Dysfunction Of Tumor-infiltrating Lymphocytes Research Articles

Heteroantigen-assembled nanovaccine enhances the polyfunctionality of TILs against tumor growth and metastasis

The dysfunction of tumor infiltrating lymphocytes (TILs) directly correlates with out of control of tumor growth and metastasis. New approaches and insightful clarity for rescuing TILs dysfunction are urgently needed. Here, we design two heterogenous antigens based on MHC-I epitope and MHC-II epitope from tumor, and assemble heterogenous antigens by electrostatic interactions and π-π stacking into heteroantigen-assembled nanovaccine (HANV). HANV not only significantly increases the abundance of CD8+ and CD4+ TILs, but also elicits stronger polyfunctionality of CD8+ and CD4+ TILs in vivo. Enhanced polyfunctionality of CD8+ and CD4+ TILs positively correlate to suppression of tumor growth and metastasis in melanoma-bearing mouse models. We also validate that nucleotide-binding oligomerization domain-containing protein 2 (NOD2) dominantly enhances anti-tumor capacity of TILs in a temporal immunoregulation manner. This work presents a new insight in developing HANV as a rational strategy to shape TILs polyfunctionality for tumor growth and metastasis.

Cell surface marker-based capture of neoantigen-reactive CD8+ T-cell receptors from metastatic tumor digests

BackgroundCellular immunotherapies using autologous tumor-infiltrating lymphocytes (TIL) can induce durable regression of epithelial cancers in selected patients with treatment-refractory metastatic disease. As the genetic engineering of T cells with tumor-reactive...

Integrated single-cell profiling dissects cell-state-specific enhancer landscapes of human tumor-infiltrating CD8+ T cells

Despite extensive studies on the chromatin landscape of exhausted Tcells, the transcriptional wiring underlying the heterogeneous functional and dysfunctional states of human tumor-infiltrating lymphocytes (TILs) is incompletely understood. Here, we identify gene-regulatory landscapes in a wide breadth of functional and dysfunctional CD8+ TIL states covering four cancer entities using single-cell chromatin profiling. We map enhancer-promoter interactions in human TILs by integrating single-cell chromatin accessibility with single-cell RNA-seq data from tumor-entity-matching samples and prioritize cell-state-specific genes by super-enhancer analysis. Besides revealing entity-specific chromatin remodeling in exhausted TILs, our analyses identify a common chromatin trajectory to TIL dysfunction and determine key enhancers, transcriptional regulators, and deregulated genes involved in this process. Finally, we validate enhancer regulation at immunotherapeutically relevant loci by targeting non-coding regulatory elements with potent CRISPR activators and repressors. In summary, our study provides a framework for understanding and manipulating cell-state-specific gene-regulatory cues from human tumor-infiltrating lymphocytes.

Abstract 630: Dynamic imaging of T cell surveillance in live tumor fragments using camelid nanobodies

Abstract Immunotherapies (ITx) have revolutionized the oncology landscape. However, predicting patient responses to ITx is difficult based solely on static correlates such as TIL localization and molecular signatures. Anti-tumor immune response depends on motile surveillance by tumor infiltrating lymphocytes (TIL) which recognize antigenic determinants and engage target cells in serial stop-and-go interactions that result in cell killing. However, the hostile tumor microenvironment (TME) can cause TIL dysfunction and lack of cytotoxicity which is manifested as either suppressed or aimless TIL motility. To determine which ITx works best for a given patient, we are developing a diagnostic platform using live tumor fragments (LTF) that preserves the TME and its immune cells. Antibody-based labeling of live tissues is hampered by slow diffusion and function-altering cross-linking. To overcome these limitations, we have used small camelid-derived monovalent antibodies (nanobodies) to monitor TIL motility in LTFs. Human tumor excisions were cut into 300 x 300 x 200 µm LTFs, sorted into glass bottom multi-well plates and cultured. CD8+ cells in LTFs were labeled using an anti-hCD8a camelid VHH nanobody covalently labeled with AF594. The same reagent or a mouse anti-hCD8a whole IgG antibody was used to stain human peripheral blood mononuclear cells, and staining patterns were compared by flow cytometry. Immune responsiveness of LTFs to ITx was ascertained by flow cytometry and secreted protein assays (data shown in a companion abstract). Multiphoton microscopy revealed LTF collagen fibrils and cellular autofluorescence. A fluorescent anti-CD8a nanobody, but not a similarly labeled whole IgG, yielded good contrast and fast staining of two cell subsets. The smaller cells were 12 µm in diameter, cell surface-stained, and lacking autofluorescence, consistent with T cells. The larger cells were elongated, ramified, intracellularly stained, and distinctly autofluorescent, consistent with macrophages. 3D motility tracking revealed characteristic translational motility of the smaller cells at ~10 µm/min along collagenous structures. In contrast, larger cells exhibited only slow motility. Our results show that the motility of human CD8+ T lymphocytes can be revealed in LTF culture using a fluorescent CD8-binding camelid nanobody, likely due to its small size and monovalent binding. Further examination is needed to understand if T cell function is altered. The autofluorescence of larger, immotile cells was consistent with tumor-associated macrophages. Based on this distinction, T cells could be distinguished from the macrophages clearly. Our results support the use of camelid-derived VHH and other small monovalent reagents for live tissue lymphocyte tracking, possibly to evaluate TIL response to ITx in an LTF assay. Citation Format: Leung Kau Tang, Kelsey Tweed, Christina Scribano, David Wahl, Christin Johnson, M. Anna Zal, Jonathan Oliner, Tomasz Zal. Dynamic imaging of T cell surveillance in live tumor fragments using camelid nanobodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 630.

