Function Of Tumor-infiltrating Lymphocytes Research Articles

MP48-20 EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES (TIL) FROM PRIMARY BLADDER TUMORS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2017MP48-20 EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES (TIL) FROM PRIMARY BLADDER TUMORS Michael Poch, Maclean Hall, Krithika Kodumudi Kodumudi, Cortlin Croft, Mayer Fishman, John Mullinax, Amod Sarniak, James Mule, and Shari Pilon-Thomas Michael PochMichael Poch More articles by this author , Maclean HallMaclean Hall More articles by this author , Krithika Kodumudi KodumudiKrithika Kodumudi Kodumudi More articles by this author , Cortlin CroftCortlin Croft More articles by this author , Mayer FishmanMayer Fishman More articles by this author , John MullinaxJohn Mullinax More articles by this author , Amod SarniakAmod Sarniak More articles by this author , James MuleJames Mule More articles by this author , and Shari Pilon-ThomasShari Pilon-Thomas More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1501AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Patients with advanced bladder cancer have limited therapeutic options resulting in a median overall survival (OS) between 12 and 15 months. At our institution, adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) has resulted in a durable median OS of 52 months in patients with metastatic melanoma. Immune-mediated anti-tumor responses have been previously shown in bladder cancer, therefore we investigated the phenotype and function of TIL expanded from bladder tumors to establish feasibility of ACT for the treatment of bladder cancer. METHODS Tumor specimens, including primary bladder tumors and lymph node metastases, were collected from 29 bladder cancer patients having standard of care tumor resection, who also had consented to an IRB-approved protocol for TIL generation. The tissue was minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. TIL were considered expanded if they propagated to fill =2 wells. The remaining tumor material was digested into a single cell suspension and frozen. TIL were phenotyped by flow cytometry and assessed for autologous tumor reactivity through co-culture with tumor digest and IFN-gamma ELISA. RESULTS Twenty-seven of 29 tumors were harvested from radical cystectomy patients. Urothelial cell bladder tumors were cultured from 23 patients, of whom 19 (83%) demonstrated TIL expansion. Among these were 9/12 with preceding chemotherapy and 10/11 which had been chemotherapy naive. Expanded TIL were predominantly CD3+ (median 63%, range 10-87%) with a median of 30% CD8+ T cells (range 5-70%). Eight of 15 tested samples (53%) contained TIL that secreted IFN-gamma in response to autologous tumor. Microbial contamination was observed in six specimens. All lymphatic (5/5 samples) tissue collected demonstrated TIL expansion. CONCLUSIONS The study establishes the practical first step towards an autologous TIL therapy process for therapeutic testing in patients with bladder cancer. Human bladder cancer tissue can be used to expand tumor-specific TIL in vitro. TIL were also able to be expanded from patients that received chemotherapy prior to tumor resection. Future efforts will explore the ability to further expand bladder TIL cultures to clinically meaningful numbers to develop novel ACT strategies for patients with this diagnosis. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e645 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Michael Poch More articles by this author Maclean Hall More articles by this author Krithika Kodumudi Kodumudi More articles by this author Cortlin Croft More articles by this author Mayer Fishman More articles by this author John Mullinax More articles by this author Amod Sarniak More articles by this author James Mule More articles by this author Shari Pilon-Thomas More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Evaluating the antitumor role of B cells in patients with non-small cell lung cancer.

