Tissue-resident Memory Phenotype Research Articles

Abstract 2636: Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck squamous cell carcinoma

Abstract Background Head and neck cancer is the 8th most common cancer worldwide. Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) overall survival rates remain low (25-40%) despite advances in treatments. CD8+ T cell differentiation into activated/cytotoxic (CTL), exhausted (TEX) and tissue-resident memory (TRM) phenotypes is a major determinant of successful cancer immunotherapy, which relies on the ability of CD8+ T cells to detect and eliminate cancer cells. Recent evidence suggests the importance of epigenetic regulation in the development of CD8+ T cell exhaustion and tissue residency; yet the epigenetic mechanisms that govern the transition between CTL, TEX, and TRM CD8+ T cell states remain unclear. This study investigates the epigenetic modifications associated with CTL, TEX, and TRM states using CD8+ T cells from healthy donors and HPV-negative HNSCC tumors. Methods An in vitro human CD8+ T cell stimulation assay using healthy donor cells was utilized to generate CTL, TEX and TRM phenotypes. Naïve T cells underwent CD3/28, TCR-independent stimulation for 9 days to promote CTL (3 days) and TEX (9 days) phenotypes, and TGF-β exposure (days 3-9) occurred to induce a TRM phenotype. Flow cytometry to confirm expression of cell-surface and intracellular markers for each phenotype is ongoing. Genome-wide mapping for activating (H3K4me1, H3K4me3) and repressive (H3K9me3, H3K27me3, H4K20me3) histone marks with CUT&Tag assays will be pursued for each of the in vitro CTL, TEX and TRM states, along with RNA-seq. CUT&Tag and RNA-seq will also characterize the epigenetic profiles of CD8+ T cells from single-cell suspensions generated from HPV-negative HNSCC tumors (n=5). Findings will be correlated with results from our in vitro T cell states. Results Preliminary flow cytometry confirmed increased expression of CD25, PD-1, Tim3 after 9 days of in vitro stimulation, supporting the induction of a CD8+ TEX phenotype. CD103 was also highly expressed in CD8+ T cells exposed to TGF-β, supporting the induction of the CD8+ TRM phenotype. Validation of the CTL state is ongoing. Phenotypic validation of cell states with flow cytometry (18 cell surface and intracellular markers) is ongoing. Feasibility of genome-wide mapping for activating and repressive histone marks with CUT&Tag on naïve CD8+ T cells has been confirmed. Conclusion This study investigates epigenetic changes that characterize the transition between CTL, TEX, and TRM states of CD8+ T cells using healthy donor cells through an in vitro assay and CD8+ T cells isolated from HPV-negative HNSCC tumors. Understanding the epigenetic profile of T cell states can help determine the major epigenetic regulators that lead to exhaustion and tissue residency, paving the way for improved T cell-based immunotherapies. Future experiments aim to validate our findings using TCR-specific in vitro and in vivo models. Citation Format: Marie K. Luff, Marco Craviero, Mohd Saleem Dar, Katherine McKinnon, Amir Kaskas, Clint T. Allen, Vassiliki Saloura. Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2636.

Mucosal tumor vaccination delivering endogenous tumor antigens protects against pulmonary breast cancer metastases

BackgroundGenerally, early-stage breast cancer has a good prognosis. However, if it spreads systemically, especially with pulmonary involvement, prospects worsen dramatically. Importantly, tumor-infiltrating T cells contribute to tumor control, particularly intratumoral...

Single-cell characterization of human GBM reveals regional differences in tumor-infiltrating leukocyte activation.

Glioblastoma (GBM) harbors a highly immunosuppressive tumor microenvironment (TME) which influences glioma growth. Major efforts have been undertaken to describe the TME on a single-cell level. However, human data on regional differences within the TME remain scarce. Here, we performed high-depth single-cell RNA sequencing (scRNAseq) on paired biopsies from the tumor center, peripheral infiltration zone and blood of five primary GBM patients. Through analysis of >45,000 cells, we revealed a regionally distinct transcription profile of microglia (MG) and monocyte-derived macrophages (MdMs) and an impaired activation signature in the tumor-peripheral cytotoxic-cell compartment. Comparing tumor-infiltrating CD8+ T cells with circulating cells identified CX3CR1high and CX3CR1int CD8+ T cells with effector and memory phenotype, respectively, enriched in blood but absent in the TME. Tumor CD8+ T cells displayed a tissue-resident memory phenotype with dysfunctional features. Our analysis provides a regionally resolved mapping of transcriptional states in GBM-associated leukocytes, serving as an additional asset in the effort towards novel therapeutic strategies to combat this fatal disease.