Deciphering the Labyrinthine System of the Immune Microenvironment in Recurrent Glioblastoma: Recent Original Advances and Lessons from Clinical Immunotherapeutic Approaches.

Simple SummaryActive communication between GBM cells and tumor-infiltrating immune components contributes to establishing an immunosuppressive environment where T cells are scarce and exhausted. This condition is particularly exacerbated upon recurrence, which is almost inevitable for GBM patients. Immunotherapeutic approaches, including checkpoint inhibitors, have demonstrated limited efficacy and failed to prolong survival or recurrent GBM patients. Nevertheless, many studies have shown that T cell priming is possible in the GBM microenvironment, and that T cell exhaustion or dysfunction can be reprogrammed. We will revisit data from the literature and report original results obtained from recurrent GBM patients treated with dendritic cells to demonstrate the role of the microenvironment in predicting immunotherapy response and influencing decisions for personalized therapies.The interpretation of the presence and function of immune infiltration in glioblastoma (GBM) is still debated. Over the years, GBM has been considered a cold tumor that is less infiltrated by effector cells and characterized by a high proportion of immunosuppressive innate immune cells, including GBM-associated microglia/macrophages (GAMs). In this context, the failure of checkpoint inhibitors, particularly in recurrent GBM (rGBM), caused us to look beyond the clinical results and consider the point of view of immune cells. The tumor microenvironment in rGBM can be particularly hostile, even when exposed to standard immunomodulatory therapies, and tumor-infiltrating lymphocytes (TILs), when present, are either dysfunctional or terminally exhausted. However, after checkpoint blockade therapy, it was possible to observe specific recruitment of adaptive immune cells and an efficient systemic immune response. In this review article, we attempt to address current knowledge regarding the tumor and immune microenvironment in rGBM. Furthermore, immunosuppression induced by GAMs and TIL dysfunction was revisited to account for genetic defects that can determine resistance to therapies and manipulate the immune microenvironment upon recurrence. Accordingly, we reevaluated the microenvironment of some of our rGBM patients treated with dendritic cell immunotherapy, with the goal of identifying predictive immune indicators of better treatment response.

JAML promotes CD8 and γδ T cell antitumor immunity and is a novel target for cancer immunotherapy.

T cells are critical mediators of antitumor immunity and a major target for cancer immunotherapy. Antibody blockade of inhibitory receptors such as PD-1 can partially restore the activity of tumor-infiltrating lymphocytes (TILs). However, the activation signals required to promote TIL responses are less well characterized. Here we show that the antitumor activity of CD8 and γδ TIL is supported by interactions between junctional adhesion molecule-like protein (JAML) on T cells and its ligand coxsackie and adenovirus receptor (CXADR) within tumor tissue. Loss of JAML through knockout in mice resulted in accelerated tumor growth that was associated with an impaired γδ TIL response and increased CD8 TIL dysfunction. In mouse tumor models, therapeutic treatment with an agonistic anti-JAML antibody inhibited tumor growth, improved γδ TIL activation, decreased markers of CD8 TIL dysfunction, and significantly improved response to anti-PD-1 checkpoint blockade. Thus, JAML represents a novel therapeutic target to enhance both CD8 and γδ TIL immunity.

CRISPR/Cas9-Mediated Diacylglycerol Kinase Knockout Potentiates Anti-Tumor Efficacy of Human Chimeric Antigen Receptor T-Cells

