Human Tumor-infiltrating Lymphocytes Research Articles

Abstract 1993: A comprehensive benchmarking of paired whole exome and transcriptome biomarker analysis of response to cancer immunotherapy

Abstract Recent advances in cancer immunotherapy have revolutionized cancer treatment. Notably, pembrolizumab - an anti-PD1 checkpoint inhibitor - has been approved as first-line treatment for metastatic non-small cell lung cancer. Clinical response to checkpoint inhibitors has been demonstrated in other cancer types; however, durable response is not observed in all patients. As a result, development of new biomarkers for response to checkpoint inhibitors has become an active area of research. Due to the complexity of the anti-tumor immune response, a single biomarker may not accurately predict patient response to immunotherapy. In parallel, dramatic decreases in the costs of next generation sequencing (NGS) have enabled broad whole exome sequencing (WES) and whole transcriptome sequencing (WTS)-based studies, enabling a new phase in both academic and clinical cancer research. Despite the proven utility of NGS-based biomarker analysis, the accuracy of combined WES/WTS results compared to other assays, specifically in clinical tumor tissues, has not been demonstrated. In this study, we perform comprehensive benchmarking of several immuno-oncology (IO) biomarkers on cell lines, fresh frozen tumor tissues, and formalin-fixed paraffin-embedded (FFPE) tumor tissues. WES and WTS libraries were generated using Illumina Nextera Flex for Enrichment, TruSight Oncology, and TruSeq Stranded Total RNA library prep methods, and sequenced on a NovaSeq 6000. Using an internally developed bioinformatics pipeline, the accuracy of several IO biomarker measurements was evaluated. Specifically, we investigated the performance of tumor-only (T) and/or paired tumor-normal (TN) WES/WTS in assessing tumor mutational burden (TMB), microsatellite instability (MSI), tumor purity, copy number variants (CNV), human leukocyte antigen (HLA) type, fusions, and tumor infiltrating lymphocytes (TILs) levels, among other features. Our benchmarking results demonstrated correlations of 0.99 and 0.98 for TMB as measured by TN and T, respectively (truth: FOCR reported TMB); 100.0% PPV and NPV for MSI status as measured by both TN and T (truth: MSI-PCR); 0.86 correlation for tumor purity estimates based on TN (0.64 based on T; truth: cell-line titration levels); 100% PPV for both TN and T CNV calling (truth: ddPCR); 92% accuracy for both TN and T HLA typing (truth: IHW HLA types); 92.9% sensitivity for fusion calling (truth: ddPCR); and R2 values of 0.87, 0.70, and 0.48 for CD19+, CD4+, and CD8+ TIL levels, respectively (truth: FACS). Taken together, this study demonstrates WES/WTS can not only be utilized for broad exome- and transcriptome-wide biomarker discovery, but can also be an accurate, comprehensive alternative to iteratively testing IO biomarkers. Future studies should further standardize assay and analysis approaches for NGS-based biomarker measurements to ensure consistent and accurate reporting. Citation Format: Mahdi Golkaram, Michael Salmans, Raakhee Vijayaraghavan, Shannon Kaplan, Robert Haigis, Joyee Yao, Kristina Kruglyak, Li Liu, Traci Pawlowski, Sven Bilke, Shile Zhang. A comprehensive benchmarking of paired whole exome and transcriptome biomarker analysis of response to cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1993.

Sirt2 Inhibition Enhances Metabolic Fitness and Effector Functions of Tumor-Reactive T Cells

Dysregulated metabolism is a key driver of maladaptive tumor-reactive T lymphocytes within the tumor microenvironment. Actionable targets that rescue the effector activity of antitumor Tcells remain elusive. Here, we report that the Sirtuin-2 (Sirt2) NAD+-dependent deacetylase inhibits Tcell metabolism and impairs Tcell effector functions. Remarkably, upregulation of Sirt2 in human tumor-infiltrating lymphocytes (TILs) negatively correlates with response to TIL therapy in advanced non-small-cell lung cancer. Mechanistically, Sirt2 suppresses Tcell metabolism by targeting key enzymes involved in glycolysis, tricarboxylic acid-cycle, fatty acid oxidation, and glutaminolysis. Accordingly, Sirt2-deficient murine Tcells exhibit increased glycolysis and oxidative phosphorylation, resulting in enhanced proliferation and effector functions and subsequently exhibiting superior antitumor activity. Importantly, pharmacologic inhibition of Sirt2 endows human TILs with these superior metabolic fitness and effector functions. Our findings unveil Sirt2 as an unexpected actionable target for reprogramming Tcell metabolism to augment a broad spectrum of cancer immunotherapies.

