HIVEP3 inhibits fate decision of CD8⁺ invariant NKT cells after positive selection

CD8(+) invariant natural killer T (iNKT) cells are functionally different from other iNKT cells and are enriched in human but not in mouse. To date, their developmental pathway and molecular basis for fate decision remain unclear. Here, we report enrichment of CD8(+) iNKT cells in neonatal mice due to their more rapid maturation kinetics than CD8(-) iNKT cells. Along developmental trajectories, CD8(+) and CD8(-) iNKT cells separate at stage 0, following stage 0 double-positive iNKT cells, and differ in HIVEP3 expression. HIVEP3 is lowly expressed in stage 0 CD8(+) iNKT cells and negatively controls their development, whereas it is highly expressed in stage 0 CD8(-) iNKT cells and positively controls their development. Despite no effect on IFN-gamma, HIVEP3 inhibits granzyme B but promotes interleukin-4 production in CD8(+) iNKT cells. Together, we reveal that, as a negative regulator for CD8(+) iNKT fate decision, low expression of HIVEP3 in stage 0 CD8(+) iNKT cells favors their development and T helper 1-biased cytokine responses as well as high cytotoxicity. As a negative regulator for CD8(+) iNKT fate decision, a low HIVEP3 level in CD8(+) iNKT cells favors their development, Th1-biased responses, and high cytotoxicity.