Abstract B025: CD28-negative T cells from head and neck cancer patients are functionally frail but not senescent

Abstract Introduction: T cells lacking costimulatory receptors CD27/28, with high levels of differentiation markers CD57 and KLRG1, may accumulate with aging. These T cells have been described as “senescent” (unable to proliferate) with high cytolytic enzyme expression. However, robust functional studies on these cells are lacking. Further, emerging studies on the implications of these cells in the context of prognosis and responses to immunotherapy have shown mixed results. To better understand the role of these cells in the pathogenesis of cancer, a better understanding of their functional potential is needed. Methods and Results: We collected blood from 46 patients with previously untreated, locally advanced head and neck squamous cell carcinoma (HNSCC), aged 47-89, undergoing surgery. Matched tumor tissue was collected from 29 patients. Samples were analyzed by flow cytometry. There was correlation between the percentage of CD27/28-CD57+ (CD28neg) T cells in the blood vs. tumor (R2 = 0.51 for CD8+, 0.41 for CD4+; p<0.001). The CD28neg proportion among CD8+ T cells was significantly higher in patients aged 70 or greater vs. patients under age 70. CD28neg cells from blood and tumors expressed very high levels of granzyme B and perforin but were not enriched for senescence markers p16 and beta-galactosidase. Additional CD8+ cells from the peripheral blood were sorted into CD28neg (CD27/28-CD57+) vs. CD27/28+ for comparative functional studies. Proliferation assays showed a stepwise reduction in CellTrace Violet in both the CD28neg and CD27/28+ populations over 5 days in culture with IL-2 and CD2/3/28 stimulation cocktail. While the percentage of CD28neg cells that had completed 3 or more cell divisions was significantly reduced from that of the CD27/28+ population, approximately 40% of the CD28neg cells had divided at least three times over 5 days in culture. Sorted CD28neg cells retained high levels of granzyme B/perforin, and PMA/ionomycin stimulation induced increased CD107a expression and granzyme B secretion, suggesting that these cells do possess enhanced cytolytic capacity. CD28neg cells also expressed higher levels of surface phosphatidylserine, which has been associated with cellular stress in viable cells. CD28neg cells stimulated with CD3 beads secreted higher levels of TNF-a, but not IFN-g or IL-2, vs. CD27/28+ cells. Conclusions: CD28neg T cells sampled from HNSCC patients have altered functionality, including a reduced (but intact) capacity to proliferate and increased phosphatidylserine expression. However, they also have enhanced capacity to secrete cytolytic enzymes and TNF-a, suggesting they may have differentiated to perform those specific functions. We propose that these T cells are more appropriately described as “frail,” and “immunofrailty” may be a more appropriate term than “immunosenescence.” Additional prospective clinical studies are needed to elucidate the effects of these cells on prognosis and treatment responses in cancer patients. Citation Format: Brendan L.C. Kinney, Connor Parrish, Vikash Kansal, Zachary S. Buchwald, Haydn T. Kissick, Amy Y. Chen, Nicole C. Schmitt. CD28-negative T cells from head and neck cancer patients are functionally frail but not senescent [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B025.