Humanized Rag2 mice as patient-derived orthotopic mouse models for immune checkpoint blockade and conventional chemotherapeutic drug treatment for human solid tumors

Abstract To evaluate therapeutic efficacy of immune checkpoint blockade (ICB) and conventional chemotherapy in pre-clinical models, we developed humanized patient-derived orthotopic xenograft (PDOX) models for colorectal cancer (CRC) and renal cell carcinoma (RCC). Rag2 mice were humanized by donor peripheral blood mononuclear cells (PBMC) transfer. Luciferase-tagged CRC and RCC tumor cells were injected intra-rectally and intra renal subcapsular, respectively. Groups of mice (n=5–10) received ICB, anti-PD-1 and anti-PD-L1 antibodies (Nivolumab and Atezolizumab, 200 μg each/mouse) for 4 weeks, and conventional therapy of 5FU (200 mg/kg, for CRC) alone or in combination. Tumor growth was measured by weekly bioluminescent image (BLI) and tumor weight at necropsy. The presence of human immune cells and tumor infiltrating lymphocytes were confirmed by flow cytometry and immunohistochemistry staining. Humanization was evidenced by >45% HLA-ABC+CD45+ circulating human cells in both CRC and RCC models. CRC tumor weight showed significant reduction in group treated with combination of ICB and 5FU compared with untreated controls or 5FU monotherapy group (p<0.05) using patient tumor cell CoCa302. The lung/liver metastasis detected by ex vivo BLI were reduced from 50% of mice in control group to 33% and 11%, respectively. For RCC, tumor size was reduced by >30% from controls in ICB treated group using patient tumor cell KiCa118 and cell line SN12K1. The later had 20% reduction of liver metastasis. The combination of ICB and conventional chemotherapies for CRC and RCC can be tested in our humanized PDOX mouse models. The effect of the combination therapy using MHC matched donors or patient autologous PBMC will be further investigated.

Abstract A41: Antitumor reactivity of human breast tumor-infiltrating lymphocytes (TILs)

Abstract Background: Clinical significance of tumor-infiltrating lymphocytes (TILs) was demonstrated in many tumor types. In the clinical trials, immune checkpoint inhibitors showed significant clinical efficacy in some tumor types with high mutation burden such as melanoma, bladder cancer, and smoking-induced lung cancer, while there were little effects in cancers with lower mutation rates such as breast cancer. Recently, selection and adoptive transfer of autologous mutation specific TILs showed successful regression of metastatic cancer in an estrogen receptor-positive breast cancer patient. In this study, we analyzed in vitro reactivity of cultured TILs against autologous breast cancer cells. Methods: We cultured TILs and cancer cells derived from 31 breast tumor tissues obtained from the surgery (13 of hormone receptor-positive, one of HER2-positive, and 17 of triple-negative breast cancer [TNBC]). Reactivity of TILs against cancer cells was determined by IFN-gamma ELISA analysis after co-culture of TILs and cancer cells for 24 hours. Epithelial cell markers (cytokeratin and EpCAM) for cancer cells and T cell markers [memory types (CD45RO, CCR7, and CD62L), T-cell activation and exhaustion markers (PD-1, 4-1BB, OX40, CD39, CD107a, TIM3, TIGIT, KLRG1, and LAG3), and regulatory T cells markers (CD25, CD127, and FOXP3)] for expanded TILs were analyzed by flow cytometry. Results: Seven cases showed in vitro reactivity, which was defined by increased secretion (more than twice) of IFN-gamma from TILs upon cancer cell interaction. Interestingly, most of reactive TILs were from TNBC (6/7) and all of TNBCs showing in vitro reactivity were derived from resection specimens after neoadjuvant chemotherapy (NAC, 6/11). The remaining one case with in vitro reactivity was derived from HER2-positive breast cancer. TNBC cancer cells of cases with in vitro reactivity had more EpCAM-positive cells (p = 0.02) than those of nonreactive cases. Most T cells in TIL cultures showed effector memory phenotype (CD45RO+/CCR7-; CD4 T cells, median 97.4 % (73.7-99.9); CD8 T cells, median 98.8% (93.8-99.8)). TILs of TNBC cases with in vitro reactivity expressed higher levels of PD-1 (p value < 0.01) on CD4 T cells. PD-1 expression levels on CD8 T cells in reactive group tended to be higher than nonreactive group (p = 0.05). Expression of other markers was not different between reactive and nonreactive groups. Conclusion: Among 17 TNBC cases, 35% showed in vitro TIL reactivity against autologous tumor cells. All reactive TNBCs were NAC-resistant tumors, which have high risk of tumor recurrence and high mortality rates. Adoptive TIL therapy might be a promising therapeutic modality for TNBC patients. Citation Format: Heejae Lee, Young-Ae Kim, Hye Seon Park, Won Seon Bang, In Ah Park, Miseon Lee, Gyungyub Gong, Hee Jin Lee. Antitumor reactivity of human breast tumor-infiltrating lymphocytes (TILs) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A41.

