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Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103 + CD39 + tumor-infiltrating CD8 T cells (CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103 + CD39 + CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103 + CD39 + CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103 + CD39 + CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies. Identifying and enumerating tumor-specific CD8 T cells are important for assessing cancer prognosis and therapy efficacy. Here the authors show that CD39 and CD103 mark a subset of tumor-infiltrating CD8 T cells that are tumor-reactive and exhibit characteristics of exhausted or tissue-resident memory T cells.

BackgroundTumor Infiltrating Lymphocyte (TIL) therapies have shown significant solid tumor activity in patients, but current TIL compositions require patient lymphodepletion and high dose IL-2 after cell infusion to support clinical activity. Removing this requirement through ex vivo engineering of the TIL product with mRNA could enhance potency, expand the potential patient population, and potentially allow for repeat dosing and concomitant treatment with checkpoint therapies.MethodsTo transiently overexpress both membrane-bound cytokines and costimulatory molecules, we used microfluidic cell squeezing (Cell Squeeze®) to deliver mRNA directly to the cytosol of expanded tumor reactive CD8 human TILs (AGX-148). After mRNA delivery, the TILs were cultured in media with varying levels of exogenous IL-2 and characterized by flow cytometry.ResultsWe demonstrated that multiple mRNA constructs delivered simultaneously by microfluidic cell squeezing to human TILs are highly expressed (>80% of cells) for multiple days while maintaining high viability (>80%) in vitro. Membrane bound cytokines are able to support cell expansion in the absence of exogenous IL-2 at rates comparable to control cells incubated with a high concentration of IL-2 for up to 3 days. Furthermore, we have identified a membrane-bound cytokine that alters the TIL phenotype as quantified by multiple markers, including increased L-selectin (CD62L), which is an indicator of central memory T cells.ConclusionsThrough microfluidic cell squeeze delivery of mRNAs, we have created enhanced TILs with high levels of membrane-bound cytokines and/or costimulatory molecules in vitro. These cells are able to proliferate without exogenous IL-2 and have an improved phenotype.

The ability of memory CD8+ T cells to rapidly proliferate and acquire cytolytic activity is critical for protective immunity against intracellular pathogens. The signals that control this recall response remain unclear. We show that CD40L production by memory CD8+ T cells themselves is an essential catalyst for secondary expansion when systemic inflammation is limited. Secondary immunization accompanied by high levels of systemic inflammation results in CD8+ T cell secondary expansion independent of CD4+ T cells and CD40-CD40L signaling. Conversely, when the inflammatory response is limited, memory CD8+ T cell secondary expansion requires CD40L-producing cells, and memory CD8+ T cells can provide this signal. These results demonstrate that vaccination regimens differ in their dependence on CD40L-expressing CD8+ T cells for secondary expansion, and propose that CD40L-expression by CD8+ T cells is a fail-safe mechanism that can promote memory CD8+ T cell secondary expansion when inflammation is limited.

BackgroundTumor Infiltrating Lymphocyte (TIL) therapy has proven effective for patients with stage IV melanoma, however there are critical issues that can limit the efficacy of standard TIL therapy across a wide range of different malignancies. We and others have shown that some tumor types contain a low percentage of tumor-specific T cells. We hypothesize that most of the patients that do not respond to TIL therapy are likely receiving a low percentage of tumor-reactive T cells and therefore a high percentage of non-therapeutic bystander TIL. We have developed a streamlined method that expands a highly enriched fraction of tumor-reactive T cells contained within the CD39+CD103+CD8+ TIL in greater than 90% of patient samples from a wide variety of malignancies (melanoma, colon cancer, head and neck cancer, etc.). This TIL product displays a broad repertoire of tumor-specific TCRs. The expanded CD39/CD103 TIL can kill autologous tumors in vitro, but the possibility remains that they could revert to a suppressed or exhausted state when they reach the tumor microenvironment upon transfer back into patients. To mitigate the suppressive effects of the tumor microenvironment we have evaluated Phio Pharmaceutical’s self-delivering RNAi INTASYL(TM) platform to silence PD-1 in the expanded TIL product.MethodsThe TIL product was treated during the rapid expansion phase of the protocol with either nontargeting control compounds or PD-1 targeting INTASYL™ compounds. PD-1 protein levels and TIL functionality were assessed via flow cytometry and cytokine bead array.ResultsSilencing of PD-1 expression in the expanded TIL product was obtained by adding the self-delivering RNAi compounds to the cell culture media, without needing transfection media, delivery formulations or electroporation. The RNAi-treated TIL product showed increased IFN-?? TNF-α and Granzyme B expression.ConclusionsThese data highlight a promising combination to improve the activity of tumor-reactive TIL in future human clinical trials.

