Single-cell Proteogenomics Reveals That SLAM/SAP Signaling Regulates the Development of Innate-Like Γδ T Cell Subsets with Distinct TCR Repertoires

γδ T cells are non-conventional T cells that are highly enriched in the mucosal tissues where they play critical roles in immunity. We recently demonstrated that the SLAM/SAP signaling pathway regulates the thymic development of innate-like γδT17 and γδTIFN subsets, in addition to γδNKT cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. This analysis not only confirmed our previous finding that SLAMF1 and SLAMF6 expression marks γδT17 and γδTIFN subsets, respectively, it also identified SLAMF7 as a novel marker of the SAP-dependent innate-like CD44+ CD45RB+ γδTIFN cells in both the thymus and periphery. Next, our data indicated that disruption of SAP-dependent signaling impaired γδT17 development at a very early (CD24high CD73−) stage, and was associated with the decreased expression of critical regulators of γδT17 development such as Blk and c-Maf. In contrast, while SAP also impaired γδTIFN development at an early (CD24high CD73+) stage, SAP-deficient γδTIFN cells exhibited increased expression of genes associated with TCR signaling and thymic export such as Prkch, Dgka, Klf2, and S1p1r. Finally, our analysis revealed significant alterations in the γδT17 TCR repertoire in both embryonic/neonatal thymus and the lung, as well as the presence of a SAP-dependent IFN-γ-producing lung Vγ4 population that preferentially utilized TRDV7. Altogether, these data suggest that SLAM/SAP signaling acts during the very early stages of γδ T cell development where it regulates critical pathways in both γδT17 and γδTIFN development, and influences the development of the innate-like γδ TCR repertoire. Supported by NIH (R03AI153902, P30GM118228), AAI Careers in Immunology Fellowship