Abstract B85: Combination therapy using the programmed death receptor-1 and the programmed death-ligand 1 inhibitors and Fluorouracil in human colorectal carcinoma in a humanized patient-derived orthotopic mouse model

Abstract Background: One of the recently understood mechanisms associated with the progression of cancer is the immune checkpoint pathway. Many tumors can stimulate the expression of immune checkpoint molecules, resulting in a phenotype of exhausted T cells that cannot restrain tumor progression. One such inhibitory ligand and receptor pair in solid tumors are the programmed death-ligand 1 (PD-1) and programmed death receptor-1 (PD-L1). They prevent the killing of cancer cells by cytotoxic T-lymphocytes. PD-1 receptor is expressed by activated T cells among other cells, while PD-L1 is overexpressed on many tumor types including colorectal carcinoma (CRC). We have developed a humanized patient-derived orthotopic xenograft (hPDOX) model for CRC. Here, we investigate the potential efficacy of combination of immune checkpoint inhibitors (ICIs) and fluorouracil (5FU) in this CRC hPDOX model. Methods: Humanized mice were established by IP injection of donor peripheral blood mononuclear cells (PBMCs) into recombinase activating gene 2 (Rag2) and common cytokine receptor gamma chain gene (IL2Rγ) double knockout (Rag2-/-/IL2Rγ-/-) Rag2 mice. Luciferase-tagged patient tumor CoCaPt302 cells were injected intrarectally into Rag2 mice. Groups of mice (n=7-10) received ICIs, anti-PD-1 and anti-PD-L1 antibodies (nivolumab, 200 μg/mouse and atezolizumab, 200 μg/mouse), and 5FU (200 mg/kg) once a week for 4 weeks, IV injection alone or in combination. Tumor growth was measured weekly by bioluminescent imaging (BLI). At necropsy, tumor weights were measured. Human HLA-ABC+CD45+ hematopoietic cells and CD4+ or CD8+ T cells were detected in peritoneal lavage, blood, and spleen by flow cytometry. The presence of human immune cells (humanization) and tumor-infiltrating lymphocytes was confirmed by immunohistochemistry staining. Results: Humanization was evidenced by mouse blood containing more than 45% HLA-ABC+CD45+ human cells when tested by flow cytometry. Tumor weight showed significant reduction in group treated with combination of ICIs and 5FU compared with untreated controls (p=.0038). Tumors were also significantly smaller than ICIs (p=.0123) or 5FU (p=.0427) monotherapy groups. In addition, the lung and liver metastasis detected by ex vivo BLI were reduced from 50% of mice in control group to 33% and 11%, respectively, and exhibited better antitumor response as well as less development of metastases. CD3+ T cells, especially CD4+, CD8+ T cells in blood and spleen, were reduced in the ICIs only and combination treatment groups, but not in 5FU group compared to the controls. Conclusion: Our study provides preclinical evidence of establishment of humanized Rag2 mice to be used as a hPDOX model for CRC; and treatment efficacy including inhibiting tumor growth and distant organ metastasis was significantly enhanced through ICI and 5FU combination therapy. The effect of the combination therapy using CRC patient-derived specimens and PBMCs from MHC matched donors or autologous PBMCs will be further investigated. Citation Format: Xin Zhang, Grace Maresh, Linh Hellmers, Henry Yip, Lara McKean Baste, Heather Green, David Margolin, Li Li. Combination therapy using the programmed death receptor-1 and the programmed death-ligand 1 inhibitors and Fluorouracil in human colorectal carcinoma in a humanized patient-derived orthotopic mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B85.

Inhibition of Haspin Kinase Promotes Cell-Intrinsic and Extrinsic Antitumor Activity.

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo-expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages. SIGNIFICANCE: HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor-resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.