Single-cell characterization of human GBM reveals regional differences in tumor-infiltrating leukocyte activation

Glioblastoma (GBM) harbors a highly immunosuppressive tumor microenvironment (TME) which influences glioma growth. Major efforts have been undertaken to describe the TME on a single-cell level. However, human data on regional differences within the TME remain scarce. Here, we performed high-depth single-cell RNA sequencing (scRNAseq) on paired biopsies from the tumor center, peripheral infiltration zone and blood of five primary GBM patients. Through analysis of >45,000 cells, we revealed a regionally distinct transcription profile of microglia (MG) and monocyte-derived macrophages (MdMs) and an impaired activation signature in the tumor-peripheral cytotoxic-cell compartment. Comparing tumor-infiltrating CD8+ T cells with circulating cells identified CX3CR1high and CX3CR1int CD8+ T cells with effector and memory phenotype, respectively, enriched in blood but absent in the TME. Tumor CD8+ T cells displayed a tissue-resident memory phenotype with dysfunctional features. Our analysis provides a regionally resolved mapping of transcriptional states in GBM-associated leukocytes, serving as an additional asset in the effort towards novel therapeutic strategies to combat this fatal disease.

SARS-COV-2 specific t-cells in patients with thyroid disorders related to COVID-19 are enriched in the thyroid and acquire a tissue-resident memory phenotype

BackgroundSARS-CoV-2 infections have been associated with the onset of thyroid disorders like classic subacute thyroiditis (SAT) or atypical SAT upon severe COVID disease (COV-A-SAT). Little is known about thyroid anti-viral immune responses. ObjectivesTo define the role of T-cells in COV-A-SAT. MethodsT-cells from COV-A-SAT patients were analyzed by multi-dimensional flow cytometry, UMAP and DiffusionMap dimensionality reduction and FlowSOM clustering. T-cells from COVID-naïve healthy donors, patients with autoimmune thyroiditis (ATD) and with SAT following COVID vaccination were analyzed as controls. T-cells were analyzed four and eight months post-infection in peripheral blood and in thyroid specimen obtained by ultrasound-guided fine needle aspiration. SARS-COV2-specific T-cells were identified by cytokine production induced by SARS-COV2-derived peptides and with COVID peptide-loaded HLA multimers after HLA haplotyping. ResultsCOV-A-SAT was associated with HLA-DRB1*13 and HLA-B*57. COV-A-SAT patients contained activated Th1- and cytotoxic CD4+ and CD8+ effector cells four months post-infection, which acquired a quiescent memory phenotype after eight months. Anti-SARS-CoV-2-specific T-cell responses were readily detectable in peripheral blood four months post-infection, but were reduced after eight months. CD4+ and CD8+ tissue-resident memory cells (TRM) were present in the thyroid, and circulating CXCR3+T-cells identified as their putative precursors. SARS-CoV-2-specific T-cells were enriched in the thyroid, and acquired a TRM phenotype eight months post-infection. ConclusionsThe association of COV-A-SAT with specific HLA haplotypes suggests a genetic predisposition and a key role for T-cells. COV-A-SAT is characterized by a prolonged systemic anti-viral effector T-cell response and the late generation of COVID-specific TRM in the thyroid target tissue.

Single-cell RNA and T cell receptor (TCR) sequencing in patients with HPV-positive head and neck squamous cell carcinoma (HNSCC) after induction CTLA4 and PD-1 immune checkpoint blockade (ICB).