75 Background: The focus of immunotherapy has been on CD8 and CD4 tumor infiltrating lymphocytes (TILs), however, tumor infiltrating B cells (TIL-Bs) have been reported in tertiary lymphoid structures (TLS) with CD4 TILs, and both TIL-Bs and TLS correlate with NSCLC patient survival. While TIL-Bs have been identified in NSCLC patients, their function in the tumor microenvironment has been understudied with no focus on their role as antigen presenting cells (APCs) and their influence on CD8 and CD4 TILs. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer by presenting tumor antigens to CD4 TILs. Methods: We used freshly isolated B cells from NSCLC patient tumor and tumor adjacent tissue to determine TIL-B function. We generated a specific in vitro antigen presentation assay to determine APC function and immunoregulation by TIL-Bs, and we correlated our findings with tumor-free survival. Results: We observed that the total number of B cells at the site of the tumor versus the tumor-adjacent tissue was increased compared to other immune subsets. Further, we observed three types of CD4 TIL responses when TIL-Bs presented autologous tumor antigens. There were activated responder CD4 TILs that proliferated when combined with TIL-Bs alone, which indicates stimulation with endogenous tumor antigens. There were antigen-associated responders that required exogenous autologous tumor lysate to elicit a CD4 TIL response, and there were patient CD4 TILs that did not respond to antigen presentation by TIL-Bs. Within the activated and antigen-associated responders, the TIL-B phenotype influenced the CD4 TIL phenotype; if the TIL-Bs were activated (CD69+CD27+CD21+), the CD4 TILs were T helper (anti-tumor) CD4 T cells and if the TIL-Bs were exhausted (CD69-CD27-CD21-), the CD4 TILs were T regulatory cells (pro-tumor). These data suggest that TIL-Bs influence the phenotype and function of CD4 TILs in NSCLC patient tumors. Conclusions: In conclusion, determining if TIL-Bs are activated or exhausted in NSCLC patients will determine the extent of their anti-tumor function in cancer. Ultimately, results from this study will help predict how to target TIL-B functions in future immunotherapies for NSCLC patients.

Expansion of tumor infiltrating lymphocytes (TIL) from bladder cancer.

142 Background: Patients with advanced bladder cancer have limited therapeutic options resulting in a median overall survival (OS) between 12 and 15 months. At our institution, adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) has resulted in a durable median OS of 52 months in patients with metastatic melanoma. Immune-mediated anti-tumor responses have been previously shown in bladder cancer, therefore we investigated the phenotype and function of TIL expanded from bladder tumors to establish feasibility of ACT for the treatment of bladder cancer. Methods: Tumor specimens, including primary bladder tumors and lymph node metastases, were collected from 29 bladder cancer patients having standard of care tumor resection, who also had consented to an IRB-approved protocol for TIL generation. The tissue was minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. TIL were considered expanded if they propagated to fill ≥2 wells. The remaining tumor material was digested into a single cell suspension and frozen. TIL were phenotyped by flow cytometry and assessed for autologous tumor reactivity through co-culture with tumor digest and IFN-gamma ELISA. Results: Transitional cell bladder tumors were cultured from 23 patients, of whom 19 (83%) demonstrated TIL expansion. Microbial contamination precluded TIL growth in six specimens. TIL were cultured from 9/12 (75%) patients with preceding chemotherapy and 10/11 (91%) who were chemotherapy naive. Expanded TIL were predominantly CD3+(median 63%, range 10-87%) with a median of 30% CD8+ T cells (range 5-70%). Eight of 15 tested samples (53%) contained TIL that secreted IFN-gamma in response to autologous tumor. Conclusions: The study establishes the practical first step towards an autologous TIL therapy process for therapeutic testing in patients with bladder cancer. Human bladder cancer tissue can be used to expand tumor-specific TIL in vitro. TIL were also expanded from patients that received chemotherapy prior to tumor resection. Future efforts will explore the ability to further expand bladder TIL cultures to clinically meaningful numbers to develop novel ACT strategies for patients with this diagnosis.

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.

The Characteristics of Naive-like T Cells in Tumor-infiltrating Lymphocytes From Human Lung Cancer.

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TILs) or genetically modified lymphocytes from TILs is a new effective approach, but the application of TIL immunotherapy is still limited in many solid tumors. Knowledge of the classification and function of TILs is important to develop personalized immunotherapy with TILs in non-small lung cancer (NSCLC). In this study, we show the characteristics of T-cell subsets in TILs isolated from NSCLC. CD3 CD8 CD45RA T cells outnumbered CD3 CD4 CD45RA T cells in CD45RA TILs, but it was the opposite in CD45RO TILs. Effector memory CD4 T cells predominated in CD4 TILs; about 10% of the stem cell-like memory T cells (Tscm) were detected in TILs. To further analyze their functions, we stimulated TILs from NSCLC patients by mitogens to examine cytokine production. Our data demonstrated that naive-phenotype T cells in TILs secret IFN-γ in abundance; TNF-α-producing T cells were significantly increased in TILs; there were more IL-17-expressing CD4 Tscm cells than other subtypes of CD4T cells in TILs. Our findings indicate that the CD4/CD8 naive-phenotype T cells and Tscm cells in TILs from lung cancer exhibit distinct composition and strong cytokine production. Attributes of Tscm cells from a naive-like T-cell population in TILs are the promising cell type for adoptive cell therapy in human lung cancer.

