) APC conjugated anti-Tim-3 (Clone F38- 2E2)(Biolegend) In situ Immunofluorescence analysis CD8-PD-1-Tim-3 Rabbit anti-CD8 (Clone P17-V)(Novus) Cyan 5 conjugated donkey anti- Cy3 labeled rabbit (Jackson Immunoresearch) streptavidin (Amesham) Mouse anti-PD-1 (Clone NAT)(Abcam) Biotynalted F(ab2) donkey anti- mouse IgG (Jackson Immunoresearch Goat anti-Tim-3 (R&D) Alexa Fluor R 488 conjugated donkey anti-goat IgG (Abcam)

TABLE-US-00005 TABLE S4 Correlation between the total number (Nb) of CD8+T cells and those expressing Tim-3 and/or PD-1. Nb Nb Nb Nb CD8.sup.+T PD-1.sup.+CD8.sup.+T PD-1.sup.+Tim-3.sup.+CDB.sup.+T PD1.sup.+Tim3.sup.CD8.sup.+T cell cell cell cell Furhman 0.029 0.0055 0.0024 0.1 UISS 0.17 0.0229 0.012 0.17

Results

[0098] 1) Detection and Characterization of CD8+T Cells Co-Expressing or Not PD-1 and Tim-3 by Automated in Situ Immunofluorescence Spectral Imaging.

[0099] To characterize the CD8+T cells infiltrating renal cell carcinoma and expressing PD-1 and Tim-3, we set up a multifluorescence in situ technique with automated counting. We first showed that about half of CD8+T cell express PD-1 (mean 53.9%; SE: 30.49%). This population could be divided into two groups: i) one corresponding to double positive PD-1+Tim-3+ within CD8+T cells with a mean percent of 38.16% (SE: 28.11%) i) a second population of CD8+T cells expressing PD-1 without Tim-3 (mean: 15.77% ; SE: 8.62%). Thus within tumor microenvironment, most PD-1+CD8+T cell coexpressed Tim-3, whereas

[0100] Tim-3 without PD-1 was detected in less than 3% of CD8+T cells (data not shown). The mean number of total CD8+T cells, PD-1+CD8+T cells, PD-1+Tim-3+ and PD-1+Tim-3 negative CD8+T cells were 116.5 (SE: 216), 89.32 (SE: 191.8), 66.9 (SE: 143), 22.38 (SE: 57.5) respectively. As expected we also observed the expression of Tim-3 on non CD8+T cells.

[0101] 2) Clinical Significance of the Co-Expression of PD-1 and Tim-3 on CD8+T Cells in Situ.

[0102] Various criteria (TNM, Fuhrman grade, size of the tumor, UISS score) have been proposed to define the prognostic value of primary RCC. The percent or the number of intratumor CD8+T cells expressing PD-1 without Tim-3 did not correlate with any criteria of aggressivity defined above (Table 1 and Table S4). In contrast, a positive relationship was observed between the percent of intratumor CD8+T cells expressing PD-1 (i.e. Tim-3+ or Tim-3neg) or co-expressing PD-1 and Tim-3 and the TNM stage, the Fuhrman grade and the UISS score (Table 1). Coexpression of PD-1 and Tim-3 was also associated with a larger tumor size. In addition, the number of tumor infiltrating CD8+T cells expressing PD-1 or co-expressing PD-1 and Tim-3 correlated with the Fuhrman grade and the UISS score (Table S4). In line with this more pejorative phenotype, RCC patients whose CD8+T cells co-express PD-1 and Tim-3 above the median (34.7) are more likely to relapse (p=0.046 ; HR 2.9; 95% confidence interval (CI): 1.02-8.21) (FIG. 1A). A correlation was also shown between the percent of CD8+T cells coexpressing PD-1 and Tim-3 and 36 months overall survival (FIG. 1B). This same group of patients had also a trend toward a poorer overall survival when median was selected as a cut-off (p=0.079; HR 2.16; 95% CI: 0.91-5.1). When all other CD8 subsets were set up as a continuous variable or binary variable defined by the median, no significant statistical correlation were found with progression free survival (PFS) or overall survival (OS). Only the percent of the co-expression of PD-1 and Tim-3 on CD8+T cells had an impact on the clinical outcome of patients. In a multi-variate model including the percent of the double positive PD-1+Tim-3+CD8+T cells, the TNM classification and the size of the tumor, only the size of the tumor remains significantly associated with the PFS (p=0.0099).

[0103] 3) Assessment of the Co-Expression of PD-1 and Tim-3 on CD8+T Cells by Cytometry and its Clinical Significance.

