Tumor-specific Tumor-infiltrating Lymphocytes Research Articles

Abstract B12: Immune checkpoint blockade to improve T cell infiltration and function in colorectal cancer

Abstract Adoptive transfer of tumor infiltrating lymphocytes (TIL) has emerged as a promising approach to induce effective anti-tumor immunity and tumor regression in various types of cancers. Current studies suggest that the presence of TIL play a role in patient outcomes and can predict tumor stage, node positivity, and in some reports, disease free and overall survival in patients with colorectal cancer. TIL may be inactivated in patients due to factors in the local tumor microenvironment, such as regulatory T cells, myeloid derived suppressor cells and the expression of a number of co-inhibitory immune checkpoints including PD-L1, BTLA, PD-1, CTLA-4, Lag-3 and Tim-3. We first examined the feasibility of expanding TIL from the tumors of three colorectal patients. Tumors were minced into fragments and cultured in media supplemented with 6000 IU IL2 / mL. TIL growth was observed in tumor fragments from each of the three patients with a total of 12 out of 41 fragments demonstrating TIL expansion. TIL grown out of these fragments were predominantly CD3+ with 32% CD4+T cells and 46% CD8+ T cells and 18.5% NK cells. Seven of the 12 fragments tested were tumor specific as demonstrated by IFN-gamma production in response to autologous tumor. CD8+ T cells within the patient TIL expressed PD-1, BTLA, Lag-3 and Tim-3 and a co-stimulatory molecule, 41BB. In addition, we found high levels of PD-L1 expression on tumors of colorectal patients and human colorectal cell lines. Since we observed high levels of immune checkpoints expression in colorectal patients, we wanted to develop a preclinical colorectal model to investigate the effect of immune checkpoint blockade on tumor growth and TIL function. A previous study from our lab has shown that TIL from our preclinical MC38 colorectal murine model express high levels of co-inhibitory checkpoints and that blockade of PD-L1 leads to enhanced infiltration and anti-tumor specificity of TIL. However, given the dynamic nature of immune responses to tumors and expression of multiple immune checkpoints, it may be necessary to block additional checkpoints to improve clinical efficacy. In this study, we examined whether blockade of the immune checkpoint receptor BTLA had an effect on TIL infiltration and tumor growth. Mice bearing MC-38 tumors treated with anti-BTLA antibody demonstrated a delay in tumor growth (150mm2, p<0.01) on day 28 post tumor injection compared to isotype control (300mm2). We observed an increase in CD8+ T cell infiltration (30%, p<0.001) in the tumors of anti-BTLA antibody treated mice compared to mice treated with isotype control (13%). TIL isolated from the anti-BTLA antibody treated mice demonstrated increased IFN-gamma and granzyme B secretion compared to isotype controls (p<0.001). Dual immune checkpoint blockade with anti-PD-L1 and anti-BTLA antibodies had a synergistic effect in delaying tumor growth. We also measured a significant increase in CD8+CD107a+IFN-gamma+ secreting T cells (37%) compared to TIL from mice treated with anti-PD-L1 (27%) or anti-BTLA antibody alone (25%) (p<0.002). These findings suggest that targeting immune checkpoint molecules can improve T cell infiltration and anti-tumor immunity in a preclinical model of colorectal cancer. In addition, we demonstrate that tumor-specific TIL can be expanded from the tumors of colorectal patients. This study presents a rationale for the potential synergy between blockade of multiple immune checkpoints and adoptive transfer of TIL for the treatment of colorectal cancer. Citation Format: Krithika N. Kodumudi, James Charles, Carrie Luu, John Mullinax, Julie Li, MacLean Hall, Amy Weber, Amod A. Sarnaik, Shari Pilon-Thomas. Immune checkpoint blockade to improve T cell infiltration and function in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B12.

Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model.