TGFβ1-Mediated SMAD3 Enhances PD-1 Expression on Antigen-Specific T Cells in Cancer.

Engagement of the coinhibitory receptor PD-1 or its ligand, PD-L1, dramatically inhibits the antitumor function of TILs within the TME. Our findings represent a novel immunosuppressive function of TGFβ and demonstrate that TGFβ1 allows tumors to evade host immune responses in part through enhanced SMAD3-mediated PD-1 expression on TILs. Cancer Discov; 6(12); 1366-81. ©2016 AACRThis article is highlighted in the In This Issue feature, p. 1293.

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.

Abstract B30: Discovery and functional characterization of novel anti-PD-1 antibodies using ex vivo cell-based assays, single-cell immunoprofiling, and in vivo studies in humanized mice

Abstract Given recent approvals of anti-PD-1 inhibitors Keytruda (pembrolizumab) and Opdivo (nivolumab) for treatment of metastatic melanoma and non-small cell lung cancer NSCLC, we asked whether additional anti-PD-1 inhibitors with different epitopes or potentially differentiated mechanisms of action can provide clinical benefit beyond the two marketed therapies. Using Enumeral's proprietary single cell technology, we generated more than 300 anti-PD-1 monoclonal antibodies from primary B cells. Bioinformatics analysis of sequences show these antibodies comprise 26 distinct clades, or families, that bind to PD-1. Results from binding studies further indicate we have discovered a family of novel antibodies that do not appear to compete with currently marketed antibodies for binding to PD-1, nor do they appear to compete with PD-L1, suggesting a differentiated mechanism of action. We have humanized two lead antibodies for preclinical testing and in preparation for clinical studies. Here we describe functional characterization of the two lead antibodies using cell-based ex vivo assays, patient-derived tumor profiling assays using our single-cell platform, and in vivo studies in humanized NSG mice. Results show that the two lead anti-PD-1 antibodies exhibit a higher level of T cell activation in mixed lymphocyte reaction (MLR) assays using primary human immune cells. Second, these antibodies also demonstrated dose-dependent increases in T cell CD25 expression. Next, to test the effect of the two lead anti-PD-1 antibodies on tumor growth, we are conducting a study using a patient-derived tumor (PDX) model on humanized NSG mice. NSG mice reconstitute a full human immune system including a functional T, B cell repertoire, enabling the direct, in vivo evaluation of our human anti-PD-1 antibodies. Effects of drug treatment on tumor growth, transcriptome (RNA seq) and protein expression (IHC) will be described. Finally, results from ex vivo tumor profiling comparing the immmunomodulatory effects of our anti-PD-1 antibodies on tumor-infiltrating lymphocyte function will also be presented. Citation Format: Felix Scheuplein, Sheila Ranganath, Thomas McQuade, Lei Wang, Vikki Spaulding, Sri Vadde, Jennifer Watkins-Yoon, Bin Feng, Shanu Mehta, Maria Isabel Chiu, Cokey Nguyen. Discovery and functional characterization of novel anti-PD-1 antibodies using ex vivo cell-based assays, single-cell immunoprofiling, and in vivo studies in humanized mice. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B30.

Galectin-3 inhibition using novel inhibitor GR-MD-02 improves survival and immune function while reducing tumor vasculature