IntroductionTumors can evade immune surveillance by deactivating T cells via various immunosuppressive ligands and soluble factors. The inhibitory signals from tumors turn on or off specific signaling pathways of T cells, making T-cell therapy inefficient. Many efforts for efficient adoptive T-cell therapy have focused on blocking or inhibiting CD28/B7 pathway, signal2, by targeting PD1 or CTLA4, but robust therapeutic outcomes have remained challenging. Diacylglycerol (DAG) is an essential signal transduction molecule cascading CD3 signal, signal1, and DGK is an enzyme that phosphorylates DAG to phosphatidic acid (PA). In many tumors, DGK was found to be highly expressed in tumor infiltrating lymphocytes (TILs) and inhibition of DGK recover the dysfunction of TILs. In this study, we sought to invigorate the T-cell function by deleting Diacylglycerol kinase (DGKs) using CRISPR/Cas9. We found that ablation of DGKs potentiates anti-tumor function and proliferation of CAR-T cell, by strengthening the CD3 distal signals. Also, DGKαζ (two isoforms) knockout (KO) CAR-T showed no significant elevation of exhaustion markers, and was less responsive to immunosuppressive soluble factors, such as TGF-beta and prostaglandin E2. Collectively, these findings highlight the utility of DGK KO by CRISPR/Cas9 to reinforce the effector function of T-cell. The observed biological effects of DGK KO may also yield new strategies for improving the efficacy of T-cell therapy in combination with signal 2 checkpoint inhibitors in clinical applications.MethodsHuman pan-T cells were purchased from STEMCELL TECHNOLOGIES. X-VIVO15 media was supplemented with hIL-2 (50 U/mL) and hIL-7 (5 ng/mL) for culture, and activation was performed by using anti-CD3/28 Dynabeads (ThermoFisher SCIENTIFIC) at bead-to-cell ratio of 3:1. After 24 hours of activation, T-cells were mixed with 139-CAR lentivirus plates coated with Retronectin for 48 hours. 40 μg of recombinant S. pyogenes Cas9 (Toolgen) and 10 μg of chemically synthesized tracr/crRNA (Integrated DNA Technologies) were electroporated using 4D-Nucleofecter (Lonza).For in vitro assays, U87vIII, pre-stained with Cell Trace (ThermoFisher Scientific), was co-cultured with 139 CAR-T at indicated ratio, supplemented with or without TGF-β1 at 10 ng/mL and PGE2 at 0.5 ug/mL. Following co-incubation, cells were stained with 7-aminoactinomycin for cytotoxicity assays. Sample were acquired on Attune NxT Acoustic Focusing Cytometer, and analyzed with FlowJo. Cytotoxicity was calculated as [(%lysis sample-% lysis minimum)/(%lysis max [100%]-%lysis minimum]×100%. The supernatant of co-culture was analyzed to measure IL-2 and IFN-γ using ELISA Kit(Biolegend). For proliferation assays, distribution of CellTrace in 139 CAR-T cells was assessed using flow cytometry.ResultsIn following experimental scheme (Fig 1A), delivery of single Cas9/gRNA Ribonucleoprotein (RNP) complex targeting DGKα or DGKζ resulted in 75.9% and 93.5% of Indel, respectively in 139 CAR-T (Fig 1B). To generate DGKα and DGKζ double-negative 139 CAR-T cells, two gRNAs targeting DGKα and DGKζ were electroporated, which resulted in 49.2% and 92.4% indel, respectively (Fig 1B). No significant off-targets were detected by targeted deep-sequencing (Fig 1C). DGKα, DGKζ, and DGKαζ KO 139 CAR-T showed dramatic increase in cytotoxicity, multiple cytokine production, and proliferation capacity compared to wild-type 139 CAR-T (Fig 2A, B, C). Interestingly, DGKαζ KO 139 CAR-T showed further increase in cytokine release compared to DGKα or DGKζ single KO one, implying a synergistic effect of DGKα and DGKζ. The increase in effector function of DGK KO 139 CAR-T attributed to amplified CD3-distal signal, ERK1/2, and higher CAR expression after antigen exposure (Fig 3A, B). Despite of the strong activation signals in DGKs KO 139 CAR-T, no significant tonic activation was observed as indicated by basal IFN-γ secretion level (Fig 4A). Also, in comparison with 139 CAR-T, PD1 and TIM3 expression were not elevated in DGKs KO 139 CAR-T, suggesting that DGKs KO does not facilitate T-cell exhaustion after long term antigen exposure (Fig 4B). In contrast that anti-tumor effect of 139 CAR-T was markedly impaired with treatment of signal1 immunosuppressive inhibitors such as TGF-β1 and PGE2, DGKαζ KO 139 CAR-T maintained its cytotoxicity and cytokine release in the presence of the inhibitory soluble factors. (Fig 5 A, B) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tet2 Inactivation Enhances the Antitumor Activity of Tumor-Infiltrating Lymphocytes.

Inactivation of tumor-infiltrating lymphocytes (TIL) is one of the mechanisms mitigating antitumor immunity during tumor onset and progression. Epigenetic abnormalities are regarded as a major culprit contributing to the dysfunction of TILs within tumor microenvironments. In this study, we used a murine model of melanoma to discover that Tet2 inactivation significantly enhances the antitumor activity of TILs with an efficacy comparable to immune checkpoint inhibition imposed by anti-PD-L1 treatment. Single-cell RNA-sequencing analysis suggested that Tet2-deficient TILs exhibit effector-like features. Transcriptomic and ATAC-sequencing analysis showed that Tet2 ablation reshapes chromatin accessibility and favors binding of transcription factors geared toward CD8+ T-cell activation. Furthermore, the ETS family of transcription factors contributed to augmented CD8+ T-cell function following Tet2 depletion. Overall, our study establishes that Tet2 constitutes one of the epigenetic barriers that account for dysfunction of TILs and that Tet2 inactivation could promote antitumor immunity to suppress tumor growth. SIGNIFICANCE: This study suggests that ablation of TET2+ from TILs could promote their antitumor function by reshaping chromatin accessibility for key transcription factors and enhancing the transcription of genes essential for antitumor activity.