Abstract 2706: Comprehensive immune profiling of primary tumor-infiltrating lymphocytes isolated from primary non-small cell lung cancer specimens

Abstract In the past years, multiple inhibitors of the immune checkpoint receptor (ICR) PD-1 or its ligand PD-L1 have been approved in the U.S. in a selected, yet growing number of indications including non-small cell lung cancer (NSCLC). In patients with advanced NSCLC, checkpoint inhibition significantly prolongs overall survival and, due to its overall responsiveness, additional novel immunotherapeutic combinations are being investigated. Rational design of future combination treatment with immunomodulatory agents like PD-1 inhibitors will greatly benefit from a detailed understanding of the immune contexture and the functional state of tumor infiltrating lymphocytes (TILs) within NSCLC. To this end, we characterized the immune infiltrate in 13 primary NSCLC resections using multiple orthogonal techniques: (1) surface expression of multiple ICRs on various T cell subsets as well as the functional markers granzyme B and Ki-67 were assessed by flow cytometry; (2) the spatial localization of immune infiltrates was analyzed by 7-color multiplex immunohistochemistry (mIHC); (3) gene expression profiling enabled in depth molecular characterization of TILs and indicated their functional status. Flow cytometry revealed differential contributions of ICR expression in various T cell compartments. Of note, around 30% (median=29.1%; 95% CI, 20.3-38.5%) of CD8+ T cells co-expressed CD39 and CD103, which represents a recently described population of tumor-reactive CD8+ T cells. Furthermore, approximately 90% of these double-positive CD8+ T cells expressed PD-1 (median=91.3%; 95% CI, 78.3-96.4%) and about 50% were granzyme B-positive (median=51.8%; 95% CI, 32.5-63.95%), indicative of functionally active effector status. Interestingly, more than 90% of PD-1+ CD8+ T cells co-expressed at least one more ICR (TIM-3 and/or LAG-3) (median=90.6%; 95% CI, 70.3-95%), suggesting ongoing development of T cell exhaustion in response to chronic T cell activation. In contrast, the CD4+ T cell compartment was dominated by PD-1 single-positive cells and contained a median of 15.4% (95% CI, 11.7-20%) regulatory T cells.The overall composition of the immune infiltrates in these patients were corroborated by mIHC, revealing strong immune infiltration within the tumor stroma. Furthermore, immune cell deconvolution of bulk RNA-seq data from FFPE tissues revealed a high concordance with mIHC. In conclusion, we established a pipeline for high content immune profiling of human TIL enabling correlative analyses of proteomic and transcriptomic data. The high proportion of markers indicative of TIL with anti-tumor effector potential was in line with the responsiveness of NSCLC to immunotherapy. Integration of additional gene expression data, mutational burden, and T cell clonality will deepen our understanding of the NSCLC tumor microenvironment. Citation Format: Yoshinobu Koguchi, William Miller, Brian D. Piening, Venkatesh Rajamanickam, Brady Bernard, Zhaoyu Sun, Yaping Wu, Johanna K. Kaufmann, William L. Redmond. Comprehensive immune profiling of primary tumor-infiltrating lymphocytes isolated from primary non-small cell lung cancer specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2706.

Function of Human Tumor-Infiltrating Lymphocytes in Early-Stage Non-Small Cell Lung Cancer.

Cancer progression is marked by dysfunctional tumor-infiltrating lymphocytes (TIL) with high inhibitory receptor (IR) expression. Because IR blockade has led to clinical responses in some patients with non-small cell lung cancer (NSCLC), we investigated how IRs influenced CD8+ TIL function from freshly digested early-stage NSCLC tissues using a killing assay and intracellular cytokine staining after in vitro T-cell restimulation. Early-stage lung cancer TIL function was heterogeneous with only about one third of patients showing decrements in cytokine production and lytic function. TIL hypofunction did not correlate with clinical factors, coexisting immune cells (macrophages, neutrophils, or CD4+ T regulatory cells), nor with PD-1, TIGIT, TIM-3, CD39, or CTLA-4 expression. Instead, we found that the presence of the integrin αeβ7 (CD103), characteristic of tissue-resident memory cells (TRM), was positively associated with cytokine production, whereas expression of the transcription factor Eomesodermin (Eomes) was negatively associated with TIL function. These data suggest that the functionality of CD8+ TILs from early-stage NSCLCs may be influenced by competition between an antitumor CD103+ TRM program and an exhaustion program marked by Eomes expression. Understanding the mechanisms of T-cell function in the progression of lung cancer may have clinical implications for immunotherapy.

PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+ T-cell Function.