[0104] To validate these results, we measure the expression of PD-1 and Tim-3 on CD8+T cell by cytometry in a series of 42 fresh tumors derived from RCC patients. As previously observed with the multiparametric immunofluorescence in situ technique, about half of CD8+T cells express PD-1 (mean 50.8+20.76) and none CD8+T cells express Tim-3 without PD-1. In the whole population, 15% of CD8+T cells co-express PD-1 and Tim-3 and this percent increases to 17.42% in the restricted CRCC patients. Although, the two series of patients were independent, we were surprised that the percent of PD-1 expression on CD8+T cells fit with the two techniques but not those of Tim-3 expression. We first confirmed that this difference was not secondary to the independent series of patients tested. Indeed, FIG. 6A showed that for the same group of 10 RCC patients for whom both TIL and frozen section were available, the percent of PD-1+Tim-3+ on CD8+T cells was higher when detected by in situ immunofluorescence technique than by cytometry (p=0.049). We further explain this discordance by showing that TIL treated by collagenase have a decrease in the expression of Tim-3 compared to the use of mechanical method of dissociation (p=0.028) (FIG. 6B). Unfortunately, this mechanical method has a low yield of recovered cells compared to the use of collagenase (data not shown). The expression of Tim-3 on activated PBMC were also diminished, when they were treated by collagenase compared to the mechanical dissociation exposure or the absence of treatment (p=0.029) (FIG. 6). Interestingly, collagenase did not affect the PD-1 expression of CD8+T cells explaining the concordance for this marker between these two techniques. These results support the clinical value of in situ immunofluorescence multiparametric technique to avoid this kind of bias. Regarding the clinical significance of results obtained by cytometry, we confirmed that the percent of CD8+T cells expressing PD-1 without Tim-3 by cytometry analysis did not correlate with any prognostic criteria (TNM, Fuhrman grade, size of the tumor, UISS score), while patients whose CD8+T cells co-express PD-1 and Tim-3 had a more advanced TNM stage (p=0.021), a tumor with a larger size (p=0.021) and a higher UISS score (p=0.049). Interestingly, the co-expression of Tim-3 and PD-1 on CD8+T cells was higher in the group patients with a CRCC histology which is considered as a more aggressive cancer than the chromophobe or tubulo-papillary or oncocytoma subgroups (FIG. 5). Since the cytometry study was a prospective analysis, the follow up was not long enough to assess the PFS and the OS of this group of patients. So except for the absolute percent of the co-expression of PD-1 and Tim-3 on CD8+T cells, the other parameters measured and their clinical significance were very similar between the two series.

[0105] 4) Phenotypic and Functional Characterization of the Population of CD8+T Cells Co-Expressing PD-1 and Tim-3

[0106] Since it has been shown that the levels of PD-1 on T cells correlated with the exhaustion of T cells defined by multiple expression of inhibitory receptors (15), we quantitated in a series of 16 patients the expression of PD-1 on CD8+T cells co-expressing or not Tim-3 by cytometry. Patients were selected based on the coexpression of PD-1 and Tim-3 at least of 10% of the total CD8+T cell population. In the patient shown in FIG. 2A, we observed an increased expression of the mean fluorescence intensity (MFI) of PD-1, when it co-expressed Tim-3 (MFI: 10.4) compared to its expression alone (5.88). For the whole population, the MFI of PD-1 on CD8+T cells co-expressing Tim-3 (mean 19.13+8.8) was also higher than the MFI observed on CD8+T cells not expressing Tim-3 (mean 11.58+5.2) (p=0.0063) (FIG. 2A). This result was confirmed when in situ immunofluorescence analysis was performed and the intensity levels of PD-1 compared, when it was co-expressed or not with Tim-3 on CD8+T cells. Indeed, in the absence of Tim-3, the mean of PD-1 fluorescence intensity was measured at 0.31 (+0.15) and it increased to 0.489 (+0.19), when it was co-expressed with Tim-3 (FIG. 2B) (p=0.0418). Thus by using two different techniques we demonstrated that the co-expression of PD-1 and Tim-3 was associated with an increased levels of PD-1 on CD8+T cells. To determine the putative difference in term of functionality between the PD-1+Tim-3+CD8+T cells and the PD-1+Tim-3negCD8+T cell population, we selected two patients which coexpress PD-1 and Tim-3 and sorted the three following populations: PD-1negTim-3negCD8+T cells, PD-1+Tim-3negCD8+T cells and PD-1+Tim-3+CD8+T cells (FIG. 3). After a stimulation with anti-CD3 and anti-CD28 mAb we showed that the PD1-Tim3 and the PD1+Tim3 population secrete large amount of IFN in their supernatants with no significant difference between these two subpopulations. In contrast, the production of IFN was significantly decreased in the CD8+T cells co-expressing PD1 and Tim-3 compared to the other two populations (FIG. 3).