Abstract PO-005: Tet2 inactivation enhances the anti-tumor efficiency of tumor-infiltrating lymphocytes (TILs)

Abstract Inactivation of tumor infiltrating lymphocytes (TILs) is one of the mechanisms mitigating anti-tumor immunity during tumor onset and progression. Epigenetic abnormalities are regarded as a major culprit contributing to the dysfunction of TILs within tumor microenvironments. In this study, we used a murine model of melanoma to discover that Tet2 inactivation significantly enhances the anti-tumor activity of TILs, with the efficacy comparable to immune checkpoint inhibition imposed by anti-PD-L1 treatment. Single-cell RNA-seq analysis further revealed that Tet2-deficient TILs exhibit effector-like features. Transcriptomic and ATAC-seq analysis further demonstrated that Tet2 deletion reshapes the chromatin accessibility and favors the binding of transcription factors geared toward CD8+ T cell activation. In summary, our study establishes that Tet2 constitutes one of the epigenetic barriers contributing to dysfunction of TILs, and that Tet2 inactivation could benefit anti-tumor immunity to boost tumor suppression. Citation Format: Minjung Lee, Jiangfang Li, Shaohai Fang, Wei Han, Margarita Moczygemba, Yubin Zhou, Jia Li, Deqiang Sun, Yun Huang. Tet2 inactivation enhances the anti-tumor efficiency of tumor-infiltrating lymphocytes (TILs) [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-005.

Tet2 Inactivation Enhances the Anti-Tumor Activity of Tumor-Infiltrating Lymphocytes (TILs) to Curtail Melanoma Growth

Inactivation of tumor infiltrating lymphocytes (TILs) is one of the mechanisms mitigating anti-tumor immunity during tumor onset and progression. Epigenetic abnormalities are regarded as a major culprit contributing to the dysfunction of TILs within tumor microenvironments. In this study, we used a murine model of melanoma to discover that Tet2 inactivation significantly enhances the anti-tumor activity of TILs, with the efficacy comparable to immune checkpoint inhibition imposed by anti-PD-L1 treatment. Single-cell RNA-seq analysis further revealed that Tet2-deficient TILs exhibit effector-like features. Transcriptomic and ATAC-seq analysis further demonstrated that Tet2 deletion reshapes the chromatin accessibility and favors the binding of transcription factors geared toward CD8+ T cell activation. In summary, our study establishes that Tet2 constitutes one of the epigenetic barriers contributing to dysfunction of TILs, and that Tet2 inactivation could benefit anti-tumor immunity to boost tumor suppression. Disclosures No relevant conflicts of interest to declare.

Abstract A224: Bone marrow T-cells are tumor-infiltrating T-cells in pediatric patients with acute leukemia and their phenotype reflects immune evasion of leukemic blasts

Abstract Object: Acute leukemia is the most common malignancy in children. Despite recent therapeutic advances patients with relapsed or refractory disease require new treatment options. While synthetic immunotherapies such as chimeric antigen receptor (CAR) T-cells have shown impressive efficacy in B-precursor acute lymphoblastic leukemia (BCP-ALL) patients, the interaction between leukemic blasts and bone marrow T-cells remains largely unknown. Therefore, the role for immune response amplifiers in leukemia patients is currently unclear. Leukemia outgrowth leads to low frequency of physiologic bone marrow populations such as T-cells. Those T-cells are consequently within the site of tumor development and can thus be defined as tumor-infiltrating lymphocytes (TILs). Dysfunction of TILs has been described in a variety of solid and in some hematologic malignancies. To determine the changes driven by leukemia blasts we analyzed T-cells in bone marrow samples from pediatric patients with BCP-ALL, T-precursor ALL (TCP-ALL) and acute myelogenous leukemia (AML) at the time of diagnosis and relapse in comparison to healthy bone marrow donors. Material and Methods: In pilot experiments, any artificial changes in marker expression due to cryopreservation and thawing were excluded (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (n=23) were identified in our local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T-cells in primary (n=49) and relapse leukemia samples (n=28). Results: The frequency of bone marrow T-cells was reduced in patients with acute leukemia in comparison with healthy controls (5.9% vs. 24.4%, mean values, p<0.001). This reduction was more pronounced in BCP-ALL than in AML (0.9% vs. 8.4%, p<0.001). The CD4/CD8 ratio of bone marrow T-cells in leukemia patients was not altered compared with healthy controls (1.27 vs. 1.09, p=0.82). The frequency of regulatory T-cells (Tregs, defined as CD4+ CD25+ CD127low T-cells) was decreased in leukemic bone marrow (7.5% vs. 9.8%, p=0.022). However, while BCP-ALL samples did not show a difference in Treg frequency between initial diagnosis and relapse (8.0 vs. 7.2, p=0.86), there was an increase of Tregs at relapse in AML samples (9.5% vs. 6.2%, p=0.004). Surface markers of T-cell exhaustion such as PD1, TIM-3 and LAG3 were found to be consistently more highly expressed on T-cells of leukemia patients than in healthy controls, both on CD4 and CD8 T-cells. PD1 was more highly expressed in relapse samples than in primary diagnosis samples than in healthy controls: (CD4: 42.3% vs. 28.9% vs. 19.8%, p<0.001; CD8: 45.2% vs. 33.3% vs. 26.5%, p=0.002). This observation was consistent for relapse samples in all three different leukemia subtypes both on CD4 and CD8 T-cells. LAG3 expression on T-cells was increased in leukemia patients vs. healthy controls (CD4: 2.6% vs. 0.7%, p<0.001; CD8: 8.6% vs. 2.2%, p<0.001). The same was observed for TIM3 (CD4: 3.7% vs. 1.3%, p=0.002; CD8: 8.5% vs. 3.3%, p<0.001). However, no difference in LAG3 or TIM-3 expression could be observed between primary disease and relapse. Conclusion: By analyzing bone marrow samples from pediatric leukemia patients and healthy controls, we confirm that bone marrow T-cells of leukemia patients show significant changes compared to healthy individuals. Clinical parameters such as relapse status or leukemia subtype are associated with changes in the T-cell phenotype. Most importantly, PD1 surface expression on T-cells was identified as a marker that correlates with disease status (relapse > primary > healthy). These findings could reflect insufficient immune surveillance of pediatric leukemia by bone marrow T-cells and may provide a rationale for future therapeutic interventions. Citation Format: Semjon Manuel Willier, Paula Rothaemel, Jonas Wilhelm, Dana Stenger, Theresa Käuferle, Irene Schmid, Michael H. Albert, Vera Binder, Franziska Blaeschke, Tobias Feuchtinger. Bone marrow T-cells are tumor-infiltrating T-cells in pediatric patients with acute leukemia and their phenotype reflects immune evasion of leukemic blasts [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 A224.