Mechanisms of self-tolerance often result in CD8(+) tumor-infiltrating lymphocytes (TIL) with a hypofunctional phenotype incapable of tumor clearance. Using a transplantable colon carcinoma model, we found that CD8(+) T cells became tolerized in <24 h in an established tumor environment. To define the collective impact of pathways suppressing TIL function, we compared genome-wide mRNA expression of tumor-specific CD8(+) T cells from the tumor and periphery. Notably, gene expression induced during TIL hypofunction more closely resembled self-tolerance than viral exhaustion. Differential gene expression was refined to identify a core set of genes that defined hypofunctional TIL; these data comprise the first molecular profile of tumor-specific TIL that are naturally responding and represent a polyclonal repertoire. The molecular profile of TIL was further dissected to determine the extent of overlap and distinction between pathways that collectively restrict T cell functions. As suggested by the molecular profile of TIL, protein expression of inhibitory receptor LAG-3 was differentially regulated throughout prolonged late-G1/early-S phase of the cell cycle. Our data may accelerate efficient identification of combination therapies to boost anti-tumor function of TIL specifically against tumor cells.

Selection of CD8+PD-1+ Lymphocytes in Fresh Human Melanomas Enriches for Tumor-reactive T Cells

CD8+ tumor-infiltrating lymphocytes (TILs) in human melanomas express high levels of PD-1 and are functionally impaired. However, adoptive cell therapy using in vitro-expanded TIL can be a highly effective therapy for patients with advanced melanoma. This discrepancy led us to further analyze the CD8+PD-1+ TILs. We found that the percentage of PD-1-expressing CD8+ T cells was higher in the tumor digests that generate tumor-reactive TILs after in vitro culture in interleukin-2 (P=0.0007). Also sorted and expanded CD8+PD-1+ T cells in tumor digests showed much higher tumor-specific interferon-γ production compared with CD8+PD-1⁻ T cells. These results suggested that tumor-specific CD8+ T cells in melanoma tumor digests are largely PD-1, and this population can recover function after culturing in interleukin-2. PD-1 has been reported as an inhibitory receptor on T cells. We found that the in vitro functional suppression of cultured-TILs from native levels of PD-L1 expression on melanomas was minimal, and moreover expression level of PD-1 on CD8+ tumor-specific TILs decreased during the culture. As a consequence, the PD-1 receptor can be a useful biomarker for enriching tumor-specific T cells from fresh melanomas.

Cancer Metastasis Is Accelerated through Immunosuppression during Snail-Induced EMT of Cancer Cells

Epithelial-mesenchymal transition (EMT) is a key step toward cancer metastasis, and Snail is a major transcription factor governing EMT. Here, we demonstrate that Snail-induced EMT accelerates cancer metastasis through not only enhanced invasion but also induction of immunosuppression. Murine and human melanoma cells with typical EMT features after snail transduction induced regulatory T cells and impaired dendritic cells in vitro and in vivo partly through TSP1 production. Although Snail(+) melanoma did not respond to immunotherapy, intratumoral injection with snail-specific siRNA or anti-TSP1 monoclonal antibody significantly inhibited tumor growth and metastasis following increase of tumor-specific tumor-infiltrating lymphocytes and systemic immune responses. These results suggest that inhibition of Snail-induced EMT could simultaneously suppress both tumor metastasis and immunosuppression in cancer patients.

Persistence of tumor infiltrating lymphocytes in adoptive immunotherapy correlates with telomere length.

Transfer of autologous tumor-specific tumor infiltrating lymphocytes (TILs) in adoptive immunotherapy can mediate the regression of tumor in patients with metastatic melanoma. In this procedure, TILs from resected tumors are expanded in vitro, then administered to patients and further stimulated to proliferate in vivo by the administration of high dose IL-2. After in vitro expansion, TILs are often dominated by a few specific clonotypes, and recently it was reported that the persistence in vivo of one or more of these clonotypes correlated with positive therapeutic response. We and others have previously shown that repeated in vitro stimulation and clonal expansion of normal human T lymphocytes results in progressive decrease in telomerase activity and shortening of telomeres, ultimately resulting in replicative senescence. In the studies reported here, we therefore compared telomerase activity and telomere length in persistent and nonpersistent TIL clonotypes before transfer in vivo, and found a correlation between telomere length and clonal persistence. We also observed that TILs proliferate extensively in vivo in the days after transfer, but fail to induce substantial telomerase activity, and undergo rapid decreases in telomere length within days after transfer. Thus, in vivo loss of telomeres by clonotypes that have the shortest telomeres at the time of administration may drive these clones to replicative senescence, whereas cells with longer telomeres are able to persist and mediate antitumor effects. These findings are relevant both to predicting effectiveness of adoptive immunotherapy and in deriving strategies for improving effectiveness by sustaining telomere length.