6056 Background: Combination CTLA-4 and PD-1 ICB results in durable responses in patients with recurrent and metastatic HNSCC and therefore justifies further investigation in the induction or neoadjuvant setting. Methods: We investigated features of the tumor immune microenvironment (TIME) associated with MHR after induction CTLA-4 and PD-1 ICB in patients with newly diagnosed HPV-positive HNSCC. Tumor samples were collected at baseline and on treatment from 31 patients enrolled in a phase 2 trial of a 6-week cycle of CTLA-4 and PD-1 ICB induction followed by dose/volume-adapted IMRT (50-66Gy) concurrent with cycle 2. Tumors were evaluated for histological response and single-cell RNA sequencing (scRNA-seq) and scTCR-seq were performed on the 10X genomics platform. After stringent clustering and annotation of scRNA-seq data, 76,319 CD8+ and 78,622 CD4+ T cells with paired TCR sequence data were evaluable for transcriptomic, TCR repertoire and clonotype differentiation analysis. Results: Percent reduction in tumor viability on histology correlated significantly with reduction in tumor cell proportion in scRNA-seq data (R2= 0.7). Thirteen (48%) of 27 patients with paired samples (missing data due to COVID) had MHR, defined as ≤10% residual tumor viability on ICB treatment. Through TCR tracing, over 100 T cell clonotypes that expanded significantly (p < 0.05, Fisher’s exact) post-ICB were identified. Highly expanded clonotypes resided mainly in exhausted CD8+ T cell clusters and exhibited a tissue resident memory (TRM) phenotype. Examination of their functional states revealed a variety of T cell modulatory trajectories, including reverse transition from exhausted to less exhausted states and from memory to functional states. When compared to patients without MHR, those with MHR had significantly higher ICB-induced TCR clonotype expansion (p = 0.025, Wilcoxon), reductions of activated Tregs (p = 0.003, Wilcoxon) and terminally exhausted CD8+ T cells (p = 0.003, Wilcoxon), and increase of polyfunctional CD8+ T cells (p = 0.014, Wilcoxon). A lasso regression model associated T cell expansion, basal 7-gene TRM and 5-gene effector scores, and on-treatment proliferation score and effector cell proportion with percent reduction in tumor viability (R2 = 0.66). Conclusions: In HPV-HNSCC, induction CTLA4 and PD1 ICB lead to extensive reinvigoration of CD8+ T cell clonotypes with both an exhausted and TRM phenotype, revealing a high degree of plasticity of distinct T cell clones. Paired RNA and TCR profiling facilitated identification of potentially tumor reactive and ICB-responsive T cells and clonotypes and identified several TIME features associated with tumor cell death. Clinical trial information: NCT03799445 .

Intratumoral CD8+ Tcells with a tissue-resident memory phenotype mediate local immunity and immune checkpoint responses in breast cancer.

CD8+ tumor-infiltrating lymphocytes with a tissue-resident memory T (TRM) cell phenotype are associated with favorable prognosis in patients with triple-negative breast cancer (TNBC). However, the relative contribution of CD8+ TRM cells to anti-tumor immunity and immune checkpoint blockade efficacy in breast cancer remains unknown. Here, we show that intratumoral CD8+ Tcells in murine mammary tumors transcriptionally resemble those from TNBC patients. Phenotypic and transcriptional studies established two intratumoral sub-populations: one more enriched in markers of terminal exhaustion (TEX-like) and the other with a bona fide resident phenotype (TRM-like). Treatment with anti-PD-1 and anti-CTLA-4 therapy resulted in expansion of these intratumoral populations, with the TRM-like subset displaying significantly enhanced cytotoxic capacity. TRM-like CD8+ Tcells could also provide local immune protection against tumor rechallenge and a TRM gene signature extracted from tumor-free tissue was significantly associated with improved clinical outcomes in TNBC patients treated with checkpoint inhibitors.

Runx3-overexpression cooperates with ex vivo AKT inhibition to generate receptor-engineered T cells with better persistence, tumor-residency, and antitumor ability

BackgroundSolid tumors pose unique roadblocks to treatment with chimeric antigen receptor (CAR) T cells, including limited T-cell persistence, inefficient tumor infiltration, and an immunosuppressive tumor microenvironment. To date, attempts to...

