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T lymphocytes expressing CD57 and lacking costimulatory receptors CD27/CD28 have been reported to accumulate with aging, chronic infection, and cancer. These cells are described as senescent, with inability to proliferate but enhanced cytolytic and cytokine-producing capacity. However, robust functional studies on these cells taken directly from cancer patients are lacking. We isolated these T cells and their CD27/28+ counterparts from blood and tumor samples of 50 patients with previously untreated head and neck cancer. Functional studies confirmed that these cells have enhanced ability to degranulate and produce IFN-γ. They also retain the ability to proliferate, thus are not senescent. These data suggest that CD27/28-CD57+ CD8+ T cells are a subset of highly differentiated, CD45RA+ effector memory (T EMRA ) cells with retained proliferative capacity. Patients with > 34% of these cells among CD8+ T cells in the blood had a higher rate of locoregional disease relapse, suggesting these cells may have prognostic significance., Competing Interests: NCS discloses consulting fees from Sensorion, Regeneron, and GeoVax in addition to research funding from Astex Pharmaceuticals. The authors have no conflicting financial interests related to this work., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Inhibitor of apoptosis protein (IAP) antagonists have shown activity in preclinical models of head and neck squamous cell carcinoma (HNSCC), and work across several cancer types has demonstrated diverse immune stimulatory effects including enhancement of T cell, NK cell, and dendritic cell function. However, tumor-cell-intrinsic mechanisms for this immune upregulation have been largely unexplored. In this study, we show that ASTX660, an antagonist of cIAP1/2 and XIAP, induces expression of immunogenic cell death (ICD) markers in sensitive HNSCC cell lines in vitro . Experiments in syngeneic mouse models of HNSCC showed that ASTX660 can also enhance radiation-induced ICD in vivo . On a functional level, ASTX660 also enhanced killing of multiple murine cell lines by cytotoxic tumor-infiltrating lymphocytes, and when combined with XRT, stimulated clonal expansion of antigen-specific T lymphocytes and expression of MHC class I on the surface of tumor cells. Flow cytometry experiments in several human HNSCC cell lines showed that MHC class I (HLA-A,B,C) was reliably upregulated in response to ASTX660 + TNFα, while increases in other antigen processing machinery (APM) components were variable among different cell lines. These findings suggest that ASTX660 may enhance anti-tumor immunity both by promoting ICD and by enhancing antigen processing and presentation., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The role of T cell receptor (TCR) signaling for adaptive immune responses is essential. The ability to respond to a broad spectrum of tumor antigens requires an adaptive selection of various TCR. So far, little is known about the role of TCR richness and clonality in the cellular immune response to head and neck cancer (HNC), though the Endothelial Growth Factor Receptor (EGFR)-specific CD8 + T cell response can be enhanced by cetuximab therapy. Therefore, we investigated differences in TCR sequences between human papillomavirus (HPV) + and HPV - HNC patients, as well as differences in TCR sequence characteristics between T cells of peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL). Additionally, we were able to investigate the TCR richness and clonality in samples pre- and post- treatment in a prospective clinical trial of neoadjuvant cetuximab. Interestingly, HPV + and HPV - HNSCC did not significantly differ in the extent of TCR clonality and richness in PBMC or TIL. However, neoadjuvant cetuximab treatment increased the number of unique TCR sequences in PBMC (p = 0.0003), which was more prominent in the clinical responder patients compared to non-responders (p = 0.04). A trend toward TCR gene focusing was observed in TIL (p = 0.1) post-treatment. Thus, an increase in richness of TCR sequences in the periphery with a focusing at the tumor site is associated with an improved treatment response, suggesting an influence of peripheral quantity and intratumoral quality on adaptive immunity in cetuximab treated patients.

Head and neck squamous cell carcinomas (HNSCCs) frequently harbor genomic mutations in cell death pathways. Nearly 30% of HNSCCs overexpress Fas-Associated Death Domain (FADD), with or without BIRC2/3 genes encoding cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2), critical components of the Tumor Necrosis Factor (TNF) Receptor signaling pathways. ASTX660 is a novel non-peptidomimetic antagonist of cIAP1/2 and XIAP under evaluation in a clinical trial for advanced solid tumors and lymphomas. Herein, we show that ASTX660, at nanomolar concentrations, sensitized Murine Oral Cancer (MOC1) cells to TNFα. Using syngeneic mouse models, ASTX660 showed additive anti-tumor activity with radiation therapy (XRT), cisplatin chemotherapy, and PD-1 blockade to significantly delay or eradicate MOC1 tumors. These combinations significantly increased CD8 + T cells and dendritic cells, as well as T cell activity. ASTX660 stimulated cytotoxic T lymphocyte (CTL) killing of MOC1 cells expressing ovalbumin. Early stages of CTL killing were predominantly mediated by perforin/granzyme B, whereas later stages were mediated by death ligands TNFα, TRAIL, and FasL. Correspondingly, depletion of CD8 + T cells and NK cells in vivo revealed both types of immune cells to be important components of the complete anti-tumor response enhanced by ASTX660+XRT. These findings serve to inform future studies of IAP inhibitors and support the potential for future clinical trials investigating ASTX660 with XRT and immunotherapies like PD-1/PD-L1 blockade in HNSCC.