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Tumor antigen-specific CD8+ T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer
Accepted Manuscript Research paper Tumor antigen-specific CD8+ T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer Xu Lu, Lin Yang, Daxing Yao, Xuan Wu, Jingpo Li, Xuesong Liu, Lijuan Deng, Caiting Huang, Yue Wang, Dan Li, Jingwei Liu PII: DOI: Reference:
S0008-8749(17)30001-1 http://dx.doi.org/10.1016/j.cellimm.2017.01.001 YCIMM 3618
To appear in:
Cellular Immunology
Received Date: Revised Date: Accepted Date:
14 December 2016 27 December 2016 1 January 2017
Please cite this article as: X. Lu, L. Yang, D. Yao, X. Wu, J. Li, X. Liu, L. Deng, C. Huang, Y. Wang, D. Li, J. Liu, Tumor antigen-specific CD8+ T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer, Cellular Immunology (2017), doi: http://dx.doi.org/10.1016/j.cellimm.2017.01.001
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Tumor antigen-specific CD8+ T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer Xu Lu1, Lin Yang2, Daxing Yao3, Xuan Wu1, Jingpo Li1, Xuesong Liu1, Lijuan Deng1, Caiting Huang1, Yue Wang1, Dan Li1, and Jingwei Liu 1 1
Department of Oncology, Beijing Biohealthcare Biotechnology Co.,Ltd, China Department of internal medicine, the Chinese Academy of Medical Sciences Tumor Hospital, China 3 Department of Agricultural Science Research Institute, Guangzhou Zengcheng District, China 2
Correspondence to: Jingwei Liu, email:[email protected] Abstract: Cytotoxic CD8 T lymphocytes that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally important in eliciting tumor rejection. NY-ESO-1 is a major target of CD8+ T cell recognition in gastric cancer (GC) and is among the most immunogenic tumor antigens defined to date. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8 + T cell dysfunction may reveal effective strategies for immunotherapy. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8+ T cells derived from peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) of GC patients. Here, we report that Tim-3 expression defines a subpopulation of PD-1+ exhausted NY-ESO-1-specific CD8 + T cell and
PD-1 +Tim-3+ CD8+ T cells represented
the
largest subset of
NY-ESO-1-specific CD8+ T cells in GC patients. Functionally, CD8 +PD-1+Tim-3 + T cells were more impaired in IFN-γ, TNF-α and IL-2 production compared with PD-1+Tim-3 - or PD-1-Tim-3- subsets. Dual blockade of Tim-3 and PD-1 during T-cell priming
efficiently
augmented
proliferation
and
cytokine
production
by
NY-ESO-1-specific CD8 + T cells could potentially be improved by therapeutic targeting of these inhibitory receptors, indicating that antitumor function of NY-ESO-1-specific CD8 + T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.
Keywords PD-1; Tim-3; CD8+ T cells; tumor-associated lymphocytes
1. Introduction: Certain immunotherapeutic strategies have been designed to activate and expand tumor-reactive T cells and have experienced major progress in the last decade. Although several lines of evidence have shown that even in the face of strongly induced antitumor T cell responses, there are distinct mechanisms that allow tumors to escape immune destruction. This is probably a reflection of several in vivo immunosuppressive mechanisms in patients with advanced cancer. Therefore, understanding factors that regulate the functions of tumor antigen-specific CD8 + T cells is critical for effective control of tumor recurrence. The NY-ESO-1 tumor antigen is a major target of CD8 + T cell recognition in advanced (stage III and IV) gastric cancer (GC), eliciting both cellular and humoral immune responses in a proportion of patients with advanced NY-ESO-1 expressing tumors [2]. However, chronic antigens stimulation may result in exhaustion of antigen-specific CD8 + T cells and loss of ability to produce key cytokines that are critical for the maintenance of CD8 + T cell memory and effective antitumor T cell immune responses [3]. In this regard, tumor expression of programmed death ligand 1 (PD-L1, B7-H1), an immune inhibitory receptor belonging to the CD28:B7 family of costimulatory molecules, which is expressed on activated T cells, B cells and myeloid cells [4, 5], may contribute to the downregulation of immune responses by limiting the expansion or survival of effector T cells [6]. Tumor antigen (TA)-specific CTLs present in PBLs or at tumor sites have been shown to upregulate PD-1 expression [7, 8]. Expression of the PD-1 ligand PD-L1 in gastric cancer has been shown to inversely correlate with TA-specific CD8 + T cells response [9], suggesting a pivotal role for the PD-1/PD-L1 pathway in initiating and maintaining tumor-antigen-specific T-cell hyporesponsiveness in the disease. Whereas, whether distinct TA-specific CD8 + T cell subsets exhibit variable levels of dysfunction and express different sets of
inhibitory molecules has not been determined yet. These may provide novel therapeutic targets to reverse tumor-induced T cell dysfunction in patients with advanced cancers. To address this question, we have investigated the expression of the T-cell Ig- and mucin-domain-containing molecule-3 (Tim-3), a member of the tumor necrosis (TNF) family, is a negative regulator of CD4+ T helper 1 (Th1) and CD8 + T cytotoxic 1 (Tc1) cells [10, 11], in combination with PD-1 on spontaneous NY-ESO-1-specific CD8+ T cells from patients with advanced GC. Recently, it was reported that Tim-3 is highly expressed on ‘exhausted’ or impaired CD8 + T cells obtained from peripheral blood lymphocytes (PBLs) [12, 13], suggesting a relationships between PD-1 and Tim-3 expression and immune evasion in patients with GC. Because Tim-3 and PD-1 are expressed on activated T cells, we predicted that both molecules could coordinately mediate the inability of tumor-antigen-specific T cells to efficiently control GC. Furthermore, we observed that whereas NY-ESO-1-specific CD8+ T cells were readily detectable ex vivo in tumor-associated lymphocytes (TALs) of patients with spontaneous humoral immunity NY-ESO-1, they were only detectable in PBLs following in vitro stimulation. Whereas tumor-derived NY-ESO-1-specific CD8 + T cells enriched coexpression of PD-1 and Tim-3 demonstrated impaired effector function. Here, we report that when CD8+ cells were cocultured with tumor-derived APCs, the frequency and quality of NY-ESO-1-specific CD8+ T cells significantly decreased compared with those stimulated with PBL-derived APCs. Moreover, PD-1 blockade acts in combination with Tim-3 blockade to further enhance NY-ESO-1 specific CD8+ T cell expansion and function. Together, our data point to a coordinate negative role of PD-1 and Tim-3 in regulating the functional properties of NY-ESO-1 specific CD8 + T cells in GC. This understanding could ultimately lead to interventions to restore the effector function of NY-ESO-1 specific CD8 + T cells in human GC.
2. Materials and Methods 2.1. Study subjects Peripheral bloods and ascites fluids samples were obtained from eleven HLA-A2 +
patients with NY-ESO-1-expressing stage IV GC who exhibited spontaneous NY-ESO-1-specific CD8+ T cell responses assessed ex vivo by flow cytometry using APC-labeled HLA-A2/NY-ESO-1157-165 tetramers, under an approved protocol from the Institute Ethical Committee of the Affiliated Hospital of Capital Medical University (China). Written informed consents were obtained from all patients. PBLs from healthy donors and PBLs and/or ascites fluids used in this study were obtained from patients with no prior immunotherapy. 2.2. Phenotypic analysis Cells were stained with tetramers and mAbs against CD8 (BD Biosciences), PD-1 (BD Biosciences), and Tim-3 (BD Biosciences). Cytokine production from tetramer+ cells was determined as described previously [14]. NY-ESO-1-specific CD8+ T cells in PBLs were determined as described previously [14]. For the analysis of CD8 + T cells, NY-ESO-1157-165 peptide was used for in vitro stimulation in HLA-A2 patients. PBLs were collected from GC patients in sample sizes as low as 10 million cells. CD8 + T lymphocytes were separated from PBLs by antibody-coated magnetic beads (Minimacs;
Miltenyi
Biotec)
and
incubated
with
APC-labeled
HLA-A2/NY-ESO-1157-165 tetramers. The purity of CD8 + T cells was always greater than 95%. As controls, we did not observe HLA-A2/NY-ESO-1157-165 tetramer staining of CD4 + T cells purified from PBLs of GC patients (data not shown). Tetramers were provided by the department of Agricultural Science Research Institute for cancer research. TALs were isolated from ascites of GC patients and multicytokine production from CD8+ cells was assessed following stimulated for 6 hours with PMA and ionomycin.