Characterization of immune responses in gastric cancer patients: A possible impact of H. pylori to polarize a tumor-specific type 1 response?

Recently, we were able to show that Helicobacter pylori-positive gastric cancer (GC) patients have a significantly better survival after the complete resection of their tumor compared to H. pylori-negative GC patients. H. pylori is known to polarize an immune response towards a type 1 cytokine profile and tumor-specific type 1 cytokine responses are associated with protection from tumor challenge and T-cell-mediated tumor regression. Therefore, we hypothesized that the improved survival in H. pylori-positive patients may be secondary to the induction of a GC-specific type 1 T cell response. To characterize the anti-tumor immune response in GC patients we analyzed tumor-infiltrating lymphocytes (TIL) isolated from primary tumors. The CD3+ T cell population contained 50% CD4+ (range 0.4–81%) and 39% CD8+ cells (range 22–53%). The number of B cells (CD19+, P = 0.03) was significantly increased and the number of T cells (CD3+, P = 0.02) significantly decreased in intestinal compared to diffuse type of tumors. Four tumor cell lines were established from primary GCs and three from lymph node metastases. T cell cultures were established from isolated TIL from four H. pylori-positive and one H. pylori-negative GC patients and tested for tumor-specific cytokine secretion. Eight of ten T cell cultures derived from H. pylori-positive patients secreted both IFN-γ and IL-5 after restimulation with autologous tumor cells. The only tumor-specific TIL line expressing a dominant IL-5 response was derived from an H. pylori-negative patient.

The in vitro effects of interleukin-12 upon tumor-infiltrating lymphocytes derived from renal cell carcinoma.

Clinical trials utilising interleukin (IL)-2 with tumor-infiltrating lymphocytes (TIL) have demonstrated efficacy in the treatment of metastatic renal cell carcinoma (RCC). Several cytokines, as well as growth factors have demonstrated modulatory effects upon the biological properties of TIL from RCC, suggesting a potentially important role for cytokines other than IL-2 in the development of active and tumor-specific TIL. IL-12 was recently characterized as a natural-killer-cell-stimulatory factor or cytotoxic-T-cell-maturation factor. These properties of IL-12 prompted us to investigate the impact of this cytokine upon the activation of TIL from human RCC. In an attempt to enhance the in vitro growth and activity of renal TIL, we have grown eight renal TIL cultures in varying concentrations of IL-2 (8, 40, 80, 400 U/ml) and IL-12 (200 U/ml). In addition, IL-12 (200 U/ml) was added to TIL cultures pre-activated with IL-2 (400 U/ml). Growth, cell expansion, and the ability of TIL to release certain cytokines upon tumor stimulation were determined. Proliferation assays, phenotypic analysis, and cytotoxicity assays were performed at an early and a late culture stage. IL-12, alone and when added to suboptimal concentrations of IL-2, failed to induce TIL growth. While the addition of IL-12 to optimal doses of IL-2 suppressed TIL culture expansion, sequential culture exposure first to IL-2 and then to IL-2+IL-12 increased the number of cells expressing CD3+/CD56+ and these cultures demonstrated enhanced in vitro lysis of autologous tumor. IL-12 clearly demonstrated a sequence-dependent impact of the biological behaviour of TIL from RCC. The optimal use of IL-12 in the in vitro expansion of renal TIL may result in cells with an enhanced specific anti-tumor effect.

Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1.

MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1(27-35)-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2+ melanoma patients, as well as from 2 of 4 PBL from HLA-A2+ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1(27-35) peptide. These CTL lines specifically lysed and release cytokines (TNF-alpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART-1(27-35), as well as to HLA-A2+ MART-1+ melanoma cells. CTL generated with MART-1(27-35) also lysed uncultured HLA-A2+ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- to 100-fold higher lytic activity than MART-1-reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.

T cell receptor V beta 2 and V beta 6 mediate tumor-specific cytotoxicity by tumor-infiltrating lymphocytes in ovarian cancer.

The interaction between T lymphocytes and the Ag-HLA complex on tumor cells is mediated by the TCR. The diversity and the specificity of the TCR are in part secondary to the gene rearrangement of the V region on the beta-chain (V beta). To determine whether a restricted number of TCR V beta genes are utilized in the recognition of ovarian cancer, tumor-infiltrating lymphocytes (TIL) were isolated from six consecutive untreated ovarian cancer patients. TIL were also cultured using repeated autologous tumor stimulation, and by 7 wk, five of six patients produced bulk cultures consisting of > 50% CD8+ T cells and demonstrating an autologous tumor-specific pattern of cytotoxicity. TCR V beta gene usage was analyzed in the five patients yielding fresh TIL and corresponding 7-wk cultured, tumor-specific TIL; 22 primers specific for 20 TCR V beta gene families were employed and amplified by polymerase chain reaction and then quantitated by HPLC. A heterogeneous pattern of V beta usage was seen in the fresh TIL; however, V beta 2, V beta 3, V beta 6, V beta 7, V beta 8, and V beta 13.1 were found in increased proportions in at least three of five patients. In the 7-wk tumor-specific TIL, V beta analysis showed an increased usage of V beta 2, V beta 3, V beta 6, and V beta 7 in more than three of five patients. No significant change in V beta representation was seen in control populations that were not stimulated with tumor. Looking at the percent change in V beta usage between fresh and 7-wk tumor-specific cultures, V beta 2 and V beta 6 were augmented significantly in at least three of five patients (108% and 61%, respectively). To verify that the increase in representation of these V beta families was responsible for the increased cytotoxicity observed, mAb specific for V beta 2 and V beta 6 were used to block tumor lysis. Anti-V beta 6 and anti-V beta 2 significantly blocked cytotoxicity against autologous tumor cells in those TIL populations expressing increased levels of these V beta families. These data suggest that a selective repertoire of TCR V beta genes is used to recognize the Ag-HLA class 1 complexes on the surface of ovarian tumor cells, and specifically V beta 2 and V beta 6 appear to mediate antitumor activity. These findings may aid in the development of a more specific immunotherapy in ovarian cancer.

Human melanoma-mediated inhibition of autologous CD4+ helper tumor-infiltrating lymphocyte growth in vitro.

Tumor-infiltrating lymphocytes (TIL) were isolated from a human melanoma metastatic to the abdomen. The TIL were 99% CD3+ and 99% CD4+ and CD8-. They were dependent on interleukin-2 (IL-2) for growth, as measured in a thymidine uptake assay, and were not cytotoxic to autologous or allogeneic melanoma or K562. When co-cultured with irradiated autologous tumor cells, or tumor cell supernatants, the TIL not only did not respond, but the IL-2-dependent growth was inhibited significantly. Inhibition occurred during the first 24 hours of co-culture and persisted as long as the tumor was present. After being washed free of inhibitory tumor cells, the TIL again were able to grow in the presence of IL-2, indicating that the inhibition was not caused by irreversible toxicity mediated by the tumor. Addition of excess IL-2 did not reverse the inhibitory effect, but addition of indomethacin, an inhibitor of cyclooxygenase and prostaglandin synthesis, partially blocked the inhibition. These data show melanoma-mediated inhibition of induction and expansion of human T-cells in vitro, which may reflect one of the mechanisms of inhibition of cellular responses in vivo. These results stress the need to examine the techniques for optimal in vitro expansion of tumor-specific TIL or cytotoxic T-cells for adoptive immunotherapy.