Abstract Immunosuppression and reduced cytotoxic function of tumor-infiltrating lymphocytes (TIL) are major obstacles to creating effective therapies for patients. It is known that Galectin-3 (Gal3), a lectin family member, is expressed and secreted by numerous cancers and immune cell subsets. Serum Gal3 expression is higher in patients with metastatic versus non-metastatic disease, and is associated with reduced survival in metastatic melanoma. Furthermore, Gal3 has been implicated in disease progression via the promotion of angiogenesis and metastasis, the inhibition of TIL function, and promoting M2 polarization of macrophages and mobilization of myeloid cells from the bone marrow to promote a metastatic niche within the tumor. We hypothesized that Gal3 inhibition would improve TIL function while inhibiting the growth and metastasis of tumors. Through collaboration with Galectin Therapeutics, we tested Gal3 inhibition using the novel Gal3 inhibitor, GR-MD-02, with agonist anti-OX40 therapy. We observed that Gal3 inhibition/OX40 agonism improved survival in the MCA-205 sarcoma and 4T-1 mammary carcinoma models, and prolonged survival in the TRAMP-C1 prostate cancer model. Importantly, Gal3 inhibition/OX40 agonism reduced lung metastases in the 4T-1 model. Within the tumor, we observed an increase in the number of proliferating and IFNg-producing TIL. Gal3 inhibition/OX40 agonism was also associated with a decrease in tumor vasculature, as determined by CD31 staining by IHC within the tumors. These studies, along with clinical data from a liver fibrosis trial, supported testing GR-MD-02 in combination with additional immunotherapies to augment T cell function in patients with metastatic melanoma.

Immune Checkpoint Blockade to Improve Tumor Infiltrating Lymphocytes for Adoptive Cell Therapy.

Tumor-infiltrating lymphocytes (TIL) has been associated with improved survival in cancer patients. Within the tumor microenvironment, regulatory cells and expression of co-inhibitory immune checkpoint molecules can lead to the inactivation of TIL. Hence, there is a need to develop strategies that disrupt these negative regulators to achieve robust anti-tumor immune responses. We evaluated the blockade of immune checkpoints and their effect on T cell infiltration and function. We examined the ability of TIL to induce tumor-specific immune responses in vitro and in vivo. TIL isolated from tumor bearing mice were tumor-specific and expressed co-inhibitory immune checkpoint molecules. Administration of monoclonal antibodies against immune checkpoints led to a significant delay in tumor growth. However, anti-PD-L1 antibody treated mice had a significant increase in T cell infiltration and IFN-γ production compared to other groups. Adoptive transfer of in vitro expanded TIL from tumors of anti-PD-L1 antibody treated mice led to a significant delay in tumor growth. Blockade of co-inhibitory immune checkpoints could be an effective strategy to improve TIL infiltration and function.

Tolerance of Tumor-Specific T cells in Melanoma Metastases.

Tolerance of Tumor-Specific T cells in Melanoma Metastases.

Abstract 1283: In situ activity of tumor specific CTL is a prognostic factor in colorectal cancer

Abstract The extent of T cell infiltrates in the primary tumor is one of the most important prognostic factors in colorectal cancer (CRC). Still, a putative causal role of T cell infiltrates for prolonged patient survival remains unclear. To answer this question, we studied the presence and functional activity of tumor infiltrating lymphocytes (TIL) together with their specificity in CRC patients and correlated the obtained results to long term survival. We have utilized intracytoplasmic cytokine staining, conjointly with human leukocyte antigen (HLA)- multimers loaded with peptides derived from tumor antigen (TA), as well as antigen specific cytokine secretion assays, to demonstrate that tumor necrosis factor alpha (TNFα) expression characterizes a population of TA specific, in situ active cytotoxic T cells. TNFα was increased in CRC tumors only when patients develop spontaneous systemic T cell responses, as elucidated by IFN-γ Elispot, and this increase correlated with the number of TNFα expressing tumor infiltrating T cells. Based on these observations we concluded that intratumoral TNFα can be a direct indicator of the in situ functionality of tumor specific effector T cells in CRC. In accordance, intratumoral TNFα concentration was an independent long term prognostic factor in a retrospective multivariate analysis of a cohort of 102 CRC patients involving various other immune parameters, such as CD4+, CD8+ T cell infiltration, presence of regulatory T cells or mast cells. In conclusion, our findings provide evidence for a causal relationship between T cell infiltrates and improved survival in CRC patients as the prognostic impact of T cell infiltrates depends on small subpopulations of in situ active TA specific T cells. Furthermore TNFα, as an indicator for the functionality of TIL, might be better and more accurate prognostic parameter in CRC than mere T cell infiltration. Citation Format: Slava Stamova, Christoph Reissfelder, Christina Gossmann, Marion Braun, Andreas Bonertz, Ute Walliczek, Moritz Koch, Axel Benner, Dirk Jäger, Niels Halama, Khashayarsha Khazaie, Jürgen Weitz, Philipp Beckhove. In situ activity of tumor specific CTL is a prognostic factor in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1283. doi:10.1158/1538-7445.AM2015-1283