2.3. Blocking antibody treatment For in vitro stimulation assays, PBLs were cultured in complete medium containing 100IU/ml rhIL-2 with peptide NY-ESO-1157-165 (10 µg/ml), in the presence of 10 µg/ml anti-PD-1 (clone EH12.2H7; Biolegand) and/or 20µg/ml anti-Tim-3 (clone 2E2) blocking mAbs or isotype control antibodies. On day 6, cells were restimulated for 6
hours with peptide NY-ESO-1157-165. After 2 hour of incubation, Brefeldin A (Sigma-Aldrich) was added to the culture medium (10 µg/ml). After tetramer labeling, multicytokine production from tetramer+ cells was determined by intracellular cytokine staining (ICS).
2.4. CFSE proliferation assay Five million CFSE-labeled PBLs were incubated in culture medium containing 100IU/ml rhIL-2 with peptide NY-ESO-1157-165 (10 µg/ml), in the presence of 10 µg/ml anti-PD-1 and/or 20µg/ml anti-Tim-3 blocking mAbs or isotype control antibodies. On day 6, cells were stained with APC-labeled HLA-A2/NY-ESO-1157-165 tetramers, CD14-PE, CD19-PE, CD56- PE-Cy5.5, CD4-FITC, and CD8-PE-Cy7 (BD Pharmingen) conjugated antibodies and reagents. One million events were collected during flow cytometric analysis.
2.5. Statistical Analysis The data presenting the differences between paired and unpaired groups was performed using the appropriate Student’s t test. The correlation coefficient and P value between the graphs of two datasets were determined by Pearson product-moment correlation using SPSS 13.0 software. P<0.05 was defined as statistically significant.
3. Results 3.1.
Tim-3
expression
defines
a
subpopulation
of
PD-1 +
exhausted
NY-ESO-1-specific CD8+ T cell in PBLs and TALs of patients with advanced GC We first to assess antigen-specific immune responses on spontaneous ex vivo detectable NY-ESO-1-specific CD8+ T cells isolated from PBLs of eleven HLA-A*02-ESO157-165 stage IV GC patients who were serum antibody-positive for NY-ESO-1 (Supplemental data) using HLA-A2 tetramers. As shown in Fig. 1A，in vitro stimulation once with NY-ESO-1 peptides was necessary to reveal NY-ESO-1-specific CD8 + T cells from PBLs of seropositive patients. We further
observed the expression of PD-1 and Tim-3 on spontaneous ex vivo detectable NY-ESO-1-specific CD8 + T cells isolated from PBLs of eleven HLA-A*02-ESO157-165 stage IV GC patients using HLA-A2 tetramers. In all patients, the frequencies of PD-1+ and Tim-3+ cells among NY-ESO-1-specific CD8 + T cells comprise the major population with cells expressing PD-1 alone or neither Tim-3 nor PD-1 comprising smaller populations (Fig.1B). Collectively, these data indicate that PD-1 is upregulated on tumor-induced NY-ESO-1-specific CD8+ T cells isolated from PBLs of patients with advanced GC. In contrast to total CD8 + T cells, the vast majority of PD-1+NY-ESO-1-specific CD8 + T cells upregulate Tim-3 expression. PD-1 +Tim-3+ cells represent the largest exhausted subset of NY-ESO-1-specific CD8+ T cells.
3.2. Expression of coinhibitory molecules correlates with impaired effector function of tumor-associated NY-ESO-1-specific CD8+ T cell To assess antigen-specific immune responses in NY-ESO-1 seropositive GC patients, TALs were isolated from GC ascites, and the frequencies of NY-ESO-1 specific CD8+ T cells were determined directly ex vivo with HLA-A*02-ESO157-165 tetramers. As shown previously, in chronic infection, exhausted cells exhibit PD-1high and CD127low phenotype, and these cells are unable to produce IFN-γ to virus-antigen challenge [15]. Because most NY-ESO-1 tetramer+ cells expressed PD-1 (Fig 1B), we reasoned that the expression level of PD-1 and CD127 could also be related to the effector function of TALs. We analyzed PD-1 and CD127 expression on whole CD8 + or Tim-3+ CD8+ T cells (Fig 2A and B). Consistent with our findings for NY-ESO-1-specific CD8+ T cells, bulk CD8 + T cells from TALs exhibited higher frequencies of PD-1+CD8+ T cells as compared with PBLs from healthy donors or GC patients. Importantly, the subset of Tim-3 +CD8+ T cells was remarkably enriched for PD-1+ cells in PBLs of GC patients, suggesting coordinated regulation of Tim-3 and PD-1 expression. Because Tim-3 expression by NY-ESO-1-specific T cells negatively correlated with the proportion of IFN-γ+ cells [16, 17], and the majority of Tim-3 +CD8+ T cells
coexpressed the PD-1 molecule, we examined the capacity for IFN-γ and TNF-α production from four CD8 + T cell subsets according to PD-1 and Tim-3 expression. In addition, PBLs and TALs were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and the proportion of IFN-γ and TNF-α-producing cells from each population was assessed. We found that the PD-1 +Tim-3+ subset showed the fewest IFN-γ+/TNF-α+ double-positive cells among these populations in TALs (Fig. 2C), and suggest a role for Tim-3 in cooperating with PD-1 to reduce the effector function of CD8 + T cells from GC patients. Tim-3-PD-1+ cells were capable of producing high levels of IFN-γ, but the frequency of dual-IFN-γ/TNF-α-producing cells was less than Tim-3-PD-1- cells. Together, these results indicate that Tim-3 +PD-1+CD8 + cells in TALs have less capacity to produce IFN-γ and TNF-α, and suggest a role for Tim-3 in cooperating with PD-1 to reduce the effector function of CD8+ cells in human GC cancer.
3.3 Coexpression of PD-1 and Tim-3 correlates with increased levels
of
NY-ESO-1-specific CD8 + T cell dysfunction and activation We next defined the functional status of NY-ESO-1-specific CD8+ T cells isolated from PBLs of GC patients according to PD-1 and Tim-3 expression. As shown in Fig 3A, tetramer+ cells in PBLs exhibited high IFN-γ production in response to peptide antigen stimulation (range 39-83% ； mean 60.2±17.3%). After short ex vivo stimulation with cognate peptide, we assessed the percentage of cytokine-producing NY-ESO-1-specific CD8 + T cells among PD-1+Tim-3 +, PD-1+Tim-3 -, PD-1-Tim-3cells, which represent the main subsets of NY-ESO-1-specific CD8+ T cells (Fig. 3B-D). PD-1+Tim-3 + NY-ESO-1-specific CD8 + T cells produced significantly less TNF-α than PD-1+Tim-3- and PD-1-Tim-3- cells (P<0.05) (Fig. 3B). Interestingly, PD-1+Tim-3 + and PD-1+Tim-3 - NY-ESO-1-specific CD8 + T cells produced significantly less IFN-γ and IL-2 than PD-1-Tim-3 - cells (P<0.05, respectively). These observations were confirmed in a second set of independent experiments (data not shown). These results therefore indicate that functional exhaustion of NY-ESO-1-specific
CD8 + T cells in PBLs from GC patients is associated with coexpression of PD-1 and Tim-3, and that PD-1 +Tim-3+ cells exhibit a deeper exhaustion.