Discussion

[0107] Using a novel spectral multi-immunofluorescence in situ imaging technology, we showed that the clinical significance of PD-1 on CD8+T cells differs, whether it was co-expressed or not with Tim-3. Indeed, we showed that renal cell cancer patients infiltrated with CD8 cells that co-expressed PD-1 and Tim-3 had a more aggressive phenotype defined by high Fuhrman grade and tumor of larger size and advanced TNM and UISS score. In addition, this group of patients also exhibited a lower PFS and reduced overall survival at 36 months. We confirmed this aggressive phenotype by cytometry analysis, as patients whose CD8+T cells co-expressed PD-1 and Tim-3 had a more advanced TNM stage and UISS score and a larger tumor size. These data may explain some controversies in the literature about the prognostic value of PD-1 (8, 15-18) and emphasized about the critical role of the combined expression of coinhibitory receptors especially Tim-3 in the clinical significance of PD-1. This complex interpretation of the clinical value of PD-1 parallels its multiple biological significance. Indeed, PD-1 is both an activation marker and a hallmark of exhausted T cells. However, PD-1 also preserves CD8+T cells from overstimulation and the risk of accumulation of terminally differentiated exhausted CD8+T cells (19). Although Tim-3 is also induced after activation (20), its co-expression with PD-1 in the tumor microenvironment may represent a switch leading to compromised functionality of T cells (4, 20, 21). We showed that the CD8+T cell population co-expressing PD-1 and Tim-3 presented all the features of an exhausted T cell population, as they respond poorly to T cell stimulation. In addition, high levels of PD-1 expressed at their membrane are considered as a hallmark of a particularly dysfunctional T cell (11, 14). Interestingly both in vitro and in vivo, Tim-3Tim-3 ligand blockade in combination with the inhibition of the PD-1-PD-L1 pathway synergized to restore T cell function resulting in the control of chronic infection and inhibition of tumor growth (4, 5, 22, 23). Besides activation, Th1 cytokines may favor this co-expression of PD-1 and Tim-3, as type I and II IFN regulated PD-1 and IL-12 enhanced the expression of Tim-3 (20, 24). Tumor associated M2 macrophages also regulated the expression of Tim-3 on T cells derived from RCC (25). We recently showed that VEGF also enhanced the expression of PD-1 and Tim-3 after activation (12). Tim-3 could also be expressed on non T cells such as myeloid cells conferring to these cells an impaired immunosurveillance (26, 27). In RCC, Tim-3 has been shown to be expressed in macrophages and in tumor cells (28). Tim-3 promoted ccRCC invasion and rendered these cells more resistant to anti-angiogenic molecules (28). Intratumor Tim-3+CD8+T cells have been correlated with histological grade and advanced tumor stage in follicular lymphoma and NSCLC respectively but with no data on their influence on the clinical outcome (11, 20). Furthermore, higher expression of Tim-3 gene expression in kidney renal cell carcinoma was a marker for worse 5-year survival (13). In contrast to cancer, in preneoplastic lesions such as usual-type vulvar intra epithelial neoplasia, the significance of Tim-3+CD8+T cells may be less pejorative, as it was related with an absence of recurrence. However, the number of Tim-3+CD8+T cells increased in vulvar carcinoma compared to benign lesions (29). All these data converge for the targeting of Tim-3 in cancer alone or preferentially in combination with anti-PD-1/PD-L1. Other checkpoint inhibitors such as Lag-3 could be co-expressed with PD-1 on CD8+T cells as shown in RCC and other tumors and it usually correlated with an impaired effector function of these cells (9, 30). Interestingly, in NSCLC, CD8+T cells expressing Tim-3 are those which co-expressed the higher number of other inhibitory receptors compared to cells expressing other checkpoint inhibitors possibly making Tim-3 as a surrogate marker of more advanced exhausted T cells (11). One limit of this study is that we did not compartimentalize our CD8+T cell population in the tumor core or in the stroma due to difficulties to combine a homogenous tumor cell marker with our set of T cell antibodies. But, renal carcinomas are not histological entities with welldefined invasive margin as observed in some other epithelial tumors (i.e colorectal cancer). As it has been shown that the prognostic value of subpopulations of T cells depends on their location in the nest of the tumor or in the periphery, it could explain some minor discrepancies between our results and report in the literature regarding the prognosis value of the number of CD8+T cells and PD-1+T cells (8, 9, 31). It could also explain the more significant impact of the percent expression of the inhibitory receptors, PD-1 and Tim-3 over the number of cells expressing them, as this percent reflects an intrinsic status of the exhausted state of the intra-tumor CD8+T cells. The influence of the number of PD-1+Tim 3+CD8+T cells may be more dependent on their ratio with the number of tumor cells. In addition, in most reports from the literature, a monoparametric immunochemistry technique for the analysis of checkpoint inhibitor expression have been employed which differed from our focus on the characterization of checkpoint inhibitors specifically on CD8+T cells considered as one of the main effectors after immunotherapy (3). We also selected the various variables either as a continuous variable or with a median cut-off, which differs from previous studies that used optimal p value (8, 9). Novel multiparametric in situ technology set up in this study and recently described by other groups (3, 32) will allow a better characterization of CD8+T cells and other immune cells at single cell levels in the tumor microenvironment to better guide the choice of immune target for immunotherapy. We showed that for some parameters such as Tim-3, collagenase may decrease their expression when detected by cytometry which reinforces the value of our in situ multiparametric automated immunofluorescence technique to directly assess in vivo the intratumor expression of checkpoint inhibitor in untouched cells. In addition, the fact that in contrast to the PD-1+Tim-3neg CD8+T cell population, the double positive PD-1+Tim-3+CD8+T cells could not be activated in vitro with a strong stimulus suggest that it could also be difficult to revigorate them after PD-PDL-1 blockade and thus constitutes a biomarker of resistance to immunotherapy.

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