Impaired enolase 1 glycolytic activity restrains effector functions of tumor-infiltrating CD8+ T cells.

In the context of solid tumors, there is a positive correlation between the accumulation of cytotoxic CD8+ tumor-infiltrating lymphocytes (TILs) and favorable clinical outcomes. However, CD8+ TILs often exhibit a state of functional exhaustion, limiting their activity, and the underlying molecular basis of this dysfunction is not fully understood. Here, we show that TILs found in human and murine CD8+ melanomas are metabolically compromised with deficits in both glycolytic and oxidative metabolism. Although several studies have shown that tumors can outcompete T cells for glucose, thus limiting T cell metabolic activity, we report that a down-regulation in the activity of ENOLASE 1, a critical enzyme in the glycolytic pathway, represses glycolytic activity in CD8+ TILs. Provision of pyruvate, a downstream product of ENOLASE 1, bypasses this inactivity and promotes both glycolysis and oxidative phosphorylation, resulting in improved effector function of CD8+ TILs. We found high expression of both enolase 1 mRNA and protein in CD8+ TILs, indicating that the enzymatic activity of ENOLASE 1 is regulated posttranslationally. These studies provide a critical insight into the biochemical basis of CD8+ TIL dysfunction.

Chromatin State and Immunotherapy

The functional impairment of T cell-mediated immunity within the tumor microenvironment (TME) is a defining feature of many cancers. Checkpoint blockade therapy seeks to reinvigorate T cell responses by targeting inhibitory receptors such as PD-1, which are upregulated by dysfunctional TILs. However, the fundamental mechanisms underlying T cell dysfunction in the TME remain poorly understood, as are the mechanisms by which checkpoint blockade overcomes this dysfunction. Initial studies of dysfunctional CD8+ T cells in both human and mouse tumors suggested that they share features of T cell exhaustion, including co-inhibitory receptor upregulation and defects in cytokine production. However, more recent studies have suggested that TIL dysfunction is a unique state that is distinct from T cell exhaustion. Here we show that anti-PD-1 therapy acts on a specific subpopulation of CD8+ tumor-infiltrating lymphocytes (TILs) in melanoma mouse models as well as patients with melanoma. We find that dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of T cell exhaustion, mirroring those seen in chronic viral infection. Similar to chronic viral infection, exhausted CD8+ TILs contain a subpopulation of “stem-like exhausted” T cells that have a distinct regulatory state. Stem-like exhausted TILs also have critical functional attributes that are not shared by the majority “terminally exhausted” TILs: they retain more polyfunctionality, persist following transfer into tumor-bearing mice, and differentiate to repopulate terminally exhausted TILs in the TME. As a result, stem-like exhausted CD8+ TILs are better able to control tumor growth than terminally exhausted cells. Stem-like exhausted, but not terminally exhausted, CD8+ TILs can respond to anti-PD-1 therapy without reversion of their exhausted epigenetic state. CD8+ T cells with a stem-like exhausted phenotype can be found in human melanoma samples and patients with a higher fraction of this subpopulation in their tumors have a significantly longer duration of response to combination checkpoint blockade therapy. Responsiveness to checkpoint blockade is therefore restricted to a subpopulation of exhausted TILs that retain specific functional properties which enable them to control tumors. Approaches to expand stem-like exhausted CD8+ T cells in the tumor microenvironment may be an important component of improving checkpoint blockade response. DisclosuresHaining:Rheos Medicines: Consultancy; Iomx Therapeutics: Consultancy; Third Rock Ventures: Consultancy; Roche: Research Funding; Calico: Research Funding; Novartis: Research Funding; Tango Therapeutics: Consultancy, Equity Ownership.