3.4 Dual blockade of PD-1 and Tim-3 pathway during priming restores frequency and effector function of NY-ESO-1-specific CD8 + T cells Our results show a correlation between coexpression of PD-1 with Tim-3 and deeper CD8 + T cell exhaustion. However, it is not clear whether the PD-1 and Tim-3 pathways have overlapping functions or contribute independently to T cell dysfunction in GC. First, we test whether blockade of Tim-3 and/or PD-1 pathway could restore the effector function of NY-ESO-1-specific CD8 + T cells，whole PBLs from eleven GC patients with spontaneous NY-ESO-1-specific CD8 + T cells were incubated for 6 days with NY-ESO-1157-165 peptide in the presence of blocking mAbs against Tim-3 and/or PD-1 or IgG control antibodies. After 6 days, cells were briefly restimulated (6hr) with NY-ESO-1 peptide, before evaluating antigen-specific multicytokine production (IFN-γ, TNF-α and IL-2) by A2/NY-ESO-1157-165 tetramer+ CD8 + T cells. Consistent with our data from Fig. 3A NY-ESO-1-specific IFN-γ-producing cells were detectable upon peptide stimulation, and a small proportion were IFN-γ/TNF-α+ or IFN-γ/IL-2+ (Fig. 4A). Tim-3 blockade alone did not significantly increase cytokine production compared with T cells cultured without antibody. This is consistent with a previous report indicating that Tim-3 blockade did not modify the effector function of CTL [18]. In contrast, PD-1 pathway blockade significantly enhanced the total frequency of IFN-γ-producing cells, especially those that coexpressed TNF-α or IL-2. Based on our data showing impaired effector function of Tim-3 +PD-1+CD8 + T cells, we hypothesized that dual, rather than individual, blockade of PD-1 and Tim-3 pathways could enhance effector function of the Tim -3+PD-1 +CD8+ subset. These findings indicate that whereas Tim-3 plays a role in the unresponsiveness of PD-1+CD8+ T cells, direct blockade of Tim-3 on T cells does not restore the function of NY-ESO-1-specific CD8+ T cells in GC patients. Next, we evaluated the effect of Tim-3 pathway blockade alone or in combination with
PD-1
blockades
on
the
proliferative
capacity
and
expansion
of
NY-ESO-1-specific CD8 + T cells in response to cognate antigen. CFSE-labeled PBLs from eleven GC patients with spontaneous NY-ESO-1-specific CD8+ T cell response were stimulated for 6 days with NY-ESO-1 157-165 peptide in the presence of mAbs against Tim-3 and/or PD-1 or IgG control antibodies. We noted a significant increase in the frequencies of CFSElo and total A2/NY-ESO-1157-165 tetramer+ CD8+ T cells as compared with peptide and IgG control antibody, resulting in a 1.6-fold and a 1.2-fold change in the frequencies of CFSElo and total A2/NY-ESO-1157-165 tetramer+CD8 + T cells, respectively. Tim-3 blockade in combination with PD-1 blockade further increased the frequencies of CFSElo and total A2/NY-ESO-1 157-165 tetramer+ CD8 + T cells as compared to Tim-3 blockade alone (P=0.01) or PD-1 blockade alone (P=0.02), resulting in 2.3-fold and a 1.9-fold change in the frequencies of CFSElo and total A2/NY-ESO-1 157-165 tetramer+ CD8+ T cells, respectively. No significant proliferation of NY-ESO-1157-165-specific CD8 + T cells was observed in the presence of an irrelevant peptide with or without blockade (data not shown). Collectively, our findings demonstrate that PD-1 blockade enhances the proliferation and expansion of NY-ESO-1-specific CD8 + T cells. Furthermore, Tim-3 blockade acts in combination with PD-1 blockades to further increase the frequencies of proliferating and total NY-ESO-1-specific CD8+ T cells. Third, Although our results indicate that dual blockade of PD-1 and Tim-3 pathways
substantially enhanced
the
frequency and
effector
function of
NY-ESO-1-specific CD8 + T cells, it is not clear whether blockade of PD-1 and Tim-3 pathways during antigen stimulation would improve the quality of NY-ESO-1-specific T cells in the presence of tumor-derived DCs (APCs). We found that when CD8+ cells were cocultured with tumor-derived APCs, the frequency of NY-ESO-1-specific tetramer+ cells and IFN-γ+/CD107+ cells significantly decreased compared with those stimulated with PBL-derived APCs. Moreover, the combined blockade PD-1 and Tim-3 pathways not only led to a significant increase
in the frequency of
NY-ESO-1+ tetramer+ cells but also to a marked increase in the proportion of dual-functional (IFN-γ+CD107 +) tetramer+ cells compared with untreated cells or treated with a single antibody. Together, this results indicate that dual blockade of
PD-1 and Tim-3 pathways during priming of tumor-antigen-specific T cells with tumor-derived APCs efficiently restores T-cell effector function to levels observed with peripheral blood-derived APCs.
4. Discussion The identification of coinhibitory molecules that antagonize the function of tumor-antigen-specific T cells is crucial for effective tumor immunotherapy. In this study, we have focused on NY-ESO-1-specific CD8 + T cells detected directly ex vivo in human GC specimens. We first observed that NY-ESO-1-specific CD8 + T cells were readily detectable in PBLs and TALs of GC patients who were serum antibody-positive for NY-ESO-1 following in vitro stimulation. Second, we demonstrated that PD-1+Tim-3 + NY-ESO-1-specific CD8+ T cells represent a highly dysfunctional population of tumor-antigen-induced T cells in patients with advanced GC. This observation is in line with a recent report by Fourcade et al. spontaneous NY-ESO-1-specific CD8 + T cell responses were detectable directly ex vivo in peripheral blood of melanoma patients who were seropositive for NY-ESO-1 [13, 17]. The co-expression of these receptors was associated with lower T cell function and the blockade of multiple inhibitory pathways appeared to better reverse T cell dysfunction. Consistent with these observations, we have shown that a subset of highly dysfunctional NY-ESO-1-specific CD8 + T cells upregulate both PD-1 and Tim-3 in patients with advanced GC. Here we have focused on coinhibitory pathway blockade ex vivo under different stimulating conditions and differentiation states of CD8 + T cells and observed distinct outcomes. In the present study, we further demonstrate that dual blockade of PD-1 and Tim-3 pathways increased the frequency and effector function of NY-ESO-1-specific CD8+ T cells. This was most notable under stimulatory conditions where tumor-derived APCs were present, and less so in the absence of antigenic stimulation. Because coculture of peripheral blood-derived CD8 + T cells with tumor-derived APCs resulted in generation of PD-1 +Tim-3+ cells with diminished effector function, the PD-1- and/or Tim-3 positive “hyporesponsive”
phenotype appears to be acquired upon infiltration of tumor-antigen-specific T cells into the tumor site, while this phenotype could be acquired by chronic antigen stimulation [19, 20]. It has been confirmed that the intrinsic variability of effector/memory CD8 + T cell subsets contributes to significant differences in in vivo function, whereas whether there are functionally distinct subsets of tumor-antigen-specific TALs based on the expression Tim-3 and PD-1 inhibitory molecules. In an attempt to provide the basis for this functional heterogeneity, we showed that the majority of PD-1+CD8 + TALs lacked expression of CD127, and this phenotype is known to be associated with impaired effector-to-memory transition [21, 22]. Because cells that are PD-1+ but lack Tim-3 expression exhibit functional properties intermediate between Tim-3 -PD-1- and Tim-3+PD-1 + cells, we propose that PD-1 plays a critical role in reducing the effector function of Tim-3+CD8+ T cells in cancer. It is known that T cell exhaustion represents a progressive loss of T cell function occurring upon chronic exposure to high antigen load and exhausted CD8+ T cells upregulate multiple inhibitory receptors, including PD-1, CTLA-4, CD160, LAG-3 and Tim-3 [19]. In previous studies, blocking the PD-1/PD-L1 pathway in vivo incompletely restored frequency and function of LCMV-specific CD8 T cell from chronically infected mice [20, 23]. Recently, Fourcade et al. reported that PD-1 expression was up-regulated on NY-ESO-1-specific CD8 T cells in PBLs of melanoma patients, and PD-1 and Tim-3 pathway blockade enhanced the expansion and cytokine production of vaccine-induced CD8 + T cells in vitro [13]. Here we have focused on coinhibitory pathway blockade ex vivo under different stimulating conditions and differentiation states of CD8+ T cells and observed distinct outcomes. Whereas Tim-3 blockade enhanced the frequency of NY-ESO-1 tetramer+ cells and polyfunctional T cells only during priming in the presence of APCs, these effects were observed for PD-1 pathway blockade in the presence or absence of APCs. With regard to PD-1, the results are also consistent with a previous report indicating in vivo significance because expression of PD-L1, the ligand for PD-1, has been observed in melanoma and other human cancers [24, 25]. These findings are of considerable