CagA-specific Gastric CD8+ Tissue-Resident T Cells Control Helicobacter pylori During the Early Infection Phase

Background & AimsInfection with Helicobacter pylori strongly affects global health by causing chronic gastritis, ulcer disease, and gastric cancer. Although extensive research into the strong immune response against this persistently colonizing bacterium exists, the specific role of CD8+ T cells remains elusive. MethodsWe comprehensively characterize gastric H pylori–specific CD8+ T-cell responses in mice and humans by flow cytometry, RNA-sequencing, immunohistochemistry, and ChipCytometry, applying functional analyses including T-cell depletion, H pylori eradication, and ex vivo restimulation. ResultsWe define CD8+ T-cell populations bearing a tissue-resident memory (TRM) phenotype, which infiltrate the gastric mucosa shortly after infection and mediate pathogen control by executing antigen-specific effector properties. These induced CD8+ tissue-resident memory T cells (TRM cells) show a skewed T-cell receptor beta chain usage and are mostly specific for cytotoxin-associated gene A, the distinctive oncoprotein injected by H pylori into host cells. As the infection progresses, we observe a loss of the TRM phenotype and replacement of CD8+ by CD4+ T cells, indicating a shift in the immune response during the chronic infection phase. ConclusionsOur results point toward a hitherto unknown role of CD8+ T-cell response in this bacterial infection, which may have important clinical implications for treatment and vaccination strategies against H pylori.

Abstract 1354: Regulation and roles of polyamines in CD8+ T cell fate and function

Abstract CD8+ T cells play central roles in tumor immune surveillance and are major effectors in adoptive cell therapy (ACT). Thus, strategies that enhance their functions are an urgent clinical need. Metabolic reprogramming determines T cell fate and function, where glycolysis principally drives an effector phenotype while oxidative phosphorylation (OxPhos) drives T cell memory [1]. However, the roles of amino acid catabolism in controlling effector and memory CD8+ T cell fate and function are less well understood [1, 2]. Glutamine is essential for nucleotide biosynthesis and as an anaplerotic fuel source for the tricarboxylic acid (TCA) cycle. Isotope tracing experiments with 13C glutamine or 13C arginine revealed that glutamine is the major source of polyamines in activated CD8+ T cells. Further, activation of CD8+ T cells provokes marked increases in the expression and activity of enzymes that direct polyamine biosynthesis, specifically ornithine decarboxylase (Odc), spermidine synthase (Srm) and spermine synthase (Sms), as well as to increases in the polyamines putrescine, spermidine and spermine. Notably, pharmacologic inhibition of Odc using difluoromethylornithine (DFMO), or genetic deletion of Odc using CD8+ T cells from CD4-Cre;Odcfl/fl mice, augments cytokine production in activated CD8+ T cells, including IFN-γ, TNF-α and granzyme B. Further, metabolic flux analysis indicates that DFMO treatment switches the metabolism of activated CD8+ T cells to rely on OxPhos, a phenotype found in CD8+ tissue resident memory (TRM) cells that play key roles in tissue and anti-tumor immunity anti-tumor reactivity. Indeed, inhibition or loss of Odc augments other TRM phenotypes, including increases in CD69high;CD44high;S1PR1low;CD62Llow cells that define the TRM phenotype. In addition, ACT of DFMO treated CD8+ T cells augments their survival in vivo and augments the generation of CD69+CD103+CD69+CXCR6+ and Ly6C+ TRMs in the bone marrow. Finally, DFMO treatment enhances IFN-γ and TNF-α production in human TILs, and co-treatment with TGF-β polarizes TILs into CD69+CD103+ and CD69+CD49a+ TRM-like cells. We conclude that polyamines are a metabolic check point in CD8+ TRM generation and are an actionable target to improve the anti-tumor immunity of T-cell based immunotherapies. Citation Format: Aya G. Elmarsafawi, Rebecca S. Hesterberg, John L. Cleveland. Regulation and roles of polyamines in CD8+ T cell fate and function [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 1354.

PD-1 and ICOS coexpression identifies tumor-reactive CD4+ T cells in human solid tumors.

