Functional Heterogeneity of CD4⁺ Tumor-Infiltrating Lymphocytes With a Resident Memory Phenotype in NSCLC

Resident memory T cells (T_RM) inhabit peripheral tissues and are critical for protection against localized infections. Recently, it has become evident that CD103⁺ T_RM are not only important in combating secondary infections, but also for the elimination of tumor cells. In several solid cancers, intratumoral CD103⁺CD8⁺ tumor infiltrating lymphocytes (TILs), with T_RM properties, are a positive prognostic marker. To better understand the role of T_RM in tumors, we performed a detailed characterization of CD8⁺ and CD4⁺ TIL phenotype and functional properties in non-small cell lung cancer (NSCLC). Frequencies of CD8⁺ and CD4⁺ T cell infiltrates in tumors were comparable, but we observed a sharp contrast in T_RM ratios compared to surrounding lung tissue. The majority of both CD4⁺ and CD8⁺ TILs expressed CD69 and a subset also expressed CD103, both hallmarks of T_RM. While CD103⁺CD8⁺ T cells were enriched in tumors, CD103⁺CD4⁺ T cell frequencies were decreased compared to surrounding lung tissue. Furthermore, CD103⁺CD4⁺ and CD103⁺CD8⁺ TILs showed multiple characteristics of T_RM, such as elevated expression of CXCR6 and CD49a, and decreased expression of T-bet and Eomes. In line with the immunomodulatory role of the tumor microenvironment, CD8⁺ and CD4⁺ TILs expressed high levels of inhibitory receptors 2B4, CTLA-4, and PD-1, with the highest levels found on CD103⁺ TILs. Strikingly, CD103⁺CD4⁺ TILs were the most potent producers of TNF-α and IFN-γ, while other TIL subsets lacked such cytokine production. Whereas, CD103⁺CD4⁺PD-1^low TILs produced the most effector cytokines, CD103⁺CD4⁺PD-1⁺⁺ and CD69⁺CD4⁺PD-1⁺⁺ TILs produced CXCL13. Furthermore, a large proportion of TILs expressed co-stimulatory receptors CD27 and CD28, unlike lung T_RM, suggesting a less differentiated phenotype. Agonistic triggering of these receptors improved cytokine production of CD103⁺CD4⁺ and CD69⁺CD8⁺ TILs. Our findings thus provide a rationale to target CD103⁺CD4⁺ TILs and add co-stimulation to current therapies to improve the efficacy of immunotherapies and cancer vaccines.