whether other molecular pathways are involved in tumor-antigen-specific T cell dysfunction, we observed that blockade of the Tim-3-Tim-3L pathway ex vivo increased the percentages of NY-ESO-1-specific CD8 + T cells that produced cytokines, supporting the role of the Tim-3 pathway in tumor antigen-specific T cell exhaustion/dysfunction. These findings in line with our previous study demonstrating the role of Tim-3 blockade in improving tumor-induced CD8 + T cell functions in patients with CRC [26]. Our critical findings is that Tim-3 blockade adds to PD-1 blockade to enhance NY-ESO-1-specific CD8+ T cell expansion and function among total CD8+ T cells upon prolonged antigen stimulation. Tim-3 blockade, in combination with PD-1 blockade further augment the frequencies of IFN-γ/TNF-α or IFN-γ/IL-2-producing, proliferating and total NY-ESO-1-specific CD8 + T cells among total CD8 + T cells upon prolonged antigen stimulation. In summary, our results have identified PD-1 and Tim-3 as important inhibitory molecules frequently expressed by TA-specific CD8 + T cells. Tim-3 plays a role in attenuating the effector function of PD-1+CD8+ T cells, wherein effector function is most impaired in antigen-specific PD-1 +Tim-3+ CD8+ T cells. Although the precise role of PD-1 and Tim-3 pathways in CD8 T-cell exhaustion should be further investigated, these results indicate that the PD-1 and Tim-3 pathways may play independent roles in NY-ESO-1-specific CD8+ T cells exhaustion in patients with advanced GC. This can be further supported by our data showing that the population of TA-specific CD8+ T cells coexpressing PD-1 and Tim-3 exhibited a more severe defect in proliferation and production of cytokines, such as IFN-γ, TNF-α, and IL-2, than did those cells expressing only PD-1. Thus, dual blockade of PD-1 and Tim-3 pathways may allow for a more comprehensive reversal of tumor-induced T cell exhaustion, potentially leading to potent combination therapies. Because dual blockade of PD-1 and Tim-3 pathways acted additively to efficiently restore T-cell effector function, our findings indicate that antitumor T-cell function could potentially be improved by therapeutic targeting of these inhibitory receptors in human gastric cancer.
Conflict of interest statement The authors declare no conflict of interest of this work. Acknowledgements We thank Mr. Jinwan wang and Dr. Linyang of Chinese Academy of Medical Sciences Tumor Hospital, Department of internal medicine for their excellent technical support. This work was supported by the Beijing Science and Technology Plan special issue (Z14010101101). We sincerely thank all related people who provided support for this study.
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antigen-specific CD8(+) T cells induced by melanoma vaccines. Cancer research, 2014. 74: 1045-55. [14] Odunsi K, Qian F, Matsuzaki J, et al. Vaccination with an NY-ESO-1 peptide of HLA class I/II specificities induces integrated humoral and T cell responses in ovarian cancer. Proceedings of the National Academy of Sciences of the United States of America, 2007. 104: 12837-42. [15] Radziewicz H, Ibegbu CC, Fernandez ML, et al. Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression. Journal of virology, 2007. 81: 2545-53. [16] Fourcade J, Sun Z, Pagliano O, et al. CD8(+) T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1. Cancer research, 2012. 72: 887-96. [17] Fourcade J, Sun Z, Benallaoua M, et al. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. The Journal of experimental medicine, 2010. 207: 2175-86. [18] Sakuishi K, Apetoh L, Sullivan JM, et al. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. The Journal of experimental medicine, 2010. 207: 2187-94. [19] Blackburn SD, Shin H, Haining WN, et al. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nature immunology, 2009. 10: 29-37. [20] Barber DL, Wherry EJ, Masopust D, et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature, 2006. 439: 682-7. [21] Kaech SM, Tan JT, Wherry EJ, et al. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nature immunology, 2003. 4: 1191-8. [22] Conrad JA, Ramalingam RK, Smith RM, et al. Dominant clonotypes within HIV-specific T cell responses are programmed death-1high and CD127low and display reduced variant cross-reactivity. Journal of immunology, 2011. 186: 6871-85. [23] Penaloza-MacMaster P, Provine NM, Blass E, et al. CD4 T Cell Depletion Substantially Augments the Rescue Potential of PD-L1 Blockade for Deeply Exhausted CD8 T Cells. Journal of immunology, 2015. 195: 1054-63. [24] Rosenblatt J, Glotzbecker B, Mills H, et al. PD-1 blockade by CT-011, anti-PD-1 antibody, enhances ex vivo T-cell responses to autologous dendritic cell/myeloma fusion vaccine. Journal of immunotherapy, 2011. 34: 409-18. [25] Zou W, Chen L. Inhibitory B7-family molecules in the tumour microenvironment. Nature reviews Immunology, 2008. 8: 467-77. [26] Liu J, Zhang S, Hu Y, et al. Targeting PD-1 and Tim-3 Pathways to Reverse CD8 T-Cell Exhaustion and Enhance Ex Vivo T-Cell Responses to Autologous Dendritic/Tumor Vaccines. Journal of immunotherapy, 2016. 39: 171-80.
Figure Legends Fig 1 PD-1 and Tim-3 expression on NY-ESO-1-specific CD8+ T cells. (A) The frequency of NY-ESO-1 tetramer+ cells in PBLs before and after in vitro peptide presensitization. The number in the right upper quadrant indicates percentage of tetramer+ CD8+ cells. (B) Pooled data from eleven GC patients showing the distribution of NY-ESO-1-specific and total CD8+ T cells according to PD-1 and Tim-3 expression. Horizontal bars depict the mean percentage of PD-1 and/or Tim-3 expression on tetramer+ CD8+ T cells. Data shown are representative of two independent experiments. Fig 2 Tim-3 +CD8 + T cells in TALs exhibit high PD-1 expression. (A and B) The expression of PD-1 and CD127 on Tim-3+CD8 + or whole CD8+ cells in PBLs and TALs. ★P<0.05 compared with whole CD8 + cells of healthy donor’s (HD) PBLs. * P<0.05 compared with whole CD8+ cells of each corresponding tissue.(C) IFN-γ/TNF-α production from each indicated population following stimulation with PMA/ionomycin. The result shown is representative of samples from three patients. Fig 3 Impaired effector function of CD8+ T cells correlates with the expression of coinhibitory molecules. (A) NY-ESO-1-specific IFN-γ production from PBLs following in vitro peptide presensitization. The graph indicates the average and statistics of peptide-specific from eleven samples. The number in parentheses indicates percentage of IFN-γ-producing cells among NY-ESO-1-specific tetramer+ cells. (B-D) The summary data for 11 GC patients showing the percentages of cytokine-producing NY-ESO-1-specific CD8 + T cells according to PD-1 and Tim-3 expression. Values indicate the percentage of cytokine-producing CD8+ T cells among Tim-3+ and/or Tim-3- fractions of PD-1 - and PD-1+ NY-ESO-1-specific CD8 + T cells. The P values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate. Fig 4 Dual blockade of Tim-3 and PD-1 pathways increases the frequency and enhances effector function of NY-ESO-1-specific T cells. (A) Representative flow cytometry analysis the frequency of peptide-specific cytokine productions from NY-ESO-1 tetramer+ cells following in vitro presensitization with or without Tim-3 and PD-1 blockade. (B) Representative flow cytometry analysis from two GC patients showing percentage of CFSElo A2/NY-ESO-1157-165 tetramer+ CD8+ T cells among total CD8+ T cells. CFSE-labeled PBLs were incubated for 6 days with NY-ESO-1157-165 peptide and blocking mAbs against PD-1 (aPD-1) and/or Tim-3 (aTim-3) or an isotype control antibody (IgG). Fold changes of the frequencies of CFSElo (C) and total (D) A2/NY-ESO-1 157-165 tetramer+ CD8+ T cells after 6-day in vitro stimulation with cognate peptide and blocking anti-PD-1 and/or anti-Tim-3 mAbs (n=11). The ratio of the percentage of CFSElo and total A2/NY-ESO-1157-165 tetramer+ CD8+ T cells in the presence of indicated antibody treatment and isotype control antibody is shown. Data shown are representative of two independent
experiments performed in duplicate. (E) The frequency of tetramer+ cells and peptide-specific IFN-γ/CD107 expression following in vitro presensitization with or without Tim-3 and PD-1 blockade. The result shown is representative of three independent experiments from two patients.