Immunosuppressive tumor-infiltrating myeloid cells mediate adaptive immune resistance via a PD-1/PD-L1 mechanism in glioblastoma.

Adaptive immune resistance in the tumor microenvironment appears to attenuate the immunotherapeutic targeting of glioblastoma (GBM). In this study, we identified a tumor-infiltrating myeloid cell (TIM) population that expands in response to dendritic cell (DC) vaccine treatment. The aim of this study was to understand how this programmed death ligand 1 (PD-L1)-expressing population restricts activation and tumor-cytolytic function of vaccine-induced tumor-infiltrating lymphocytes (TILs). To test this hypothesis in our in vivo preclinical model, we treated mice bearing intracranial gliomas with DC vaccination ± murine anti-PD-1 monoclonal antibody (mAb) blockade or a colony stimulating factor 1 receptor inhibitor (CSF-1Ri) (PLX3397) and measured overall survival. We then harvested and characterized the PD-L1+ TIM population and its role in TIL activation and tumor cytolysis in vitro. Our data indicated that the majority of PD-L1 expression in the GBM environment is contributed by TIMs rather than by tumor cells themselves. While PD-1 blockade partially reversed the TIL dysfunction, targeting TIMs directly with CSF-1Ri altered TIM expression of key chemotactic factors associated with promoting increased TIL infiltration after vaccination. Neither PD-1 mAb nor CSF-1Ri had a demonstrable therapeutic benefit alone, but when combined with DC vaccination, a significant survival benefit was observed. When the tripartite regimen was given (DC vaccine, PD-1 mAb, PLX3397), long-term survival was noted together with an increase in the number of TILs and TIL activation. Together, these studies elucidate the role that TIMs play in mediating adaptive immune resistance in the GBM microenvironment and provide evidence that they can be manipulated pharmacologically with agents that are clinically available. Development of immune resistance in response to active vaccination in GBM can be reversed with dual administration of CSF-1Ri and PD-1 mAb.

Virus-specific CD8+ T cells infiltrate melanoma lesions and retain function despite high PD-1 expression

It is well known that CD8+ T cell infiltration of tumors is correlated with positive patient prognosis. Because of this, many viral vaccine vectors have been created to induce expansion and infiltration of tumor-specific CD8+ T cells. Cytomegalovirus (CMV) is a herpes virus that induces a persistent/latent infection for the life of the host. It is able to drive large populations of viral-specific T cells that are fully functional and infiltrate most tissues. We have established that acute infection with a recombinant murine CMV virus expressing an altered gp100 antigen (MCMV-gp100) caused expansion and tumor infiltration of gp100-specific CD8+ T cells, leading to a therapeutic delay in the growth of B16 melanomas in mice. Coincidentally, we observed that at least 20% of the CD8+ tumor infiltrating lymphocytes (TIL) were MCMV-specific. These virus-specific T cells infiltrated tumors independently of viral replication. Unexpectedly, T cell infiltration of tumors occurred even in latently infected animals, long after viral replication had been controlled. Together, these data suggest that virus-specific CD8+ T cells can become TIL, even long after the primary infection. We were curious about differences between tumor and virus-specific CD8+ TIL. As expected, after acute MCMV-gp100 infection, gp100-specific CD8+ TIL were PD-1hi and markedly dysfunctional. Virus-specific CD8+ T cells were also PD-1hi in the tumor after acute MCMV-gp100 infection, but these cells remained fully functional. PD-1 is an activation marker expressed on recently stimulated CD8+ T cells and both gp100-specific and virus-specific T cells expressed PD-1 after acute MCMV-gp100 infection. In the circulation of tumor-bearing mice or in the absence of a tumor, both gp100-specific and virus-specific CD8+ T cells lost PD-1 expression over time. However, PD-1hi cells that migrated into tumors retained expression of PD-1 regardless of specificity. Strikingly, introducing B16 tumors into MCMV-gp100 latently infected mice after PD-1 expression was lost, resulted in PD-1 up-regulation on and subsequent dysfunction of gp100-specific T cells that entered the tumor, but not virus-specific T cells found in the same tumors. Together, these data suggest that the tumor environment sustains PD-1 expression on recently activated CD8+ TIL independently of antigen in the tumor, but that the presence of antigen in the tumor drives subsequent CD8+ TIL dysfunction.