The tumor microenvironment of squamous cell carcinoma of the head and neck (SCCHN) has been shown to be immune suppressive. Therefore, strategies aimed at overcoming this issue could have a positive therapeutic impact. Hence, we investigated the expression of the known immune‐modulatory proteins OX40, programmed cell death protein 1 (PD‐1) and cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) in SCCHN on different T‐cell subsets of tumor‐infiltrating lymphocytes (TIL) to ascertain whether these proteins could potentially be targeted alone or in combination for future clinical trials. T cells from peripheral blood (PBL) and tumor were analyzed for the expression of OX40, PD‐1 and CTLA‐4 in 29 patients undergoing surgery. These proteins were all expressed significantly higher in T‐cell subsets isolated from tumors compared with PBL of the same patient. OX40 expression was significantly greater in the TIL regulatory T‐cell (Treg) population relative to conventional CD4 and CD8 TIL or the Treg isolated from PBL. PD‐1 expression was increased in all T‐cell subsets relative to PBL. CTLA‐4 was also increased in all TIL subsets relative to blood, and similar to OX40, its highest level of expression was observed in the Treg TIL. The highest frequency of PD‐1, CTLA‐4 and OX40 triple‐positive cells were found in the Treg population isolated from the tumor. We analyzed both human papilloma virus‐positive and ‐negative patients and found similar levels and expression patterns of these two patient populations for all three proteins. These data suggest that there may be therapeutic advantages of targeting these pathways independently or in combination for patients with this disease. Cancer: Vulnerabilities in head and neck tumors A survey of tumor‐associated immune cells suggests that some head and neck cancers may be vulnerable to potent ‘checkpoint inhibitor’ drugs. These therapeutic agents work by reactivating dormant host immune cells, which clinical trials have shown to subsequently eradicate various difficult‐to‐treat tumors. Researchers led by Andrew Weinberg at AgonOx, Inc., Portland, in the USA explored whether such drugs might prove effective against squamous cell carcinoma of the head and neck, an often lethal cancer. Their results demonstrated that tumor‐associated immune cells from such patients expressed higher levels of three different cell‐surface proteins targeted by existing checkpoint inhibitors. These proteins were especially abundant on a subset of cells that suppress rather than stimulate the immune response. However, checkpoint inhibitors may act differently on these cells, and the authors hope to explore this in clinical trials.