CD4+ Th cells play a key role in orchestrating immune responses, but the identity of the CD4+ Th cells involved in the antitumor immune response remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and colorectal cancers and identified a subset of CD4+ Th cells distinct from FOXP3+ Tregs that coexpressed programmed cell death 1 (PD-1) and ICOS. These tumor-infiltrating lymphocyte CD4+ Th cells (CD4+ Th TILs) had a tissue-resident memory phenotype, were present in MHC class II–rich areas, and proliferated in the tumor, suggesting local antigen recognition. The T cell receptor repertoire of the PD-1+ICOS+ CD4+ Th TILs was oligoclonal, with T cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1+ICOS+ CD4+ Th TILs were shown to recognize both tumor-associated antigens and tumor-specific neoantigens. Our findings provide an approach for isolating tumor-reactive CD4+ Th TILs directly ex vivo that will help define their role in the antitumor immune response and potentially improve future adoptive T cell therapy approaches.

Donor T and NK Cells with a Special Tissue-Resident Memory Phenotype Migrate into the Periphery of Lung Transplant Recipients - A Potential Feature for Tolerance Development

<h3>Purpose</h3> After lung transplantation (LuTx), a transient chimerism of donor cells can be observed in recipient blood due to the migration of lymphocytes from the transplanted lung into the periphery. We aimed to characterize the phenotype of donor T and NK cells and to investigate their potential origin from tissue-resident memory (TRM) T and NK cells. <h3>Methods</h3> Lymphocyte dynamics in recipient blood were determined in n=97 LuTx patients directly (T0), 24 hours (T24) and 3 (wks) weeks after LuTx using flow cytometry. Donor T and NK cells were analyzed by HLA class I allele-specific mAb in n=44 LuTx recipients. T and NK cell markers were used to discriminate circulating from TMR subsets in blood, organ storage solutions (perfusates, n=97), explanted lung parenchyma (n=28) and donor trachea (n=17). Single cell mRNA sequencing of explant lung parenchyma was conducted (n=16). <h3>Results</h3> In peripheral blood of all recipients, donor-derived T and NK cells were detected at T0, T24 and 3 wks after LuTx, showed higher CD69 expression compared to recipient cells (p=0.01 to 0.04) and were mostly CCR7⁻ memory cells. This phenotype was similar to T and NK cells in corresponding perfusates. In recipient parenchyma and donor trachea, most CD69⁺ T and NK cells showed coexpression of TRM markers (CD103, CD49a, PD-1) with significant enrichment in trachea (p<0.05). These other TRM markers were not found in circulating donor T and NK cells or in perfusates, arguing for different memory T and NK cell subsets. Sc-mRNA sequencing confirmed distinct TRM and TRM-like T cell subsets in lung parenchyma according to separate expression profiles. The presence of TRM T and NK cells did not have an impact on primary graft dysfunction (PGD) 24h after LuTx. Yet, patients with high frequencies of donor T cells showed a trend towards CLAD-free survival 2 years post LuTx, although no statistical significance was reached (p=0.15). <h3>Conclusion</h3> Our results demonstrate that donor T and NK cells found in recipient blood immediately after LuTx are separate subsets from circulating as well as pulmonary TRM T and NK cells, since they express CD69 but lack expression of other classical TRM markers. Donor T cells may be clinically relevant for tolerance induction and long-term survival after transplantation due to their unique features.

HIV-1 Vpr drives a tissue residency-like phenotype during selective infection of resting memory Tcells.

SummaryHIV-1 replicates in CD4+ T cells, leading to AIDS. Determining how HIV-1 shapes its niche to create a permissive environment is central to informing efforts to limit pathogenesis, disturb reservoirs, and achieve a cure. A key roadblock in understanding HIV-T cell interactions is the requirement to activate T cells in vitro to make them permissive to infection. This dramatically alters T cell biology and virus-host interactions. Here we show that HIV-1 cell-to-cell spread permits efficient, productive infection of resting memory T cells without prior activation. Strikingly, we find that HIV-1 infection primes resting T cells to gain characteristics of tissue-resident memory T cells (TRM), including upregulating key surface markers and the transcription factor Blimp-1 and inducing a transcriptional program overlapping the core TRM transcriptional signature. This reprogramming is driven by Vpr and requires Vpr packaging into virions and manipulation of STAT5. Thus, HIV-1 reprograms resting T cells, with implications for viral replication and persistence.