Highlights （for review） Tim-3 expression defines a subpopulation of PD-1+ exhausted NY-ESO-1-specific CD8+ T cell and PD-1+Tim-3+ CD8 + T cells represented the largest subset of NY-ESO-1-specific CD8+ T cells in GC patients. Tumor-derived NY-ESO-1-specific CD8 + T cells enriched coexpression of PD-1 and Tim-3 demonstrated impaired effector function. Dual blockade of PD-1 and Tim-3 pathways may allow for a more comprehensive reversal of tumor-induced T cell exhaustion, potentially leading to potent combination therapies. Antitumor T-cell function could potentially be improved by therapeutic targeting of these inhibitory receptors in human gastric cancer.
S0008-8749(17)30001-1 http://dx.doi.org/10.1016/j.cellimm.2017.01.001 YCIMM 3618
To appear in:
Cellular Immunology
Received Date: Revised Date: Accepted Date:
14 December 2016 27 December 2016 1 January 2017
Please cite this article as: X. Lu, L. Yang, D. Yao, X. Wu, J. Li, X. Liu, L. Deng, C. Huang, Y. Wang, D. Li, J. Liu, Tumor antigen-specific CD8+ T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer, Cellular Immunology (2017), doi: http://dx.doi.org/10.1016/j.cellimm.2017.01.001
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Tumor antigen-specific CD8+ T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer Xu Lu1, Lin Yang2, Daxing Yao3, Xuan Wu1, Jingpo Li1, Xuesong Liu1, Lijuan Deng1, Caiting Huang1, Yue Wang1, Dan Li1, and Jingwei Liu 1 1
Department of Oncology, Beijing Biohealthcare Biotechnology Co.,Ltd, China Department of internal medicine, the Chinese Academy of Medical Sciences Tumor Hospital, China 3 Department of Agricultural Science Research Institute, Guangzhou Zengcheng District, China 2
Correspondence to: Jingwei Liu, email:[email protected] Abstract: Cytotoxic CD8 T lymphocytes that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally important in eliciting tumor rejection. NY-ESO-1 is a major target of CD8+ T cell recognition in gastric cancer (GC) and is among the most immunogenic tumor antigens defined to date. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8 + T cell dysfunction may reveal effective strategies for immunotherapy. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8+ T cells derived from peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) of GC patients. Here, we report that Tim-3 expression defines a subpopulation of PD-1+ exhausted NY-ESO-1-specific CD8 + T cell and
PD-1 +Tim-3+ CD8+ T cells represented
the
largest subset of
NY-ESO-1-specific CD8+ T cells in GC patients. Functionally, CD8 +PD-1+Tim-3 + T cells were more impaired in IFN-γ, TNF-α and IL-2 production compared with PD-1+Tim-3 - or PD-1-Tim-3- subsets. Dual blockade of Tim-3 and PD-1 during T-cell priming
efficiently
augmented
proliferation
and
cytokine
production
by
NY-ESO-1-specific CD8 + T cells could potentially be improved by therapeutic targeting of these inhibitory receptors, indicating that antitumor function of NY-ESO-1-specific CD8 + T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.
Keywords PD-1; Tim-3; CD8+ T cells; tumor-associated lymphocytes
1. Introduction: Certain immunotherapeutic strategies have been designed to activate and expand tumor-reactive T cells and have experienced major progress in the last decade. Although several lines of evidence have shown that even in the face of strongly induced antitumor T cell responses, there are distinct mechanisms that allow tumors to escape immune destruction. This is probably a reflection of several in vivo immunosuppressive mechanisms in patients with advanced cancer. Therefore, understanding factors that regulate the functions of tumor antigen-specific CD8 + T cells is critical for effective control of tumor recurrence. The NY-ESO-1 tumor antigen is a major target of CD8 + T cell recognition in advanced (stage III and IV) gastric cancer (GC), eliciting both cellular and humoral immune responses in a proportion of patients with advanced NY-ESO-1 expressing tumors [2]. However, chronic antigens stimulation may result in exhaustion of antigen-specific CD8 + T cells and loss of ability to produce key cytokines that are critical for the maintenance of CD8 + T cell memory and effective antitumor T cell immune responses [3]. In this regard, tumor expression of programmed death ligand 1 (PD-L1, B7-H1), an immune inhibitory receptor belonging to the CD28:B7 family of costimulatory molecules, which is expressed on activated T cells, B cells and myeloid cells [4, 5], may contribute to the downregulation of immune responses by limiting the expansion or survival of effector T cells [6]. Tumor antigen (TA)-specific CTLs present in PBLs or at tumor sites have been shown to upregulate PD-1 expression [7, 8]. Expression of the PD-1 ligand PD-L1 in gastric cancer has been shown to inversely correlate with TA-specific CD8 + T cells response [9], suggesting a pivotal role for the PD-1/PD-L1 pathway in initiating and maintaining tumor-antigen-specific T-cell hyporesponsiveness in the disease. Whereas, whether distinct TA-specific CD8 + T cell subsets exhibit variable levels of dysfunction and express different sets of
inhibitory molecules has not been determined yet. These may provide novel therapeutic targets to reverse tumor-induced T cell dysfunction in patients with advanced cancers. To address this question, we have investigated the expression of the T-cell Ig- and mucin-domain-containing molecule-3 (Tim-3), a member of the tumor necrosis (TNF) family, is a negative regulator of CD4+ T helper 1 (Th1) and CD8 + T cytotoxic 1 (Tc1) cells [10, 11], in combination with PD-1 on spontaneous NY-ESO-1-specific CD8+ T cells from patients with advanced GC. Recently, it was reported that Tim-3 is highly expressed on ‘exhausted’ or impaired CD8 + T cells obtained from peripheral blood lymphocytes (PBLs) [12, 13], suggesting a relationships between PD-1 and Tim-3 expression and immune evasion in patients with GC. Because Tim-3 and PD-1 are expressed on activated T cells, we predicted that both molecules could coordinately mediate the inability of tumor-antigen-specific T cells to efficiently control GC. Furthermore, we observed that whereas NY-ESO-1-specific CD8+ T cells were readily detectable ex vivo in tumor-associated lymphocytes (TALs) of patients with spontaneous humoral immunity NY-ESO-1, they were only detectable in PBLs following in vitro stimulation. Whereas tumor-derived NY-ESO-1-specific CD8 + T cells enriched coexpression of PD-1 and Tim-3 demonstrated impaired effector function. Here, we report that when CD8+ cells were cocultured with tumor-derived APCs, the frequency and quality of NY-ESO-1-specific CD8+ T cells significantly decreased compared with those stimulated with PBL-derived APCs. Moreover, PD-1 blockade acts in combination with Tim-3 blockade to further enhance NY-ESO-1 specific CD8+ T cell expansion and function. Together, our data point to a coordinate negative role of PD-1 and Tim-3 in regulating the functional properties of NY-ESO-1 specific CD8 + T cells in GC. This understanding could ultimately lead to interventions to restore the effector function of NY-ESO-1 specific CD8 + T cells in human GC.