Characterizing the immunoprofile and endogenous immune response to squamous cell carcinomas of the head and neck to guide development of effective immunotherapy strategies

Squamous cell carcinoma of the head and neck (HNSCC) is the 6th leading cause of cancer by incidence worldwide with approximately 600,000 new cases per year. Unfortunately, only 40-50% of these patients will survive for 5 years. In order to study the immune response in patients with this disease we have developed a HNSCC tumor bank to compliment our Oral, Head and Neck Cancer Program. This tumor bank is cryopreserving enzymatically isolated viable cells from resected tumors (n = 128). We are also attempting to develop primary cell lines (10 lines established) and are isolating and assessing tumor-specific function of tumor-infiltrating lymphocytes (TIL) (n = 49). Since HNSCC can express immune inhibitory molecules and secrete suppressive factors, we are developing single stains and multi-spectral imaging protocols to assess the immune contexture of the tumor microenvironment by immunohistochemistry and immunoflouresence and are overlaying these studies with T cell functional activity and ultimately, with clinical outcome. Preliminary analyses suggest that tumor-specific T cells can be detected in 68% (N = 16) of patients evaluated. A goal of these studies is to identify strategies that will allow tailoring of therapy for patients with HNSCC. One component is to identify which inhibitors are present in a given tumor. Since not every tumor appears to contain TIL capable of recognizing autologous tumor, strategies to prime tumor-specific T cells represents another area of interest. DPV-001 is a microvesicle vaccine, DRibbles, that contains an average of at least 66 proteins that are overexpressed by HNSCC (TCGA provisional RNASeq n = 303 pts). The vaccine also contains multiple DAMPs and agonist activity for TLR 2, 3, 4, 7 and 9 packed into stable double membrane microvesicles that are targeted to CLEC9A+ APC. To increase potential activity against HPV positive cancers we have developed a mosaic construct encoding E6 and E7 peptides for a number of HPV strains and are evaluating both protein and gene-based HPV vaccine strategies. We are using the HNSCC TIL lines to evaluate DRibble vaccines and potential for adoptive immunotherapy trials. Support: NCI U43CA165048 and R44 CA121612 (TLH), OMS (HH, RBB), Steve and Cindy Harder, Robert W. and Elsie Franz, Wes and Nancy Lematta, Lynn and Jack Loacker, and The Chiles foundation (BAF).

A Short Treatment with Galactomannan GM-CT-01 Corrects the Functions of Freshly Isolated Human Tumor–Infiltrating Lymphocytes

Several galectins are released by tumor cells and macrophages and accumulate in the tumor microenvironment. Galectin-1 and -3 were found to bind to glycosylated receptors at the surface of tumor-infiltrating lymphocytes (TIL), forming glycoprotein-galectin lattices that could reduce the motility and therefore the functionality of surface molecules. In contrast to blood T cells, human TIL show defective IFN-γ secretion upon ex vivo stimulation. We have previously shown that extracellular galectin-3 participates in the impairment of TIL functions. Indeed, disruption of glycoprotein-galectin-3 lattices using anti-galectin-3 antibodies, or N-acetyllactosamine as a competing sugar, boosted cytokine secretion by TIL. Here we have tested a clinical grade galectin antagonist: GM-CT-01, a galactomannan obtained from guar gum reported to be safe in more than 50 patients with cancer. TIL were isolated from human tumor ascites, treated for 2 to 20 hours with galectin antagonists and tested for function. We found that GM-CT-01 boosts cytotoxicity of CD8(+) TIL and their IFN-γ secretion in a dose-dependent manner. Treating TIL obtained from patients with various cancers, during a few hours, resulted in an increased IFN-γ secretion in up to 80% of the samples. These observations pave the way for investigating the potential benefit of this galectin antagonist in patients with cancer, alone or combined with cancer vaccination, in order to correct in vivo impaired functions of TIL.