Empirical identification and validation of tumor-targeting T cell receptors from circulation using autologous pancreatic tumor organoids

BackgroundTumor-specific cytotoxic T cells and T cell receptors are effective tools for cancer immunotherapy. Most efforts to identify them rely on known antigens or lymphocytes that have infiltrated into the...

Tumour antigen-induced T cell exhaustion - the archenemy of immune-hot malignancies.

Two recent studies addressed the functional properties and clinical significance of tumour antigen-specific effector T cells in human melanomas and lung carcinomas using single-cell strategies. Herein, we discuss their findings, which expand our understanding of T cell alterations in the tumour microenvironment and demonstrate that CD8+ T cell exhaustion is mediated by exposure to tumour cell-specific antigens and is associated with a tissue-resident memory phenotype.

Donor T and NK Cells with a Special Tissue-Resident Memory Phenotype Migrate into the Periphery of Lung Transplant Recipients - A Potential Feature for Tolerance Development

Donor T and NK Cells with a Special Tissue-Resident Memory Phenotype Migrate into the Periphery of Lung Transplant Recipients - A Potential Feature for Tolerance Development

Balancing Inflammation and Central Nervous System Homeostasis: T Cell Receptor Signaling in Antiviral Brain TRM Formation and Function.

Tissue-resident memory (TRM) CD8 T cells provide early frontline defense against regional pathogen reencounter. CD8 TRM are predominantly parked in nonlymphoid tissues and do not circulate. In addition to this anatomic difference, TRM are transcriptionally and phenotypically distinct from central-memory T cells (TCM) and effector-memory T cells (TEM). Moreover, TRM differ phenotypically, functionally, and transcriptionally across barrier tissues (e.g., gastrointestinal tract, respiratory tract, urogenital tract, and skin) and in non-barrier organs (e.g., brain, liver, kidney). In the brain, TRM are governed by a contextual milieu that balances TRM activation and preservation of essential post-mitotic neurons. Factors contributing to the development and maintenance of brain TRM, of which T cell receptor (TCR) signal strength and duration is a central determinant, vary depending on the infectious agent and modulation of TCR signaling by inhibitory markers that quell potentially pathogenic inflammation. This review will explore our current understanding of the context-dependent factors that drive the acquisition of brain (b)TRM phenotype and function, and discuss the contribution of TRM to promoting protective immune responses in situ while maintaining tissue homeostasis.

Decreased Frequency of Intestinal CD39+ γδ+ T Cells With Tissue-Resident Memory Phenotype in Inflammatory Bowel Disease.

The ectoenzymes CD39 and CD73 play a major role in controlling tissue inflammation by regulating the balance between adenosine triphosphate (ATP) and adenosine. Still, little is known about the role of these two enzymes and ATP and its metabolites in the pathophysiology of inflammatory bowel disease (IBD). We isolated mononuclear cells from peripheral blood and lamina propria of the large intestine of patients diagnosed with IBD and of healthy volunteers. We then comprehensively analyzed the CD39 and CD73 expression patterns together with markers of activation (HLA-DR, CD38), differentiation (CCR7, CD45RA) and tissue-residency (CD69, CD103, CD49a) on CD4+, CD8+, γδ+ T cells and mucosa-associated invariant T cells using flow cytometry. CD39 expression levels of γδ+ and CD8+ T cells in lamina propria lymphocytes (LPL) were much higher compared to peripheral blood mononuclear cells. Moreover, the frequency of CD39+ CD4+ and CD8+, but not γδ+ LPL positively correlated with T-cell activation. The frequency of CD39+ cells among tissue-resident memory LPL (Trm) was higher compared to non-Trm for all subsets, confirming that CD39 is a marker for the tissue-resident memory phenotype. γδ+ Trm also showed a distinct cytokine profile upon stimulation – the frequency of IFN-γ+ and IL-17A+ cells was significantly lower in γδ+ Trm compared to non-Trm. Interestingly, we observed a decreased frequency of CD39+ γδ+ T cells in IBD patients compared to healthy controls (p = 0.0049). Prospective studies need to elucidate the exact role of this novel CD39+ γδ+ T-cell population with tissue-resident memory phenotype and its possible contribution to the pathogenesis of IBD and other inflammatory disorders.