2. Materials and Methods 2.1. Study subjects Peripheral bloods and ascites fluids samples were obtained from eleven HLA-A2 +
patients with NY-ESO-1-expressing stage IV GC who exhibited spontaneous NY-ESO-1-specific CD8+ T cell responses assessed ex vivo by flow cytometry using APC-labeled HLA-A2/NY-ESO-1157-165 tetramers, under an approved protocol from the Institute Ethical Committee of the Affiliated Hospital of Capital Medical University (China). Written informed consents were obtained from all patients. PBLs from healthy donors and PBLs and/or ascites fluids used in this study were obtained from patients with no prior immunotherapy. 2.2. Phenotypic analysis Cells were stained with tetramers and mAbs against CD8 (BD Biosciences), PD-1 (BD Biosciences), and Tim-3 (BD Biosciences). Cytokine production from tetramer+ cells was determined as described previously [14]. NY-ESO-1-specific CD8+ T cells in PBLs were determined as described previously [14]. For the analysis of CD8 + T cells, NY-ESO-1157-165 peptide was used for in vitro stimulation in HLA-A2 patients. PBLs were collected from GC patients in sample sizes as low as 10 million cells. CD8 + T lymphocytes were separated from PBLs by antibody-coated magnetic beads (Minimacs;
Miltenyi
Biotec)
and
incubated
with
APC-labeled
HLA-A2/NY-ESO-1157-165 tetramers. The purity of CD8 + T cells was always greater than 95%. As controls, we did not observe HLA-A2/NY-ESO-1157-165 tetramer staining of CD4 + T cells purified from PBLs of GC patients (data not shown). Tetramers were provided by the department of Agricultural Science Research Institute for cancer research. TALs were isolated from ascites of GC patients and multicytokine production from CD8+ cells was assessed following stimulated for 6 hours with PMA and ionomycin.
2.3. Blocking antibody treatment For in vitro stimulation assays, PBLs were cultured in complete medium containing 100IU/ml rhIL-2 with peptide NY-ESO-1157-165 (10 µg/ml), in the presence of 10 µg/ml anti-PD-1 (clone EH12.2H7; Biolegand) and/or 20µg/ml anti-Tim-3 (clone 2E2) blocking mAbs or isotype control antibodies. On day 6, cells were restimulated for 6
hours with peptide NY-ESO-1157-165. After 2 hour of incubation, Brefeldin A (Sigma-Aldrich) was added to the culture medium (10 µg/ml). After tetramer labeling, multicytokine production from tetramer+ cells was determined by intracellular cytokine staining (ICS).
2.4. CFSE proliferation assay Five million CFSE-labeled PBLs were incubated in culture medium containing 100IU/ml rhIL-2 with peptide NY-ESO-1157-165 (10 µg/ml), in the presence of 10 µg/ml anti-PD-1 and/or 20µg/ml anti-Tim-3 blocking mAbs or isotype control antibodies. On day 6, cells were stained with APC-labeled HLA-A2/NY-ESO-1157-165 tetramers, CD14-PE, CD19-PE, CD56- PE-Cy5.5, CD4-FITC, and CD8-PE-Cy7 (BD Pharmingen) conjugated antibodies and reagents. One million events were collected during flow cytometric analysis.
2.5. Statistical Analysis The data presenting the differences between paired and unpaired groups was performed using the appropriate Student’s t test. The correlation coefficient and P value between the graphs of two datasets were determined by Pearson product-moment correlation using SPSS 13.0 software. P<0.05 was defined as statistically significant.
3. Results 3.1.
Tim-3
expression
defines
a
subpopulation
of
PD-1 +
exhausted
NY-ESO-1-specific CD8+ T cell in PBLs and TALs of patients with advanced GC We first to assess antigen-specific immune responses on spontaneous ex vivo detectable NY-ESO-1-specific CD8+ T cells isolated from PBLs of eleven HLA-A*02-ESO157-165 stage IV GC patients who were serum antibody-positive for NY-ESO-1 (Supplemental data) using HLA-A2 tetramers. As shown in Fig. 1A，in vitro stimulation once with NY-ESO-1 peptides was necessary to reveal NY-ESO-1-specific CD8 + T cells from PBLs of seropositive patients. We further
observed the expression of PD-1 and Tim-3 on spontaneous ex vivo detectable NY-ESO-1-specific CD8 + T cells isolated from PBLs of eleven HLA-A*02-ESO157-165 stage IV GC patients using HLA-A2 tetramers. In all patients, the frequencies of PD-1+ and Tim-3+ cells among NY-ESO-1-specific CD8 + T cells comprise the major population with cells expressing PD-1 alone or neither Tim-3 nor PD-1 comprising smaller populations (Fig.1B). Collectively, these data indicate that PD-1 is upregulated on tumor-induced NY-ESO-1-specific CD8+ T cells isolated from PBLs of patients with advanced GC. In contrast to total CD8 + T cells, the vast majority of PD-1+NY-ESO-1-specific CD8 + T cells upregulate Tim-3 expression. PD-1 +Tim-3+ cells represent the largest exhausted subset of NY-ESO-1-specific CD8+ T cells.
3.2. Expression of coinhibitory molecules correlates with impaired effector function of tumor-associated NY-ESO-1-specific CD8+ T cell To assess antigen-specific immune responses in NY-ESO-1 seropositive GC patients, TALs were isolated from GC ascites, and the frequencies of NY-ESO-1 specific CD8+ T cells were determined directly ex vivo with HLA-A*02-ESO157-165 tetramers. As shown previously, in chronic infection, exhausted cells exhibit PD-1high and CD127low phenotype, and these cells are unable to produce IFN-γ to virus-antigen challenge [15]. Because most NY-ESO-1 tetramer+ cells expressed PD-1 (Fig 1B), we reasoned that the expression level of PD-1 and CD127 could also be related to the effector function of TALs. We analyzed PD-1 and CD127 expression on whole CD8 + or Tim-3+ CD8+ T cells (Fig 2A and B). Consistent with our findings for NY-ESO-1-specific CD8+ T cells, bulk CD8 + T cells from TALs exhibited higher frequencies of PD-1+CD8+ T cells as compared with PBLs from healthy donors or GC patients. Importantly, the subset of Tim-3 +CD8+ T cells was remarkably enriched for PD-1+ cells in PBLs of GC patients, suggesting coordinated regulation of Tim-3 and PD-1 expression. Because Tim-3 expression by NY-ESO-1-specific T cells negatively correlated with the proportion of IFN-γ+ cells [16, 17], and the majority of Tim-3 +CD8+ T cells
coexpressed the PD-1 molecule, we examined the capacity for IFN-γ and TNF-α production from four CD8 + T cell subsets according to PD-1 and Tim-3 expression. In addition, PBLs and TALs were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and the proportion of IFN-γ and TNF-α-producing cells from each population was assessed. We found that the PD-1 +Tim-3+ subset showed the fewest IFN-γ+/TNF-α+ double-positive cells among these populations in TALs (Fig. 2C), and suggest a role for Tim-3 in cooperating with PD-1 to reduce the effector function of CD8 + T cells from GC patients. Tim-3-PD-1+ cells were capable of producing high levels of IFN-γ, but the frequency of dual-IFN-γ/TNF-α-producing cells was less than Tim-3-PD-1- cells. Together, these results indicate that Tim-3 +PD-1+CD8 + cells in TALs have less capacity to produce IFN-γ and TNF-α, and suggest a role for Tim-3 in cooperating with PD-1 to reduce the effector function of CD8+ cells in human GC cancer.
3.3 Coexpression of PD-1 and Tim-3 correlates with increased levels
of
NY-ESO-1-specific CD8 + T cell dysfunction and activation We next defined the functional status of NY-ESO-1-specific CD8+ T cells isolated from PBLs of GC patients according to PD-1 and Tim-3 expression. As shown in Fig 3A, tetramer+ cells in PBLs exhibited high IFN-γ production in response to peptide antigen stimulation (range 39-83% ； mean 60.2±17.3%). After short ex vivo stimulation with cognate peptide, we assessed the percentage of cytokine-producing NY-ESO-1-specific CD8 + T cells among PD-1+Tim-3 +, PD-1+Tim-3 -, PD-1-Tim-3cells, which represent the main subsets of NY-ESO-1-specific CD8+ T cells (Fig. 3B-D). PD-1+Tim-3 + NY-ESO-1-specific CD8 + T cells produced significantly less TNF-α than PD-1+Tim-3- and PD-1-Tim-3- cells (P<0.05) (Fig. 3B). Interestingly, PD-1+Tim-3 + and PD-1+Tim-3 - NY-ESO-1-specific CD8 + T cells produced significantly less IFN-γ and IL-2 than PD-1-Tim-3 - cells (P<0.05, respectively). These observations were confirmed in a second set of independent experiments (data not shown). These results therefore indicate that functional exhaustion of NY-ESO-1-specific
CD8 + T cells in PBLs from GC patients is associated with coexpression of PD-1 and Tim-3, and that PD-1 +Tim-3+ cells exhibit a deeper exhaustion.