Although checkpoint inhibitors that block CTLA-4 and PD-1 have improved cancer immunotherapies, targeting additional checkpoint receptors may be required to broaden patient response to immunotherapy. PVRIG is a coinhibitory receptor of the DNAM/TIGIT/CD96 nectin family that binds to PVRL2. We report that antagonism of PVRIG and TIGIT, but not CD96, increased CD8+ T-cell cytokine production and cytotoxic activity. The inhibitory effect of PVRL2 was mediated by PVRIG and not TIGIT, demonstrating that the PVRIG-PVRL2 pathway is a nonredundant signaling node. A combination of PVRIG blockade with TIGIT or PD-1 blockade further increased T-cell activation. In human tumors, PVRIG expression on T cells was increased relative to normal tissue and trended with TIGIT and PD-1 expression. Tumor cells coexpressing PVR and PVRL2 were observed in multiple tumor types, with highest coexpression in endometrial cancers. Tumor cells expressing either PVR or PVRL2 were also present in numbers that varied with the cancer type, with ovarian cancers having the highest percentage of PVR-PVRL2+ tumor cells and colorectal cancers having the highest percentage of PVR+PVRL2- cells. To demonstrate a role of PVRIG and TIGIT on tumor-derived T cells, we examined the effect of PVRIG and TIGIT blockade on human tumor-infiltrating lymphocytes. For some donors, blockade of PVRIG increased T-cell function, an effect enhanced by combination with TIGIT or PD-1 blockade. In summary, we demonstrate that PVRIG and PVRL2 are expressed in human cancers and the PVRIG-PVRL2 and TIGIT-PVR pathways are nonredundant inhibitory signaling pathways.See related article on p. 244.

Abstract B027: Antitumor effects of the programmed death receptor-1 and the programmed death-ligand 1 blockade in human colorectal carcinoma in a humanized orthotopic mouse model

Abstract Background: The immune system plays an important role in tumor immune surveillance and progression. Immune checkpoint blockade is a new approach for cancer immunotherapy. The programmed death receptor-1 (PD-1) and the programmed death-ligand 1 (PD-L1) are immune checkpoint molecules and their expression results in negative regulation of T-cells primarily within the tumor microenvironment by preventing the killing of cancer cells by cytotoxic T-lymphocytes. Antibody blocking of the PD-1/PD-L1 signal exhibits promising therapeutic effects in non-small cell lung cancer and melanoma in patients. Due to its early success, more trials have been conducted to evaluate their efficacy for different tumors. We have developed a patient-derived orthotopic xenograft mouse model for colorectal carcinoma (CRC). Here, we investigate the potential efficacy of PD-1/PD-L1 blockade in our humanized orthotopic mouse models for human CRC. Methods: All studies were conducted under approved guidelines of the Institutional Animal Care and Use Committee and the Investigative Review Board of Ochsner Clinic Foundation. Humanized mice were established by intraperitoneal injection of donor human peripheral blood mononuclear cells (PBMCs) into recombinase activating gene 2 (Rag2) and common cytokine receptor gamma chain gene (IL2Rγ) double knockout (Rag2-/-/IL2Rγ-/-) Rag2 mice. Luciferase-tagged CRC cells were injected intra-rectally into Rag2 mice. One group of mice received a combination of anti-PD-1 and anti-PD-L1 antibodies (nivolumab, 200 µg/mouse and atezolizumab, 200 µg/mouse, intravenous injection) once a week for 3 weeks. Tumor growth was measured weekly by bioluminescent imaging (BLI). At necropsy, the CRC tumor weights were measured. Human CD45+ hematopoietic cells, CD4+ and CD8+ T-cells, and CD20+ B cells were detected in peritoneal lavage, blood, and tumor by flow cytometry. The presence of human immune cells (humanization) and tumor-infiltrating human lymphocytes was further confirmed by immunohistochemistry staining on paraffin-embedded tissue slides of mouse spleen and tumor, respectively. Results: Blood from mice receiving human PBMCs contained on average 31.4% CD45+ human cells (n=9) when tested by FACS analysis. Smaller tumor growth was observed in mice given PBMCs. This may be due to alloreactivity based on the recognition of MHC alloantigens in the transplanted tumor cells. However, mice further treated with the combination therapy of anti-PD-1 and anti-PD-L1 antibodies exhibited better antitumor response as well as less development of lung metastases compared to untreated controls. On average, tumor weight was reduced by 36% and lung metastases measured by ex-vivo BLI was reduced by 82% (n=5). In addition, circulating CD326+ tumor cells were reduced by 21% and CD3, CD4, CD8 and CD20 positive human lymphocytes were also reduced in the combination treatment group. Conclusion: Our study provides preclinical evidence of establishment of humanized Rag2 mice to be used as an orthotopic xenograft model for CRC; and treatment benefit for CRC through targeting immune checkpoint molecules PD-1 and PD-L1. The effect of immune checkpoint blockade in combination with conventional chemotherapy using CRC patient-derived specimens and PBMCs from MHC matched donors or autologous PBMCs will be further investigated. Citation Format: Li Li, Xin Zhang, Grace Maresh, Linh Hellmers, Avi Patel, Ravan Moret, Sarah Cohen, David Margolin. Antitumor effects of the programmed death receptor-1 and the programmed death-ligand 1 blockade in human colorectal carcinoma in a humanized orthotopic mouse model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B027.

