Rare subpopulations of tumor antigen-reactive memory T cells, which actively secrete type-1 effector cytokines, particularly TNF-α in situ, possess anti-tumor activity and prognostic relevance. These cells are relevant for cancer immunotherapy; however, their low frequencies make them difficult to study and novel protocols for their culture and expansion ex vivo are needed.

Here, we studied the presence of T cells secreting type-1 cytokines (Cy⁺T cells) in the blood and tumors of 24 patients with oral squamous cell carcinomas (OSCC) and explored possibilities for their isolation and expansion. More than 90% of OSCC patients contained enriched numbers Cy⁺T cells in the blood and tumors compared to healthy donors in which these were hardly detectable. The majority of TNF-α⁺T cells were CD4⁺ T helper cells while IFN-γ⁺TIL were predominantly CD8⁺. Cy⁺T helper cells in the blood were early-differentiated memory T cells while Cy⁺TIL and Cy⁺CD8⁺T cells showed advanced-differentiated memory T cell phenotypes. We explored different conditions for their in vitro culture and found that Cy⁺T cells can be efficiently expanded in vitro to similar levels as Cy⁻T cells and after expansion maintained their TNF-α secreting capacity. However, for optimal expansion they required specific culture conditions to support the maintenance of stem-like and central memory T cell phenotype. In conclusion, we show that Cy⁺T cells are enriched in OSCC patients and report a novel cell culture protocol optimized to specifically expand and functionally maintain these cells for further functional characterization or for their exploitation in immunotherapy of OSCC.

肿瘤抗原反应性记忆 T 细胞的罕见亚群，可主动分泌 1 型效应细胞因子，尤其是原位TNF-α ，具有抗肿瘤活性和预后相关性。这些细胞与癌症免疫治疗相关；然而，它们的低频使它们难以研究，因此需要新的体外培养和扩增方案。

在这里，我们研究了24 名口腔鳞状细胞癌 (OSCC) 患者的血液和肿瘤中分泌 1 型细胞因子（Cy ⁺ T 细胞）的 T 细胞的存在，并探索了它们分离和扩增的可能性。与几乎无法检测到的健康供体相比，超过 90% 的 OSCC 患者在血液和肿瘤中含有丰富数量的 Cy ⁺ T 细胞。大多数 TNF-α ⁺ T 细胞是 CD4 ⁺ T 辅助细胞，而 IFN-γ ⁺ TIL 主要是 CD8 ⁺。血液中的Cy ⁺ T 辅助细胞是早期分化的记忆 T 细胞，而 Cy ⁺ TIL 和 Cy ⁺ CD8 ⁺T 细胞显示出晚期分化的记忆 T 细胞表型。我们探索了其体外培养的不同条件，发现 Cy ⁺ T 细胞可以在体外有效扩增至与 Cy ^- T 细胞相似的水平，并且在扩增后保持其 TNF-α 分泌能力。然而，为了实现最佳扩增，它们需要特定的培养条件来支持干细胞样和中枢记忆 T 细胞表型的维持。总之，我们表明 Cy ⁺ T 细胞在 OSCC 患者中富集，并报告了一种新的细胞培养方案，该方案经过优化以特异性扩增和功能性维持这些细胞，以进一步进行功能表征或在 OSCC 的免疫治疗中利用它们。