CD24⁺ Cell Depletion Permits Effective Enrichment of Thymic iNKT Cells While Preserving Their Subset Composition

Invariant NKT (iNKT) cells are a small subset of thymus-generated T cells that produce cytokines to control both innate and adaptive immunity. Because of their very low frequency in the thymus, in-depth characterization of iNKT cells can be facilitated by their enrichment from total thymocytes. Magnetic-activated cell sorting (MACS) of glycolipid antigen-loaded CD1d-tetramer-binding cells is a commonly used method to enrich iNKT cells. Surprisingly, we found that this procedure also dramatically altered the subset composition of enriched iNKT cells. As such, NKT2 lineage cells that express large amounts of the transcription factor promyelocytic leukemia zinc finger were markedly over-represented, while NKT1 lineage cells expressing the transcription factor T-bet were significantly reduced. To overcome this limitation, here, we tested magnetic-activated depletion of CD24⁺ immature thymocytes as an alternative method to enrich iNKT cells. We found that the overall recovery in iNKT cell numbers did not differ between these 2 methods. However, enrichment by CD24⁺ cell depletion preserved the subset composition of iNKT cells in the thymus, and thus permitted accurate and reproducible analysis of thymic iNKT cells in further detail.