Background Head and neck squamous cell carcinoma (HNSCC) is caused by high tobacco and alcohol consumption and/or human papillomavirus (HPV) associated.1-3 Anti-PD-1 immunotherapy only benefits 15-20% of HNSCC patients4, 5 Thus, there is a need to identify mechanisms involved in resistance to immune checkpoint blockade. Several studies have described the transcriptional phenotypes of tumor infiltrating lymphocytes (TIL) and correlated it with clinical response to ICB.6-8 It has also been shown that majority of neoantigen-specific CD8+ T cells from the tumor have an exhausted-like phenotype 9-11, but specific targetable epitopes and whether anti-viral T cells dominate in HPV-associated HNSCC immunotherapy have not been determined. Collectively, these studies do not address neoantigen and HPV-antigen specificities of evaluated T cells and their transcriptional and functional status in the HNSCC TME, nor whether these cells re-circulate and demonstrate restoration of function once in the peripheral circulation.
Methods Single cell suspensions were prepared from tumor and blood of 21 treatment-naïve HNSCC patients. T cells were FACS-sorted using barcoded mAbs (CITE-seq) and used to generate single cell RNA sequencing (scRNA), T cell receptor (TCR) and ADT libraries using 10x Genomics workflow. A cell-based method for epitope discovery (SABRs) was used to characterize antigen specificity of selected CD8+ TILs.12
Results Clonal expanded CD8 T cells in the tumor express high levels of T cell exhaustion markers (PDCD1, HAVCR2, ENTPD1, LAG3). GSEA analysis show that highly expanded CD8+ TILs share gene signatures with neoantigen-specific CD8+ TILs identified from other cancers. There was no significant overlap observed between TCRs of CD8 T cells with high expression of immune checkpoint receptors in the tumor with peripheral blood CD8+ T cells. TCR analysis show that T cell clones shared between tumor and peripheral blood are found in a cluster of CD8 T cells with an effector memory phenotype expressing distinct granzyme expression in activated versus exhausted tumor reactive cells.
Conclusions In this study, we’d like to evaluate 1). Antigen specificities and transcriptional phenotypes of CD8+ T cells. We found that potential tumor reactive cells express high checkpoints, implying most of tumor reactive cells are dysfunctional within tumor. 2). Clonal dynamics of tumor reactive cells. We observed low clonal overlap between peripheral and tumor resident TCRs, suggesting tumor resident population expanded locally. Shared T cell clones between peripheral CD8+ T cells in effector memory population may reflect an active recruitment and/or exhange of T cells between periphery and tumor.
Acknowledgements This research was funded by R01 CA206517 (RLF), P50 CA097190 (RLF), and R01 DE031947 (RLF, LV). This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided by using the HTC cluster, supported by NIH award number S10OD028483. This research utilized the UPMC Hillman Cancer Center Flow Cytometry Core Facility, supported in part by award P30 CA047904 (RLF).
Bray F, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68(6):394–424.
Powell SF, et al. The Key Differences between Human Papillomavirus-Positive and -Negative Head and Neck Cancers: Biological and Clinical Implications. Cancers (Basel), 2021;13(20).
Chaturvedi AK, et al. Human papillomavirus and rising oropharyngeal cancer incidence in the United States. J Clin Oncol 2011;29(32):4294–301.
Cramer JD, et al. The changing therapeutic landscape of head and neck cancer. Nat Rev Clin Oncol, 2019. 16(11):669–683.
Ferris RL, et al. Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck. N Engl J Med, 2016;375(19):1856–1867.
Liu B, et al. Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer. Nat Cancer 2022;3(1):108–121.
Bassez A, et al. A single-cell map of intratumoral changes during anti-PD1 treatment of patients with breast cancer. Nat Med 2021;27(5):820–832.
Zhang Y, et al. Single-cell analyses reveal key immune cell subsets associated with response to PD-L1 blockade in triple-negative breast cancer. Cancer Cell, 2021;39(12):1578–1593 e8.
Zheng C, et al. Transcriptomic profiles of neoantigen-reactive T cells in human gastrointestinal cancers. Cancer Cell 2022;40(4):410–423 e7.
Oliveira G, et al. Phenotype, specificity and avidity of antitumour CD8(+) T cells in melanoma. Nature 2021;596(7870):119–125.
Caushi JX, et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature 2021;596(7870):126–132.
Joglekar AV, et al. T cell antigen discovery via signaling and antigen-presenting bifunctional receptors. Nat Methods 2019;16(2):191–198.
Ethics Approval Peripheral blood and tumor tissues from treatment-naïve HNSCC patients were collected with their written consent in accordance with the Declaration of Helsinki, under the University of Pittsburgh Cancer Institute Review Board–approved protocol (99-069).
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.