Immunotherapy-induced CD8+ T Cells Instigate Immune Suppression in the Tumor

Despite clear evidence of immunogenicity, cancer vaccines only provide a modest clinical benefit. To evaluate the mechanisms that limit tumor regression following vaccination, we have investigated the weak efficacy of a highly immunogenic experimental vaccine using a murine melanoma model. We discovered that the tumor adapts rapidly to the immune attack instigated by tumor-specific CD8+ T cells in the first few days following vaccination, resulting in the upregulation of a complex set of biological networks, including multiple immunosuppressive processes. This rapid adaptation acts to prevent sustained local immune attack, despite continued infiltration by increasing numbers of tumor-specific T cells. Combining vaccination with adoptive transfer of tumor-specific T cells produced complete regression of the treated tumors but did not prevent the adaptive immunosuppression. In fact, the adaptive immunosuppressive pathways were more highly induced in regressing tumors, commensurate with the enhanced level of immune attack. Examination of tumor infiltrating T-cell functionality revealed that the adaptive immunosuppression leads to a progressive loss in T-cell function, even in tumors that are regressing. These novel observations that T cells produced by therapeutic intervention can instigate a rapid adaptive immunosuppressive response within the tumor have important implications for clinical implementation of immunotherapies.

Markers of T Cell Infiltration and Function Associate with Favorable Outcome in Vascularized High-Grade Serous Ovarian Carcinoma

BackgroundWhen T cells infiltrate the tumor environment they encounter a myriad of metabolic stressors including hypoxia. Overcoming the limitations imposed by an inadequate tumor vasculature that contributes to these stressors may be a crucial step to immune cells mounting an effective anti-tumor response. We sought to determine whether the functional capacity of tumor infiltrating lymphocytes (TIL) could be influenced by the tumor vasculature and correlated this with survival in patients with ovarian cancer.Methodology and Principal FindingsIn 196 high-grade serous ovarian tumors, we confirmed that the tumor vascularity as measured by the marker CD31 was associated with improved patient disease-specific survival. We also found that tumors positive for markers of TIL (CD8, CD4 and forkhead box P3 (FoxP3)) and T cell function (granzyme B and T-cell restricted intracellular antigen-1 (TIA-1)) correlated significantly with elevated vascularity. In vitro, hypoxic CD8 T cells showed reduced cytolytic activity, secreted less effector cytokines and upregulated autophagy. Survival analysis revealed that patients had a significant improvement in disease-specific survival when FoxP3 expressing cells were present in CD31-high tumors compared to patients with FoxP3 expressing cells in CD31-low tumors [HR: 2.314 (95% CI 1.049–5.106); p = 0.0377]. Patients with high vascular endothelial growth factor (VEGF) expressing tumors containing granzyme B positive cells had improved survival compared to patients with granzyme B positive cells in VEGF-low tumors [HR: 2.522 (95% CI 1.097–5.799); p = 0.0294].SignificanceOverall, this data provides a rationale for developing strategies aimed at improving the adaptability and function of TIL to hypoxic tumor conditions.

Phenotype and Function of T Cells Infiltrating Visceral Metastases from Gastrointestinal Cancers and Melanoma: Implications for Adoptive Cell Transfer Therapy

Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3(+) T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8(+) cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ~50 × 10(9) T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3(+)CD8(+) cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I-mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I-mediated up-regulation of CD137 (4-1BB) expression on CD8(+) cells suggested that 0-3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells.

Local Administration of TLR Ligands Rescues the Function of Tumor-Infiltrating CD8 T Cells and Enhances the Antitumor Effect of Lentivector Immunization

