The human lung T cell compartment contains many CD8⁺ T cells specific for respiratory viruses, suggesting that the lung is protected from recurring respiratory infections by a resident T cell pool. The entry site for respiratory viruses is the epithelium, in which a subset of lung CD8⁺ T cells expressing CD103 (αE integrin) resides. Here, we determined the specificity and function of CD103⁺CD8⁺ T cells in protecting human lung against viral infection. Mononuclear cells were isolated from human blood and lung resection samples. Variable numbers of CD103⁺CD8⁺ T cells were retrieved from the lung tissue. Interestingly, expression of CD103 was seen only in lung CD8⁺ T cells specific for influenza but not in those specific for EBV or CMV. CD103⁺ and influenza-reactive cells preferentially expressed NKG2A, an inhibitor of CD8⁺ T cell cytotoxic function. In contrast to CD103^–CD8⁺ T cells, most CD103⁺CD8⁺ cells did not contain perforin or granzyme B. However, they could quickly upregulate these cytotoxic mediators when exposed to a type I IFN milieu or via contact with their specific antigen. This mechanism may provide a rapid and efficient response to influenza infection, without inducing cytotoxic damage to the delicate epithelial barrier.

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.

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probeH5with an optimal fit into granzyme B. We demonstrate that probeH5enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probeH5may enable the monitoring of early responses to anticancer treatments using tissue biopsies.