Developmental trajectory of unconventional T cells of the cynomolgus macaque thymus

As nonhuman primates are immunologically the closest model to humans, a comprehensive understanding of T-cell development in these species is crucial. However, the differentiation pathways in which thymocytes participate, along with their heterogeneity, remain poorly characterized. Using single-cell RNA sequencing, we thoroughly profiled the development of various T-cell lineages in the juvenile cynomolgus monkey thymus, identifying and characterizing 12 distinct thymic cell states or types. Interestingly, we identified two unexpected cell types, an agonist-selected and a memory-like cell population. The agonist-selected cell population expressed genes associated with strong TCR signaling, such as PDCD1, CD5, NFKBID, NFATC1, BCL2L11, and NR4A1 but exhibiting significantly higher PDCD1 expression compared with cells following the conventional developmental pathway. Additionally, we identified a substantial number of memory-like cell populations characterized by high CXCR3 and EOMES expression. Notably, this population also highly expressed the effector-associated markers, GZMK, NKG7, and GNLY, as well as the innate cell-associated markers, ZBTB16, TYROBP, KLRB1, KLRC1, and NCR3. The EOMES + memory-like cell population expressed highly PDCD1, indicating the presence of an agonist-selection footprint. Our findings provide insights into the agonist-selection pathway that allows self-reactive thymocytes to survive thymic selections and differentiate into various unconventional T-cell lineages.