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1022 Tumor-infiltrating CXCR6-expressing tissue-resident memory HPV-specific CD8+ T cells are terminally exhausted in HPV-associated head and neck squamous cell carcinoma
  1. Yang Cheng,
  2. Kathleen Atkinson,
  3. Talia Saal,
  4. Jean Campbell and
  5. Sara Pai
  1. Massachusetts General Hospital, Boston, MA, United States


Background Tissue-resident memory T cells (TRM) are important for controlling both infection and tumor growth. We performed comprehensive immunophenotyping of HPV-specific and non-HPV-specific viral bystander (EBV, CMV, IAV, and SARS2-CoV2) CD8+ TRM cells in HPV-associated HNSCCs.

Methods We assessed HPV-specific and non-HPV-specific viral bystander CD8+ T cell frequency and functional states in the primary tumor, tumor-involved lymph node (LN), and peripheral blood mononuclear cells (PBMC) from 34 HPV-associated HNSCC patients. CD8+ T cells were isolated and probed by 89 different pMHC tetramers across three HLA alleles (A*02:01, A*01:01 and A*24:02) using multiplexed combinatorial peptide-MHC (pMHC) tetramer staining and spectral flow cytometry. To interrogate HPV-specific responses, we analyzed 61 HPV16 E6 and E7 epitopes. To compare non-HPV bystander virus-specific T cells, we investigated 11 bystander virus (EBV, CMV and IAV) and 17 SARS2-CoV-2 (spike protein) epitopes. Tetramer+ CD8+ T cells were immunophenotyped by co-staining 20 cellular markers to probe T cell exhaustion and tissue resident phenotypes.

Results 76.4% (26 of 34) of HPV-associated HNSCCs had detectable HPV-specific T cells within their tissue and 82% (28 of 34) had bystander virus-specific T cells. Overall, we identified 26 HPV- (10 HPV16-E6 and 16 HPV16-E7), 11 bystander (non-HPV) virus-, and 8 SARS-CoV-2 spike-specific CD8+ T cell populations. We observed a broader HPV epitope repertoire within LN and PBMC as compared to tumors. However, the frequency of HPV-specific T cells infiltrating the tumor was significantly higher compared to the LN and PBMC. Within the tumor, 13 HPV- (6 HPV16-E6 and 7 HPV16-E7), 6 bystander (non-HPV) virus-, and 3 SARS-CoV-2 spike-specific CD8+ T cell populations were identified. The frequency of tumor-infiltrating HPV-specific T cells ranged from 0.02-3.01% for E6, 0.013-10.9% for E7, 0.013-1.93% for bystander (non-HPV) virus-specific T cells, and 0.014-11.5% for SARS-CoV-2 spike-specific T cells. High-dimensional analysis identified four distinct TRM subsets. Tumor-infiltrating HPV-specific T cells were enriched for two CXCR6+ TRM subsets that were also PD-1hiTIM-3+, suggesting they were terminal exhausted. In contrast, tumor- and LN-infiltrating bystander non-HPV-specific T cells were CXCR5+PD-1int TRM and not exhausted.

Conclusions We report a high frequency of HPV and bystander viral epitopes within HPV-associated HNSCCs, and identified two distinct CXCR6+ terminal exhausted HPV-specific TRM subsets within the tumor. Spatial localization of these two distinct CXCR6+ terminally exhausted TRM to myeloid populations in the primary tumor and tumor draining lymph nodes are ongoing to better understand tumor-specific T cell priming within these two sites.

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