Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+ stage of thymic ontogeny

T lineage commitment occurs in a discrete, stage-specific manner during thymic ontogeny. Intrathyrnic precursor transfer experiments and the identification of CD4(+)8(+) double-positive (DP), V alpha 14J alpha 18 natural T (iNKT) cells suggest that commitment to this lineage might occur at the DIP stage. Nevertheless, this matter remains contentious because others failed to detect V alpha 14J alpha 18-positive iNKT cells that are CD4+8+. In resolution to this issue, we demonstrate that retinoic acid receptor-related orphan receptor 7 (ROR gamma)(0/0) thymi, which accumulate immature single-positive (ISP) thymocytes that precede the DP stage, do not rearrange V alpha 14-to-J alpha 18 gene segments, suggesting that this event occurs at a post-ISP stage. Mixed radiation bone marrow chimeras revealed that ROR gamma functions in an iNKT cell lineage-specific manner. Further, introgression of a Bcl-X-L transgene into ROR gamma(0/0) mice, which promotes survival and permits secondary rearrangements of distal V alpha and J alpha gene segments at the DP stage, rescues V alpha 14-to-J alpha 18 recombination. Similarly, introgression of a rearranged V alpha 14J alpha 18 transgene into ROR gamma(0/0) mice results in functional iNKT cells. Thus, our data support the "T cell receptor-instructive (mainstream precursor) model" of iNKT cell lineage specification where V alpha 14-to-J alpha 18 rearrangement, positive selection, and iNKT cell lineage commitment occur at or after the DIP stage of ontogeny.