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.