Abstract PR-01: Distinct CD4+ T cell subpopulations as predictive biomarkers for anti-PD-1 and chemotherapy response in advanced NSCLC

Abstract Background: The effectiveness of immunotherapy varies significantly among patients with advanced non-small cell lung cancer (NSCLC). This study investigates distinct subpopulations of CD4+T cells in the peripheral blood of stage IV NSCLC patients treated with anti-PD-1 combined with chemotherapy to identify immune mechanisms and systemic markers associated with treatment response or failure. Methods: We analyzed a cohort of 33 treatment-naive stage IV NSCLC patients, categorizing them as responders or non-responders based on clinical outcomes at 9 weeks and 6 months post-therapy initiation. Peripheral blood mononuclear cells (PBMCs) were collected before treatment, and high-dimensional flow cytometry was employed to profile immune cell subpopulations. Additionally, single-cell RNA sequencing (scRNAseq) data from both the same patients in our cohort and an independent cohort were analyzed to expand and validate the findings. Results: Non-responders exhibited elevated levels of suppressive phenotype CD4+ T lymphocytes expressing IL-10, CTLA-4, and other inhibitory markers like CD161 and CD95. Conversely, responders showed higher frequencies of activated CD4+CD69+ T lymphocytes, as well as increased percentages of TCF-1+ Effector Memory, Effector, and Central Memory CD4+ T cells. Responders also demonstrated higher levels of TCF7 and CXCR3 expression, markers associated with effective T cell activation and migration to the tumor microenvironment. Furthermore, single-cell RNA sequencing from the same cohort revealed differentially expressed genes in CD4+ T cells between responders and non-responders. Responders exhibited upregulation of cytotoxic genes such as NKG7 and GNLY and downregulation of genes like IL7R. Non-responders had higher levels of CTLA4 and KLRB1 expression, markers associated with immune suppression. ROC analysis showed that the frequencies of CD4+IL-10+ and CD4+TCF-1+ T cells could predict response with an AUC of 0.84 (p < 0.007). Finally, an in vitro dual blockade study with anti-PD-1/CTLA-4 using non-responder PBMC demonstrated that combining these two drugs significantly increased IFN-gamma and granzyme B production in CD4+ and CD8+ T cells. Conclusion: This study highlights the critical role of distinct CD4+ T cell subpopulations in predicting therapeutic outcomes in NSCLC patients undergoing anti-PD-1 therapy with chemotherapy. These findings provide valuable insights into the immune mechanisms driving response to immunotherapy and propose potential biomarkers for patient stratification. Citation Format: Amanda Figueiredo, Guilherme Evangelista, Clara Cavalcanti, Kátia Morais, Thaiany Goulart Souza-Silva, Robert Balderas, Rodrigo Pestana Lopes, Juan Santos e Silva, Jonathan Avila, Helder Nakaya, Nayane Galdino, Larissa Melo Kuil, Ananda Domingues Lopes, James Turner, Helano Freitas, Jefferson Luiz Gross, Walderez O Dutra, Valdemir Lima, Kenneth J Gollob. Distinct CD4+ T cell subpopulations as predictive biomarkers for anti-PD-1 and chemotherapy response in advanced NSCLC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr PR-01.