Novel Effector Phenotype of Tim-3+ Regulatory T Cells Leads to Enhanced Suppressive Function in Head and Neck Cancer Patients.

Purpose: Regulatory T (Treg) cells are important suppressive cells among tumor-infiltrating lymphocytes (TIL). Treg cells express the well-known immune checkpoint receptor PD-1, which is reported to mark "exhausted" Treg with lower suppressive function. T-cell immunoglobulin mucin (Tim)-3, a negative regulator of Th1 immunity, is expressed by a sizeable fraction of TIL Tregs, but the functional status of Tim-3+ Tregs remains unclear.Experimental Design: CD4+CTLA-4+CD25high Treg cells were sorted from freshly excised head and neck squamous cell carcinoma (HNSCC) TIL based on Tim-3 expression. Functional and phenotypic features of these Tim-3+ and Tim-3- TIL Tregs were tested by in vitro suppression assays and multi-color flow cytometry. Gene-expression profiling and NanoString analysis of Tim-3+ TIL Treg were performed. A murine HNSCC tumor model was used to test the effect of anti-PD-1 immunotherapy on Tim-3+ Treg.Results: Despite high PD-1 expression, Tim-3+ TIL Treg displayed a greater capacity to inhibit naïve T-cell proliferation than Tim-3- Treg. Tim-3+ Treg from human HNSCC TIL also displayed an effector-like phenotype, with more robust expression of CTLA-4, PD-1, CD39, and IFN-γ receptor. Exogenous IFN-γ treatment could partially reverse the suppressive function of Tim-3+ TIL Treg. Anti-PD-1 immunotherapy downregulated Tim-3 expression on Tregs isolated from murine HNSCC tumors, and this treatment reversed the suppressive function of HNSCC TIL Tregs.Conclusions: Tim-3+ Treg are functionally and phenotypically distinct in HNSCC TIL, and are highly effective at inhibiting T-cell proliferation despite high PD-1 expression. IFN-γ induced by anti-PD-1 immunotherapy may be beneficial by reversing Tim-3+ Treg suppression. Clin Cancer Res; 24(18); 4529-38. ©2018 AACR.

Mitochondrial Morphological and Functional Reprogramming Following CD137 (4-1BB) Costimulation.

T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798-811. ©2018 AACR.

A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

BACKGROUND: Therapeutic strategies targeting immune checkpoint proteins have led to significant responses in patients with various tumor types. The success of these studies has led to the development of various antibodies/inhibitors for the different checkpoint proteins involved in immune evasion of the tumor. Adenosine present in high concentrations in the tumor microenvironment activates the immune checkpoint adenosine A2a receptor (A2aR), leading to the suppression of antitumor responses. Inhibition of this checkpoint has the potential to enhance antitumor T-cell responsiveness. METHODS: We developed a novel A2aR antagonist (PBF-509) and tested its antitumor response in vitro, in a mouse model, and in non-small cell lung cancer patient samples. RESULTS: Our studies showed that PBF-509 is highly specific to the A2aR as well as inhibitory of A2aR function in an in vitro model. In a mouse model, we found that lung metastasis was decreased after treatment with PBF-509 compared with its control. Furthermore, freshly resected tumor-infiltrating lymphocytes from lung cancer patients showed increased A2aR expression in CD4+ cells and variable expression in CD8+ cells. Ex vivo studies showed an increased responsiveness of human tumor-infiltrating lymphocytes when PBF-509 was combined with anti-PD-1 or anti-PD-L1. CONCLUSIONS: Our studies demonstrate that inhibition of the A2aR using the novel inhibitor PBF-509 could lead to novel immunotherapeutic strategies in non-small cell lung cancer.