3.4 Dual blockade of PD-1 and Tim-3 pathway during priming restores frequency and effector function of NY-ESO-1-specific CD8 + T cells Our results show a correlation between coexpression of PD-1 with Tim-3 and deeper CD8 + T cell exhaustion. However, it is not clear whether the PD-1 and Tim-3 pathways have overlapping functions or contribute independently to T cell dysfunction in GC. First, we test whether blockade of Tim-3 and/or PD-1 pathway could restore the effector function of NY-ESO-1-specific CD8 + T cells，whole PBLs from eleven GC patients with spontaneous NY-ESO-1-specific CD8 + T cells were incubated for 6 days with NY-ESO-1157-165 peptide in the presence of blocking mAbs against Tim-3 and/or PD-1 or IgG control antibodies. After 6 days, cells were briefly restimulated (6hr) with NY-ESO-1 peptide, before evaluating antigen-specific multicytokine production (IFN-γ, TNF-α and IL-2) by A2/NY-ESO-1157-165 tetramer+ CD8 + T cells. Consistent with our data from Fig. 3A NY-ESO-1-specific IFN-γ-producing cells were detectable upon peptide stimulation, and a small proportion were IFN-γ/TNF-α+ or IFN-γ/IL-2+ (Fig. 4A). Tim-3 blockade alone did not significantly increase cytokine production compared with T cells cultured without antibody. This is consistent with a previous report indicating that Tim-3 blockade did not modify the effector function of CTL [18]. In contrast, PD-1 pathway blockade significantly enhanced the total frequency of IFN-γ-producing cells, especially those that coexpressed TNF-α or IL-2. Based on our data showing impaired effector function of Tim-3 +PD-1+CD8 + T cells, we hypothesized that dual, rather than individual, blockade of PD-1 and Tim-3 pathways could enhance effector function of the Tim -3+PD-1 +CD8+ subset. These findings indicate that whereas Tim-3 plays a role in the unresponsiveness of PD-1+CD8+ T cells, direct blockade of Tim-3 on T cells does not restore the function of NY-ESO-1-specific CD8+ T cells in GC patients. Next, we evaluated the effect of Tim-3 pathway blockade alone or in combination with
PD-1
blockades
on
the
proliferative
capacity
and
expansion
of
NY-ESO-1-specific CD8 + T cells in response to cognate antigen. CFSE-labeled PBLs from eleven GC patients with spontaneous NY-ESO-1-specific CD8+ T cell response were stimulated for 6 days with NY-ESO-1 157-165 peptide in the presence of mAbs against Tim-3 and/or PD-1 or IgG control antibodies. We noted a significant increase in the frequencies of CFSElo and total A2/NY-ESO-1157-165 tetramer+ CD8+ T cells as compared with peptide and IgG control antibody, resulting in a 1.6-fold and a 1.2-fold change in the frequencies of CFSElo and total A2/NY-ESO-1157-165 tetramer+CD8 + T cells, respectively. Tim-3 blockade in combination with PD-1 blockade further increased the frequencies of CFSElo and total A2/NY-ESO-1 157-165 tetramer+ CD8 + T cells as compared to Tim-3 blockade alone (P=0.01) or PD-1 blockade alone (P=0.02), resulting in 2.3-fold and a 1.9-fold change in the frequencies of CFSElo and total A2/NY-ESO-1 157-165 tetramer+ CD8+ T cells, respectively. No significant proliferation of NY-ESO-1157-165-specific CD8 + T cells was observed in the presence of an irrelevant peptide with or without blockade (data not shown). Collectively, our findings demonstrate that PD-1 blockade enhances the proliferation and expansion of NY-ESO-1-specific CD8 + T cells. Furthermore, Tim-3 blockade acts in combination with PD-1 blockades to further increase the frequencies of proliferating and total NY-ESO-1-specific CD8+ T cells. Third, Although our results indicate that dual blockade of PD-1 and Tim-3 pathways
substantially enhanced
the
frequency and
effector
function of
NY-ESO-1-specific CD8 + T cells, it is not clear whether blockade of PD-1 and Tim-3 pathways during antigen stimulation would improve the quality of NY-ESO-1-specific T cells in the presence of tumor-derived DCs (APCs). We found that when CD8+ cells were cocultured with tumor-derived APCs, the frequency of NY-ESO-1-specific tetramer+ cells and IFN-γ+/CD107+ cells significantly decreased compared with those stimulated with PBL-derived APCs. Moreover, the combined blockade PD-1 and Tim-3 pathways not only led to a significant increase
in the frequency of
NY-ESO-1+ tetramer+ cells but also to a marked increase in the proportion of dual-functional (IFN-γ+CD107 +) tetramer+ cells compared with untreated cells or treated with a single antibody. Together, this results indicate that dual blockade of
PD-1 and Tim-3 pathways during priming of tumor-antigen-specific T cells with tumor-derived APCs efficiently restores T-cell effector function to levels observed with peripheral blood-derived APCs.
4. Discussion The identification of coinhibitory molecules that antagonize the function of tumor-antigen-specific T cells is crucial for effective tumor immunotherapy. In this study, we have focused on NY-ESO-1-specific CD8 + T cells detected directly ex vivo in human GC specimens. We first observed that NY-ESO-1-specific CD8 + T cells were readily detectable in PBLs and TALs of GC patients who were serum antibody-positive for NY-ESO-1 following in vitro stimulation. Second, we demonstrated that PD-1+Tim-3 + NY-ESO-1-specific CD8+ T cells represent a highly dysfunctional population of tumor-antigen-induced T cells in patients with advanced GC. This observation is in line with a recent report by Fourcade et al. spontaneous NY-ESO-1-specific CD8 + T cell responses were detectable directly ex vivo in peripheral blood of melanoma patients who were seropositive for NY-ESO-1 [13, 17]. The co-expression of these receptors was associated with lower T cell function and the blockade of multiple inhibitory pathways appeared to better reverse T cell dysfunction. Consistent with these observations, we have shown that a subset of highly dysfunctional NY-ESO-1-specific CD8 + T cells upregulate both PD-1 and Tim-3 in patients with advanced GC. Here we have focused on coinhibitory pathway blockade ex vivo under different stimulating conditions and differentiation states of CD8 + T cells and observed distinct outcomes. In the present study, we further demonstrate that dual blockade of PD-1 and Tim-3 pathways increased the frequency and effector function of NY-ESO-1-specific CD8+ T cells. This was most notable under stimulatory conditions where tumor-derived APCs were present, and less so in the absence of antigenic stimulation. Because coculture of peripheral blood-derived CD8 + T cells with tumor-derived APCs resulted in generation of PD-1 +Tim-3+ cells with diminished effector function, the PD-1- and/or Tim-3 positive “hyporesponsive”
