Single-cell analysis uncovers differential regulation of lung γδ T cell subsets by the co-inhibitory molecules, PD-1 and TIM-3

ABSTRACT IL-17A-producing γδ T cells within the lung consist of both Vγ6 + tissue-resident cells and Vγ4 + circulating cells that play important roles in homeostasis, inflammation, infection, tumor progression and metastasis. How these γδ T cell subsets are regulated in the lung environment during homeostasis and cancer remains poorly understood. Using single-cell RNA sequencing and flow cytometry, we show that lung Vγ6 + cells express a repertoire of cell surface molecules distinctive from Vγ4 + cells, including PD-1 and ICOS. We found that PD-1 functions as a co-inhibitory molecule on Vγ6 + cells to reduce IL-17A production, whereas manipulation of ICOS signaling fails to affect IL-17A in Vγ6 + cells. In a mammary tumor model, ICOS and PD-1 expression on lung Vγ6 + cells remained stable. However, Vγ6 + and Vγ4 + cells within the lung pre-metastatic niche increased expression of IL-17A, IL-17F, amphiregulin (AREG) and TIM-3 in response to tumor-derived IL-1β and IL-23, where the upregulation of TIM-3 was specific to Vγ4 + cells. Inhibition of either PD-1 or TIM-3 in mammary tumor-bearing mice further increased IL-17A by Vγ6 + and Vγ4 + cells, indicating that both PD-1 and TIM-3 function as negative regulators of IL-17A-producing γδ T cell subsets. Together, these data demonstrate how lung γδ T cell subsets are differentially controlled by co-inhibitory molecules in steady-state and cancer.