BackgroundStandard tumor-infiltrating lymphocyte (TIL) therapies use heterogeneous T cell populations that include both tumor-reactive and bystander cells. CD39+CD103+ double-positive (DP) CD8+ T cells are a phenotypically exhausted yet highly enriched subset of tumor-reactive lymphocytes within solid tumors. Despite their exhaustion markers, these cells retain potent cytotoxic activity when expanded ex vivo. AGX-148 is a selected TIL product designed to selectively isolate and expand tumor-reactive CD8+ T cells.MethodsThis ongoing Phase I dose-escalation trial (NCT05902520) is evaluating AGX-148 in adults with metastatic solid tumors that have progressed after standard and experimental therapies. Patients undergo tumor harvest, lymphodepleting chemotherapy, adoptive transfer of AGX-148 and high-dose IL-2 for one week, with subsequent low-dose IL-2 for 1, 2, or 3 weeks depending on cohort. Primary endpoints are safety and determination of the optimal biological dose; secondary endpoints include clinical response, T cell persistence, and immune correlates.ResultsAGX-148 manufacturing consistently yields billions of enriched tumor-reactive TIL. There have been no dose-limiting toxicities from AGX-148 encountered thus far. Toxicity has been as anticipated from lymphodepleting chemotherapy and IL-2. Clinical activity has been observed in solid tumors including melanoma, thyroid cancer, and oral head and neck squamous cell carcinoma with responses lasting over 600 days in some patients. TCR sequencing demonstrated long-term persistence of the infused product T cell clones in peripheral blood and metastatic tumor sites, confirming successful engraftment and tumor infiltration.ConclusionsAGX-148 offers a novel, biomarker selection strategy enriching for tumor-reactive CD8+ T cells for adoptive transfer. Preliminary data show clinical responses with tolerability and sustained DP TIL persistence. These findings support continued clinical development of AGX-148 and highlight the therapeutic potential of targeting tumor-specific T cell subsets in solid tumors. Updated safety, efficacy, and correlative immune data will be presented.Trial RegistrationThis trial is registered at ClinicalTrials.gov (NCT05902520).Ethics ApprovalThis clinical trial was approved by the Providence Institutional Review Board (Protocol #2023000082) and conducted under FDA IND#029-111. All participants provided written informed consent before study participation.

Identifying tumor antigen-specific T cells from cancer patients has been a goal of tumor immunologists for several decades. Here we show that co-expression of CD103 and CD39 on CD8 TIL highly enriched for tumor-reactive T cells. This cell population, which is only present in TIL from primary and metastatic tumors, exhibited features of exhausted cells and displayed characteristics of tissue-resident memory T cells. CD39+CD103+ CD8 TIL had a distinct TCR repertoire compared to other CD8 TIL subsets and were clonally expanded within the tumor. They were highly enriched for tumor antigen recognition and efficiently killed autologous tumor cells. Finally, patients with head and neck cancer whose CD8 TIL contained a higher frequency of CD39+CD103+ cells experienced a greater overall survival. This work describes a simple method for detecting tumor-reactive CD8 TIL, which should help define mechanisms of current immunotherapies and may lead to the development of future immunotherapies.

Background We examined the phenotype and function of lymphocytes collected from the peripheral blood (PBL) and tumor (TIL) of patients with two different solid malignancies: colorectal cancer liver metastases (CRLM) and ovarian cancer (OVC). Methods Tumor and corresponding peripheral blood were collected from 16 CRLM and 22 OVC patients; immediately following resection they were processed and analyzed using a multi-color flow cytometry panel. Cytokine mRNA from purified PBL and TIL CD4 + T cells were also analyzed by qPCR. Results Overall, we found similar changes in the phenotypic and cytokine profiles when the TIL were compared to PBL from patients with two different malignancies. The percentage of Treg (CD4 + /CD25 + /FoxP3 + ) in PBL and TIL was similar: 8.1% versus 10.2%, respectively in CRLM patients. However, the frequency of Treg in primary OVC TIL was higher than PBL: 19.2% versus 4.5% (p <0.0001). A subpopulation of Treg expressing HLA-DR was markedly increased in TIL compared to PBL in both tumor types, CRLM: 69.0% versus 31.7% (p = 0.0002) and OVC 74.6% versus 37.0% (p <0.0001), which suggested preferential Treg activation within the tumor. The cytokine mRNA profile showed that IL-6, a cytokine known for its immunosuppressive properties through STAT3 upregulation, was increased in TIL samples in patients with OVC and CRLM. Both TIL populations also contained a significantly higher proportion of activated CD8 + T cells (HLA-DR + /CD38 + ) compared to PBL (CRLM: 30.2% vs 7.7%, (p = 0.0012), OVC: 57.1% vs 12.0%, (p <0.0001)). Conclusion This study demonstrates that multi-color flow cytometry of freshly digested tumor samples reveals phenotypic differences in TIL vs PBL T cell sub-populations. The TIL composition in primary and metastatic tumors from two distinct histologies were remarkably similar, showing a greater proportion of activated/suppressive Treg (HLA-DR + , CD39 + , CTLA-4 + and Helios + ) and activated cytotoxic T cells (CD8 + /HLA-DR + /CD38 + ) when compared to PBL and an increase in IL-6 mRNA from CD4 TIL.