K+ Channel Activation Promotes Tumor infiltrating Lymphocyte (TIL) Expansion and Enhances Expression of CCR7

Abstract Only two K+ channels are known to be expressed by T-cells. Activated effector T cells express high levels of Kv1.3; activated naïve and central memory T-cell subsets express high levels of KCa3.1. Overexpression of Kv1.3 in murine T-cells increases K+ efflux, improves effector function (IFNγ production) and promotes anti-tumor activity1. Inhibition of KCa3.1 suppresses murine T-cell proliferation and cytokine production. In human PBMCs and TIL, KCa3.1 expression was relatively low with upregulation observed within 24 h following stimulation with anti-CD3 and anti-CD28. The addition of the KCa3.1 agonist SKA-31 did not alter KCa3.1 expression but enhanced CCR7 expression significantly on TIL and human PBMCs. TILs propagated in the Rapid Expansion Protocol (REP) had a 1.42-fold greater expansion (p=0.002) in the presence of SKA-31 and significant increases in the CD8+CD28+ (p=0.04), CD8+CD27+(p=0.04), and CD8+CD27+CD28+subsets (p=0.002), consistent with a less differentiated phenotype. We thus demonstrate that SKA-31 treatment enhances CCR7 expression associated with memory cells, promotes TIL expansion, and attenuates T-cell differentiation. Targeting the KCa3.1 channel is a novel strategy to expand and sustain less differentiated TILs and may improve the clinical application of adoptive T-cell therapy. 1: Eil R, Vodnala SK, Clever D, Klebanoff CA, Sukumar M, Pan JH, Palmer DC, Gros A, Yamamoto TN, Patel SJ, Guittard GC, Yu Z, Carbonaro V, Okkenhaug K, Schrump DS, Linehan WM, Roychoudhuri R, Restifo NP. Ionic immune suppression within the tumour microenvironment limits T cell effector function. Nature. 2016;537):539–543.

Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model.

It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade. We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγnull (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo. [89Zr] Keytruda (groups=2; NSG-ctl (control) and hNSG-nblk (non-blocking), n=3-5, 3.2±0.4MBq/15-16μg/200μl) and [64Cu] Keytruda (groups=3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n=4, 7.4±0.4MBq /20-25μg/200μl) were administered in mice. PET-CT scans were performed over 1-144h ([89Zr] Keytruda) and 1-48h ([64Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g±SD) of 3.8±0.4 ([89Zr] Keytruda), and 6.4±0.7 ([64Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p=0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144h ([89Zr] Keytruda) and 48h ([64Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively. Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.

Adaptive resistance to anti-PD1 therapy by Tim-3 upregulation is mediated by the PI3K-Akt pathway in head and neck cancer

ABSTRACTProgrammed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune checkpoint receptors that are expressed on tumor-infiltrating lymphocytes (TIL) in tumor-bearing mice and humans. As anti-PD-1 single agent response rates are only <20% in head and neck squamous cell carcinoma (HNSCC) patients, it is important to understand how multiple inhibitory checkpoint receptors maintain suppressed cellular immunity. One such receptor, Tim-3, activates downstream proliferative pathways through Akt/S6, and is highly expressed in dysfunctional TIL. We observed that PD-1 and Tim-3 co-expression was associated with a more exhausted phenotype, with the highest PD-1 levels on TIL co-expressing Tim-3. Dampened Akt/S6 phosphorylation in these PD-1+Tim-3+ TIL, when the PD-1 pathway was ligated, suggested that signaling cross-talk could lead to escape through Tim-3 expression. Indeed, PD-1 blockade of human HNSCC TIL led to further Tim-3 upregulation, supporting a circuit of compensatory signaling and potentially permitting escape from anti-PD-1 blockade in the tumor microenvironment. Also, in a murine HNC tumor model that is partially responsive to anti-PD-1 therapy, Tim-3 was upregulated in TIL from persistently growing tumors. Significant antitumor activity was observed after sequential addition of anti-Tim-3 mAb to overcome adaptive resistance to anti-PD-1 mAb. This increased Tim-3-mediated escape of exhausted TIL from PD-1 inhibition that was mediated by phospho-inositol-3 kinase (PI3K)/Akt complex downstream of TCR signaling but not cytokine-mediated pathways. Taken together, we conclude that during PD-1 blockade, TIL upregulate Tim-3 in a PI3K/Akt-dependent manner, providing further support for dual targeting of these molecules for more effective cancer immunotherapy.

Autocrine Complement Inhibits IL10-Dependent T-cell-Mediated Antitumor Immunity to Promote Tumor Progression.