Cancer vaccines, to date, have shown limited effect to control the growth of established tumors due largely to effector failure of the antitumor immune responses. Tumor lesion is characterized as chronic indolent inflammation in which the effector function of tumor-infiltrating lymphocytes (TILs) is severely impaired. In this study, we investigated whether the effector function of CD8 TILs could be rescued by converting the chronic inflammation milieu to acute inflammation within tumors. We found that injection of TLR3/9 ligands (polyI:C/CpG) into a tumor during the effector phase of lentivector (lv) immunization effectively rescued the function of lv-activated CD8 TILs and decreased the percentage of T regulatory within the tumor, resulting in a marked improvement in the antitumor efficacy of lv immunization. Mechanistically, rescue of the effector function of CD8 TILs by TLR3/9 ligands is most likely dependent on production, within a tumor, of type-1 IFN that can mature and activate tumor-infiltrating dendritic cells. The effector function of CD8 TILs could not be rescued in mice lacking intact type I IFN signaling. These findings have important implications for tumor immunotherapy, suggesting that type I IFN-mediated activation of tumor-infiltrating dendritic cells within a tumor will most likely restore/enhance the effector function of CD8 TILs and thus improve the antitumor efficacy of current cancer vaccines.

Tumor-derived exosome alters the proliferation and function of T lymphocytes in nasopharygneal carcinoma (P2111)

Abstract Background: Tumor-derived exosomes take an important role in the inflammatory responses and anti-tumor immune responses in tumor microenvironments. Here, we used exosomes from NPC tumor cell line TW03-EBV+ (exosome1) and TW03-EBV- (exosome2) to evaluate the impact of exosome1 and exosome2 on the proliferation of CD4+ and CD8+ T lymphocytes, the differentiation of CD4+ naïve T cells mediated by NPC tumor cells, and the lysis function of tumor-infiltrating lymphocytes (TILs) to the tumor cells from NPC. Results: Exosome1 and exosome2 all could promote the proliferation of CD4+ and CD8+ lymphocytes stimulated by OKT3 or PHA through up-regulated the phosphorylation of Erk-1,2; however exosome1 and exosome2 inhibited the proliferation of CD4+ and CD8+ lymphocytes in IL-2 medium; Exosome1 and exosome2 inhibited the Th1 and Th17 cell differentiation mediated by NPC tumor cells through changing the phosphorylation of STAT1 and STAT3; Exosome1 and exosome2 inhibited the lysis function of TIL to EBV-positive NPC tumor cell line TW03. Conclusions: The tumor-derived exosomes can alter the cross talk between lymphocytes and tumor cells in NPC, and maybe be a potential tool for NPC immunotherapy in the near future.

Abstract A18: Mouse models of autochthonous cancer to study local immune subversion.

Abstract The tumor environment delivers suppressive signals to the immune system, which impair the effector function of tumor-infiltrating lymphocytes. The identity of these signals, their targets and their mode of action, however, are largely unresolved. We use TRAMP mice, a well-known autochthonous murine model for prostate cancer, to study the interaction of the adaptive immune system and cancer cells in order to investigate which cell types and molecules are crucial in deviating immune surveillance. Tumorigenesis in TRAMP mice is driven by SV40 large T (SV40LT), which allows monitoring of specific CD8 T cell responses to the tumor. We analyzed quantitative and qualitative aspects of the tumor-specific T cell response in the spleen, the draining and non-draining lymph nodes and in the tumor of TRAMP mice. Interestingly, this mouse model shows CD8 T cell tolerance towards epitope IV but not epitope I of SV40LT when infected with recombinant vaccinia viruses expressing these epitopes. However, when transferring TCR transgenic CD8 T cells specific for epitope I, we observed rapid local and peripheral immune subversion of adoptively transferred cells in tumor-bearing animals. Homing and proliferation were not affected, but effector functions of these cells were greatly impaired. Furthermore, this immunosuppression was more severe locally at the tumor site than in the periphery. When using a combination of blocking antibodies for co-inhibitory molecules (PD-1, CTLA-4 and TIM-3), we could restore to some extent the function of transferred cells in the periphery, but not at the tumor site. In parallel, we are in the process of generating an inducible CreERT/LoxP-based model to express the immunologically well-characterized oncogene SV40LT with the luciferase in a tissue specific fashion. This model will allow sensitive detection of arising tumors and precise dissection of tumor-specific immunity over time. By comparing fundamentally different target tissues, we may find mechanisms that are active in every malignancy and some that are organ-specific. Citation Format: Ali Bransi, Hideo Yagita, Alexander Knuth, Maries van den Broek. Mouse models of autochthonous cancer to study local immune subversion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A18.