phenotype appears to be acquired upon infiltration of tumor-antigen-specific T cells into the tumor site, while this phenotype could be acquired by chronic antigen stimulation [19, 20]. It has been confirmed that the intrinsic variability of effector/memory CD8 + T cell subsets contributes to significant differences in in vivo function, whereas whether there are functionally distinct subsets of tumor-antigen-specific TALs based on the expression Tim-3 and PD-1 inhibitory molecules. In an attempt to provide the basis for this functional heterogeneity, we showed that the majority of PD-1+CD8 + TALs lacked expression of CD127, and this phenotype is known to be associated with impaired effector-to-memory transition [21, 22]. Because cells that are PD-1+ but lack Tim-3 expression exhibit functional properties intermediate between Tim-3 -PD-1- and Tim-3+PD-1 + cells, we propose that PD-1 plays a critical role in reducing the effector function of Tim-3+CD8+ T cells in cancer. It is known that T cell exhaustion represents a progressive loss of T cell function occurring upon chronic exposure to high antigen load and exhausted CD8+ T cells upregulate multiple inhibitory receptors, including PD-1, CTLA-4, CD160, LAG-3 and Tim-3 [19]. In previous studies, blocking the PD-1/PD-L1 pathway in vivo incompletely restored frequency and function of LCMV-specific CD8 T cell from chronically infected mice [20, 23]. Recently, Fourcade et al. reported that PD-1 expression was up-regulated on NY-ESO-1-specific CD8 T cells in PBLs of melanoma patients, and PD-1 and Tim-3 pathway blockade enhanced the expansion and cytokine production of vaccine-induced CD8 + T cells in vitro [13]. Here we have focused on coinhibitory pathway blockade ex vivo under different stimulating conditions and differentiation states of CD8+ T cells and observed distinct outcomes. Whereas Tim-3 blockade enhanced the frequency of NY-ESO-1 tetramer+ cells and polyfunctional T cells only during priming in the presence of APCs, these effects were observed for PD-1 pathway blockade in the presence or absence of APCs. With regard to PD-1, the results are also consistent with a previous report indicating in vivo significance because expression of PD-L1, the ligand for PD-1, has been observed in melanoma and other human cancers [24, 25]. These findings are of considerable
whether other molecular pathways are involved in tumor-antigen-specific T cell dysfunction, we observed that blockade of the Tim-3-Tim-3L pathway ex vivo increased the percentages of NY-ESO-1-specific CD8 + T cells that produced cytokines, supporting the role of the Tim-3 pathway in tumor antigen-specific T cell exhaustion/dysfunction. These findings in line with our previous study demonstrating the role of Tim-3 blockade in improving tumor-induced CD8 + T cell functions in patients with CRC [26]. Our critical findings is that Tim-3 blockade adds to PD-1 blockade to enhance NY-ESO-1-specific CD8+ T cell expansion and function among total CD8+ T cells upon prolonged antigen stimulation. Tim-3 blockade, in combination with PD-1 blockade further augment the frequencies of IFN-γ/TNF-α or IFN-γ/IL-2-producing, proliferating and total NY-ESO-1-specific CD8 + T cells among total CD8 + T cells upon prolonged antigen stimulation. In summary, our results have identified PD-1 and Tim-3 as important inhibitory molecules frequently expressed by TA-specific CD8 + T cells. Tim-3 plays a role in attenuating the effector function of PD-1+CD8+ T cells, wherein effector function is most impaired in antigen-specific PD-1 +Tim-3+ CD8+ T cells. Although the precise role of PD-1 and Tim-3 pathways in CD8 T-cell exhaustion should be further investigated, these results indicate that the PD-1 and Tim-3 pathways may play independent roles in NY-ESO-1-specific CD8+ T cells exhaustion in patients with advanced GC. This can be further supported by our data showing that the population of TA-specific CD8+ T cells coexpressing PD-1 and Tim-3 exhibited a more severe defect in proliferation and production of cytokines, such as IFN-γ, TNF-α, and IL-2, than did those cells expressing only PD-1. Thus, dual blockade of PD-1 and Tim-3 pathways may allow for a more comprehensive reversal of tumor-induced T cell exhaustion, potentially leading to potent combination therapies. Because dual blockade of PD-1 and Tim-3 pathways acted additively to efficiently restore T-cell effector function, our findings indicate that antitumor T-cell function could potentially be improved by therapeutic targeting of these inhibitory receptors in human gastric cancer.
Conflict of interest statement The authors declare no conflict of interest of this work. Acknowledgements We thank Mr. Jinwan wang and Dr. Linyang of Chinese Academy of Medical Sciences Tumor Hospital, Department of internal medicine for their excellent technical support. This work was supported by the Beijing Science and Technology Plan special issue (Z14010101101). We sincerely thank all related people who provided support for this study.
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Figure Legends Fig 1 PD-1 and Tim-3 expression on NY-ESO-1-specific CD8+ T cells. (A) The frequency of NY-ESO-1 tetramer+ cells in PBLs before and after in vitro peptide presensitization. The number in the right upper quadrant indicates percentage of tetramer+ CD8+ cells. (B) Pooled data from eleven GC patients showing the distribution of NY-ESO-1-specific and total CD8+ T cells according to PD-1 and Tim-3 expression. Horizontal bars depict the mean percentage of PD-1 and/or Tim-3 expression on tetramer+ CD8+ T cells. Data shown are representative of two independent experiments. Fig 2 Tim-3 +CD8 + T cells in TALs exhibit high PD-1 expression. (A and B) The expression of PD-1 and CD127 on Tim-3+CD8 + or whole CD8+ cells in PBLs and TALs. ★P<0.05 compared with whole CD8 + cells of healthy donor’s (HD) PBLs. * P<0.05 compared with whole CD8+ cells of each corresponding tissue.(C) IFN-γ/TNF-α production from each indicated population following stimulation with PMA/ionomycin. The result shown is representative of samples from three patients. Fig 3 Impaired effector function of CD8+ T cells correlates with the expression of coinhibitory molecules. (A) NY-ESO-1-specific IFN-γ production from PBLs following in vitro peptide presensitization. The graph indicates the average and statistics of peptide-specific from eleven samples. The number in parentheses indicates percentage of IFN-γ-producing cells among NY-ESO-1-specific tetramer+ cells. (B-D) The summary data for 11 GC patients showing the percentages of cytokine-producing NY-ESO-1-specific CD8 + T cells according to PD-1 and Tim-3 expression. Values indicate the percentage of cytokine-producing CD8+ T cells among Tim-3+ and/or Tim-3- fractions of PD-1 - and PD-1+ NY-ESO-1-specific CD8 + T cells. The P values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate. Fig 4 Dual blockade of Tim-3 and PD-1 pathways increases the frequency and enhances effector function of NY-ESO-1-specific T cells. (A) Representative flow cytometry analysis the frequency of peptide-specific cytokine productions from NY-ESO-1 tetramer+ cells following in vitro presensitization with or without Tim-3 and PD-1 blockade. (B) Representative flow cytometry analysis from two GC patients showing percentage of CFSElo A2/NY-ESO-1157-165 tetramer+ CD8+ T cells among total CD8+ T cells. CFSE-labeled PBLs were incubated for 6 days with NY-ESO-1157-165 peptide and blocking mAbs against PD-1 (aPD-1) and/or Tim-3 (aTim-3) or an isotype control antibody (IgG). Fold changes of the frequencies of CFSElo (C) and total (D) A2/NY-ESO-1 157-165 tetramer+ CD8+ T cells after 6-day in vitro stimulation with cognate peptide and blocking anti-PD-1 and/or anti-Tim-3 mAbs (n=11). The ratio of the percentage of CFSElo and total A2/NY-ESO-1157-165 tetramer+ CD8+ T cells in the presence of indicated antibody treatment and isotype control antibody is shown. Data shown are representative of two independent
experiments performed in duplicate. (E) The frequency of tetramer+ cells and peptide-specific IFN-γ/CD107 expression following in vitro presensitization with or without Tim-3 and PD-1 blockade. The result shown is representative of three independent experiments from two patients.
Highlights （for review） Tim-3 expression defines a subpopulation of PD-1+ exhausted NY-ESO-1-specific CD8+ T cell and PD-1+Tim-3+ CD8 + T cells represented the largest subset of NY-ESO-1-specific CD8+ T cells in GC patients. Tumor-derived NY-ESO-1-specific CD8 + T cells enriched coexpression of PD-1 and Tim-3 demonstrated impaired effector function. Dual blockade of PD-1 and Tim-3 pathways may allow for a more comprehensive reversal of tumor-induced T cell exhaustion, potentially leading to potent combination therapies. Antitumor T-cell function could potentially be improved by therapeutic targeting of these inhibitory receptors in human gastric cancer.