In contrast to its inhibitory effects on many cells, IL10 activates CD8(+) tumor-infiltrating lymphocytes (TIL) and enhances their antitumor activity. However, CD8(+) TILs do not routinely express IL10, as autocrine complement C3 inhibits IL10 production through complement receptors C3aR and C5aR. CD8(+) TILs from C3-deficient mice, however, express IL10 and exhibit enhanced effector function. C3-deficient mice are resistant to tumor development in a T-cell- and IL10-dependent manner; human TILs expanded with IL2 plus IL10 increase the killing of primary tumors in vitro compared with IL2-treated TILs. Complement-mediated inhibition of antitumor immunity is independent of the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) immune checkpoint pathway. Our findings suggest that complement receptors C3aR and C5aR expressed on CD8(+) TILs represent a novel class of immune checkpoints that could be targeted for tumor immunotherapy. Moreover, incorporation of IL10 in the expansion of TILs and in gene-engineered T cells for adoptive cell therapy enhances their antitumor efficacy. Our data suggest novel strategies to enhance immunotherapies: a combined blockade of complement signaling by antagonists to C3aR, C5aR, and anti-PD-1 to enhance anti-PD-1 efficacy; a targeted IL10 delivery to CD8(+) TILs using anti-PD-1-IL10 or anti-CTLA4-IL10 fusion proteins; and the addition of IL10 in TIL expansion for adoptive cellular therapy. Cancer Discov; 6(9); 1022-35. ©2016 AACR.See related commentary by Peng et al., p. 953This article is highlighted in the In This Issue feature, p. 932.

A major secretory defect of tumour-infiltrating T lymphocytes due to galectin impairing LFA-1-mediated synapse completion

Surface galectin has been shown to contribute to dysfunctions of human tumour-infiltrating lymphocytes (TILs). We show here that galectin-covered CD8 TILs produce normal amounts of intracellular cytokines, but fail to secrete them because of defective actin rearrangements at the synapse. The non-secreting TILs also display reduced adhesion to their targets, together with defective LFA-1 recruitment and activation at the synapse. These defects are relieved by releasing surface galectin. As mild LFA-1 blockade on normal blood T cells emulate the defects of galectin-covered TILs, we conclude that galectin prevents the formation of a functional secretory synapse by preventing optimal LFA-1 triggering. Our results highlight a major secretory defect of TILs that is not revealed by widely used intracellular cytokine immunomonitoring assays. They also provide additional insights into the T-cell response, by showing that different thresholds of LFA-1 triggering are required to promote the intracellular production of cytokines and their secretion.

FGL2 promotes tumor progression via inducing TIGIT expression on T cells in tumor microenvironment of glioma

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor with a median survival of only 14.6 months. Our previous work shows that FGL2 promotes glioma tumor growth and the expression levels of FGL2 positively correlated with glioma grade in patients. T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here we found that there is a statistically significant positive correlation between FGL2 and TIGIT mRNA expression level in both low grade glioma database (TCGA, provisional) and glioblastoma multiforme database (TCGA, provisional). Future, FGL2 and TIGIT are coordinately expressed by human tumor-infiltrating lymphocytes of glioma as detected by flow cytometry. In addition, TIGIT expression on T cells which suppresses their function, can be induced by initial T cell activation or FGL2 in vitro. Induced TIGIT expression upon initial T cell activation is suppressed in FGL2KO T cells which has higher tumor-killing ability than WT T cells. Therefore, our results show that FGL2 induces TIGIT expression in brain-infiltrated lymphocytes which suppresses anti-tumor immune response in glioma. By this mechanism, FGL2 promotes tumor progression of glioma.

Blockade of Programmed Death 1 Augments the Ability of Human T Cells Engineered to Target NY-ESO-1 to Control Tumor Growth after Adoptive Transfer.

Tumor-infiltrating lymphocytes (TILs) become hypofunctional, although the mechanisms are not clear. Our goal was to generate a model of human tumor-induced TIL hypofunction to study mechanisms and to test anti-human therapeutics. We transduced human T cells with a published, optimized T-cell receptor (TCR) that is directed to a peptide within the cancer testis antigen, NY-ESO-1. After demonstrating antigen-specific in vitro activity, these cells were used to target a human lung cancer line that expressed NY-ESO-1 in the appropriate HLA context growing in immunodeficient mice. The ability of anti-PD1 antibody to augment efficacy was tested. Injection of transgenic T cells had some antitumor activity, but did not eliminate the tumors. The injected T cells became profoundly hypofunctional accompanied by upregulation of PD1, Tim3, and Lag3 with coexpression of multiple inhibitory receptors in a high percentage of cells. This model allowed us to test reagents targeted specifically to human T cells. We found that injections of an anti-PD1 antibody in combination with T cells led to decreased TIL hypofunction and augmented the efficacy of the adoptively transferred T cells. This model offers a platform for preclinical testing of adjuvant immunotherapeutics targeted to human T cells prior to transition to the bedside. Because the model employs engineering of human T cells with a TCR clone instead of a CAR, it allows for study of the biology of tumor-reactive TILs that signal through an endogenous TCR. The lessons learned from TCR-engineered TILs can thus be applied to tumor-reactive TILs.