Abstract A10: Constitutive HIF activity enhances T-cell antitumor immunity and memory T-cell formation in the murine adoptive cell therapy model

Abstract Purpose: To determine if unrestrained hypoxia inducible factor (HIF) activity in CD8 T cells improves antitumor response. Methods: CD8 T cells were isolated from von Hippel Lindau (VHL)-deficient (VHL KO) mice, which have unrepressed HIF1 and HIF2 activity, as well as from VHL/HIF1/HIF2 triple-deficient (TKO) and WT mice. Following in vitro activation, T cells were transferred to melanoma (B16) tumor-bearing mice and monitored for tumor progression and survival. In other studies, activated VHL KO and TKO CD8 cells were mixed with WT CD8 cells and transferred to B16 or adenocarcinoma (MC38) tumor-bearing mice; after 7-10 days, donor T cells were analyzed from tumors, spleens, and draining lymph nodes (LN). Results: Single transfer of VHL KO CD8 T cells to B16 tumor-bearing mice resulted in delayed tumor growth and prolonged survival compared to transfer of CD8 T cells from TKO or WT donors. Co-transfer of VHL KO cells with WT cells into B16 tumor-bearing mice resulted in superior accumulation of VHL KO cells in all tissues including tumors. In contrast, mixed transfer of TKO cells with WT cells to B16 tumor-bearing mice resulted in poorer accumulation of TKO cells in all tissues including tumors. VHL KO tumor-infiltrating lymphocytes (TILs) had superior expression of tissue resident memory (TRM) phenotype markers CD103 and CD69 compared to WT and TKO TILs. Similarly in the MC38 tumor model, VHL KO cells showed improved accumulation in all tissues compared to WT cells. Conclusions: Here we demonstrate that VHL deletion in CD8 T cells results in superior efficacy in delaying tumor growth and improving recipient survival, greater accumulation in multiple solid tumor models, and increased development of the TRM phenotype. Further, TKO recipients demonstrated increased tumor growth, reduced survival, reduced TIL numbers, and absence of TRM phenotype following adoptive transfer, confirming the HIF dependency of the VHL-deletion phenotype. Taken together, these data demonstrate that T cells with constitutive HIF activity provide a more robust antitumor response. Therefore, manipulation of HIF regulation could offer new tools for enhanced T-cell efficacy in adoptive cellular therapy for solid tumors. Citation Format: Colette R. Lauhan, Deborah Witherden, Ilkka Liikanan, Ananda Goldrath. Constitutive HIF activity enhances T-cell antitumor immunity and memory T-cell formation in the murine adoptive cell therapy model [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A10.

Immunotherapy of CT26 murine tumors is characterized by an oligoclonal response of tissue-resident memory T cells against the AH1 rejection antigen.

Mice bearing CT26 tumors can be cured by administration of L19-mIL12 or F8-mTNF, two antibody fusion proteins which selectively deliver their cytokine payload to the tumor. In both settings, cancer cures crucially depended on CD8+ T cells and the AH1 peptide (derived from the gp70 protein of the murine leukemia virus) acted as the main tumor-rejection antigen, with ∼50% of CD8+ T cells in the neoplastic mass being AH1-specific after therapy. In order to characterize the clonality of the Tcell response, its phenotype, and activation status, we isolated CD8+ T cells from tumors and secondary lymphoid organs and submitted them to Tcell receptor (TCR) and total mRNA sequencing. We found an extremely diverse repertoire of more than 40000 unique TCR sequences, but the ten most abundant TCRs accounted for >60% of CD8+ T-cell clones in the tumor. AH1-specific TCRs were consistently found among the most abundant sequences. AH1-specific T cells in the tumor had a tissue-resident memory phenotype. Treatment with L19-mIL12 led to overexpression of IL-12 receptor and of markers of cell activation and proliferation. These data suggest that the antitumor response driven by antibody-cytokine fusions proceeds through an oligoclonal expansion and activation of tumor-infiltrating CD8+ T cells.