AbstractOX40 is a potent co-stimulatory receptor on the surface of T lymphocytes. An OX40 agonist was recently tested in a Phase I clinical trial and was found to be well tolerated and enhanced both humoral and cellular immunity in patients with metastatic cancer. Both PD-1 and CTLA-4 blockade are showing therapeutic clinical activity in late stage cancer patients. Hence, we hypothesize that the immune suppressed tumor microenvironment that characterizes oral, head and neck squamous cell carcinoma (OHNSCC) can be overcome by strategies that favor anti-tumor inflammatory T cell responses and may be an ideal tumor site for immune modulation. This investigation aims to provide a better understanding of the expression of OX40, PD-1, CTLA-4 and CD103 in OHNSCC tumor infiltrating lymphocytes (TIL). Additionally we evaluated whether phenotypic differences existed in patients with HPV positive vs. negative status.MethodsTIL and autologous blood were isolated from 29 patients undergoing surgery for OHNSCC. A sample of tissue was obtained from the resection specimen of the primary tumor and sent to the laboratory with autologous blood. Tumor was minced and subjected to digestive enzymes. Blood and tumor samples were separated over ficoll gradient to enrich for lymphocytes. Samples were frozen at -140°c and later thawed in batches for analysis. Proteins were then labeled with fluorescently conjugated antibodies and analyzed by flow cytometry.ResultsThe percentage of FoxP3+CD25+CD4+ (Tregs) of CD4+ T cells is significantly higher in OHNSCC Tumor Infiltrating Lymphocytes (TIL) compared to blood (p < .0005), regardless of HPV status. OX40 expression is significantly higher in the TIL Treg population relative to blood Tregs (p < .0005), also independent of HPV status. PD1 expression is increased in the Tregs (p < .0005), conventional CD4 (p=.0066), and CD8 T cells (p < .0005), in TIL relative to blood. CTLA-4 expression is increased in TIL relative to blood in all subsets with the highest expression observed in the Treg population (.0056). CD103 expression is increased on CD8 T cells in TIL relative to blood (p < .0005). CD8s from HPV positive patients have higher percentage CD103 positive cells than CD8s from HPV negative patients (p=.0056). The highest percentages of PD-1, CTLA-4 and OX40 triple positive cells were found in the Treg population of TIL compared to blood (p < .0005). Due to increased expression of OX40, CTLA-4 and PD-1 in TIL of OHNSCC patients we hypothesize that future combination clinical trials that target these pathways will be therapeutically beneficial.

The ability of memory CD8+ T cells to rapidly proliferate and acquire cytolytic activity is critical for protective immunity against intracellular pathogens. The signals that control this recall response remain unclear. We show that CD40L production by memory CD8+ T cells themselves is an essential catalyst for secondary expansion when systemic inflammation is limited. Secondary immunization accompanied by high levels of systemic inflammation results in CD8+ T cell secondary expansion independent of CD4+ T cells and CD40-CD40L signaling. Conversely, when the inflammatory response is limited, memory CD8+ T cell secondary expansion requires CD40L-producing cells, and memory CD8+ T cells can provide this signal. These results demonstrate that vaccination regimens differ in their dependence on CD40L-expressing CD8+ T cells for secondary expansion, and propose that CD40L-expression by CD8+ T cells is a fail-safe mechanism that can promote memory CD8+ T cell secondary expansion when inflammation is limited.