Abstract B67: A big role in tumor immune microenvironment of a small non-coding RNA: microRNA-200

Abstract Immunosuppression of tumor-infiltrating lymphocytes (TIL) is a common feature of advanced epithelial tumors, but its biological basis has remained obscure. In addition, anti-PD-1/PD-L1 treatment has produced encouraging clinical responses, but it is still unclear which patients are likely to benefit. Thus, successful therapeutic translation will require a thorough understanding of mechanisms of treatment resistance and predictive biomarkers to select patients. Using multiple lung cancer mouse models (spontaneous and transplant models), we demonstrate a molecular link between epithelial-to-mesenchymal transition (EMT) and intra-tumoral CD8+ T cell immunosuppression, two key drivers of malignant tumor progression. We show that microRNA-200 (miR-200), a cell-autonomous suppressor of EMT and metastasis, targets PD-L1, a checkpoint inhibitor of CD8+ T cell immunity. Moreover, ZEB1, an EMT activator and transcriptional repressor of miR-200, relieves miR-200 repression of PD-L1 on tumor cells, leading to CD8+TIL dysfunction and metastasis. These findings were further supported by robust correlations between the EMT score, miR-200 levels and PD-L1 expression in multiple human lung cancer datasets: The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset and the Profiling of Resistance patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (PROSPECT) project at MD Anderson Cancer Center. In addition to revealing a link between EMT and T cell dysfunction, these findings also show that ZEB1 promotes metastasis through a heretofore unappreciated cell non-autonomous mechanism, and they suggest that subgroups of patients in whom malignant progression is driven by EMT activators may respond to treatment with PD-L1 antagonists. Our data also suggest that EMT represents an important biomarker to select the patients who may benefit from immune checkpoint blockade agents and other immunotherapies in lung cancer and possibly a broader range of other cancers. Citation Format: Limo Chen, Sangeeta Goswami, Xiaohui Yi, Lauren Byers, Lixia Diao, Jonathon Roybal, Christin Ungewiss, David Peng, Jing Wang, Ignacio Wistuba, Lieping Chen, Stephen Ullrich, John Heymach, Jonathan Kurie, Xiao-Feng Qin, Don Gibbons. A big role in tumor immune microenvironment of a small non-coding RNA: microRNA-200. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B67.

Abstract CT220: A randomized multicenter phase Ib/II study to assess the safety and the immunological effect of chemoradiation therapy (CRT) in combination with Pembrolizumab (anti-PD1) to CRT alone in patients with resectable or borderline resectable pancreatic canc

Abstract Background: Immunotherapy has recently emerged as a promising modality in cancer treatment, but little is known about the application of this modality in pancreatic cancer (PC). Tumor-infiltrating lymphocytes (TILs) play a major role in anti-tumor immune responses, and their presence is correlated with survival in a variety of tumors. These TILs do not reach the PC cells in significant numbers due to the presence of stroma and a suppressive microenvironment. One of the leading causes for immune suppression is elevated expression of PD-L1 either by the tumor cells or the surrounding regulatory cells, resulting in dysfunction of TILs. Neoadjuvant chemoradiation therapy (CRT) has been advocated as a potential way to improve outcomes of patients with resectable or borderline resectable PC. More importantly, there is recent evidence to suggest that CRT can increase the presence of TILs in the PC microenvironment (PCME), leading to production of interferon-γ (IFN-γ), which could increase the expression of PD-L1 through a negative feedback loop. Accordingly, we hypothesize that blocking the PD-1 receptor will synergize with CRT to increase the density and activation of TILs in the PCME. Methods: This is a prospective multicenter randomized trial which will accrue subjects with resectable or borderline resectable pancreatic cancer who had not received prior treatment for PC. The primary objectives of the study are: (1) to determine the safety of neoadjuvant CRT in combination with Pembrolizumab. (2) To estimate the difference in the number of TILs in pancreatic cancer subjects receiving neoadjuvant CRT in combination with Pembrolizumab to the number of TILs in subjects receiving neoadjuvant CRT alone. This study will also investigate the effect of CRT+/-anti-PD-1 on the other effector and suppressive immune cells and immune checkpoints in PCME. Eligible subjects will be randomized 2:1 to the investigational treatment (Arm A) to receive Pembrolizumab administered IV every 3 weeks on days 1, 22, and 43 during concurrent CRT with capecitabine (825 mg/m2 orally twice daily, Monday through Friday, on days of radiation only) and radiation (50.4 Gy in 28 fractions over 28 days) or Arm B to receive only concurrent CRT with capecitabine. In all subjects, restaging CT scan or MRI will be performed at 4-6 weeks after completion of neoadjuvant treatment to determine resectability. Patients without local or distant disease progression will be taken to the operative room for planned surgery (within 2 weeks of imaging). Postoperatively, resected patients will receive off study standard of care adjuvant gemcitabine (1000mg/kg IV weekly for 3 out of 4 weeks for 6 months). Post operatively resected patients will be followed for up for PFS and OS for up to 2 years. Citation Format: Matthew H.G Katz, Todd W. Bauer, Gauri Rajani Varadhachary, Reid B. Adams, Amy R. Lankford, Gina Petroni, Timothy N. Bullock, Craig L. Slingluff, Osama E. Rahma. A randomized multicenter phase Ib/II study to assess the safety and the immunological effect of chemoradiation therapy (CRT) in combination with Pembrolizumab (anti-PD1) to CRT alone in patients with resectable or borderline resectable pancreatic canc [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 CT220. doi:10.1158/1538-7445.AM2015-CT220

Progressive loss of differentiated effector CD8 T cells infiltrating melanoma (TUM7P.1021)

Abstract CD8 tumor infiltrating lymphocytes (TIL) are critical for tumor control and their presence is a positive prognostic factor. Understanding CD8 TIL dysfunction is vital for developing cancer immunotherapies. Here, using a Granzyme B Cre ROSA YFP reporter system, we characterized the differentiation of CD8 T cells over the course of tumor growth using a murine melanoma model. At early stages of the CD8 T cell response (day 10 after tumor injection), the majority of the cells had differentiated to express Granzyme B (YFP+). However, over a period of four days, most of these effector cells were replaced by CD8+ T cells that had no evidence of effector differentiation. Concurrently, we found an increase in expression of the inhibitory receptor PD-1 in differentiated (YFP+) relative to non-differentiated (YFP-) CD8 TIL in late stage tumors. This suggests that there is both a loss of differentiated CD8 TIL and that the differentiated CD8 T cells that remain are more dysfunctional in late stage tumors. To provide insight into the cause and effect of this fluctuation in differentiation, we present data establishing the contribution of checkpoint inhibitory molecules and dendritic cell activation state in the loss of effector characteristics, and whether cytokine production and other effector activities correlate with the shift in effector differentiation status.

Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors—Response

Tumor progression is facilitated by regulatory T cells (Treg) and restricted by effector T cells. In this study, we document parallel regulation of CD8(+) T cells and Foxp3(+) Tregs by programmed death-1 (PD-1, PDCD1). In addition, we identify an additional role of CTL antigen-4 (CTLA-4) inhibitory receptor in further promoting dysfunction of CD8(+) T effector cells in tumor models (CT26 colon carcinoma and ID8-VEGF ovarian carcinoma). Two thirds of CD8(+) tumor-infiltrating lymphocytes (TIL) expressed PD-1, whereas one third to half of CD8(+) TIL coexpressed PD-1 and CTLA-4. Double-positive (PD-1(+)CTLA-4(+)) CD8(+) TIL had characteristics of more severe dysfunction than single-positive (PD-1(+) or CTLA-4(+)) TIL, including an inability to proliferate and secrete effector cytokines. Blockade of both PD-1 and CTLA-4 resulted in reversal of CD8(+) TIL dysfunction and led to tumor rejection in two thirds of mice. Double blockade was associated with increased proliferation of antigen-specific effector CD8(+) and CD4(+) T cells, antigen-specific cytokine release, inhibition of suppressive functions of Tregs, and upregulation of key signaling molecules critical for T-cell function. When used in combination with GVAX vaccination (consisting of granulocyte macrophage colony-stimulating factor-expressing irradiated tumor cells), inhibitory pathway blockade induced rejection of CT26 tumors in 100% of mice and ID8-VEGF tumors in 75% of mice. Our study indicates that PD-1 signaling in tumors is required for both suppressing effector T cells and maintaining tumor Tregs, and that PD-1/PD-L1 pathway (CD274) blockade augments tumor inhibition by increasing effector T-cell activity, thereby attenuating Treg suppression.

Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors

Tumor progression is facilitated by regulatory T cells (Treg) and restricted by effector T cells. In this study, we document parallel regulation of CD8(+) T cells and Foxp3(+) Tregs by programmed death-1 (PD-1, PDCD1). In addition, we identify an additional role of CTL antigen-4 (CTLA-4) inhibitory receptor in further promoting dysfunction of CD8(+) T effector cells in tumor models (CT26 colon carcinoma and ID8-VEGF ovarian carcinoma). Two thirds of CD8(+) tumor-infiltrating lymphocytes (TIL) expressed PD-1, whereas one third to half of CD8(+) TIL coexpressed PD-1 and CTLA-4. Double-positive (PD-1(+)CTLA-4(+)) CD8(+) TIL had characteristics of more severe dysfunction than single-positive (PD-1(+) or CTLA-4(+)) TIL, including an inability to proliferate and secrete effector cytokines. Blockade of both PD-1 and CTLA-4 resulted in reversal of CD8(+) TIL dysfunction and led to tumor rejection in two thirds of mice. Double blockade was associated with increased proliferation of antigen-specific effector CD8(+) and CD4(+) T cells, antigen-specific cytokine release, inhibition of suppressive functions of Tregs, and upregulation of key signaling molecules critical for T-cell function. When used in combination with GVAX vaccination (consisting of granulocyte macrophage colony-stimulating factor-expressing irradiated tumor cells), inhibitory pathway blockade induced rejection of CT26 tumors in 100% of mice and ID8-VEGF tumors in 75% of mice. Our study indicates that PD-1 signaling in tumors is required for both suppressing effector T cells and maintaining tumor Tregs, and that PD-1/PD-L1 pathway (CD274) blockade augments tumor inhibition by increasing effector T-cell activity, thereby attenuating Treg suppression.