Third-party CD4⁺ iNKT cells are rejected early after allogeneic HCT. (A) GFP⁺CD4⁺ iNKT cells were enriched (magnetic-activated cell sorting) and sorted (fluorescence-activated cell sorting) from whole splenocytes of GFP-expressing FVB/N mice enabling distinct re-isolation from BALB/c recipient mice after adoptive transfer. Representative dot plots gated on live cells. (B) Absolute cell numbers of adoptively transferred FVB/N donor GFP⁺CD4⁺ iNKT cells re-isolated at different time points from various organs of BALB/c mice that received TCD-BM and Tcons from WT FVB/N donor mice. No GFP⁺CD4⁺ iNKT cells could be re-isolated from lymph nodes, gut, and skin (not shown). Error bars indicate SEM. Shown are cell numbers from 3 mice per time point from 1 of 2 independent experiments. (C) Fluorescence intensity deriving from 1.5 × 10⁶ adoptively transferred FVB/N donor GFP⁺CD4⁺ iNKT cells labeled with CellTrace Violet and re-isolated at days +3 and +5. Histogram gated on GFP⁺ cells pooled from the spleen and liver of one BALB/c mouse that received TCD-BM and Tcons from one WT FVB/N donor mouse. Shown is 1 of 3 independent experiments. (D) Donor and third-party model of allogeneic HCT with BALB/c (H-2K^d) mice receiving TCD-BM and Tcons from WT FVB/N (H-2K^q) and C57BL/6 (H-2K^b) mice, respectively. Adoptively transferred CD4⁺ iNKT cells derived from GFP-expressing FVB/N (H-2K^q) mice and were re-isolated from recipient livers at days +5 and +10. Shown are representative dot plots gated on live cells from 1 of 2 independent experiments. The third row shows T-cell chimerism from the same sample. Comparable results were obtained for re-isolation of GFP⁺ cells from recipient spleens (not shown). LVR, liver; SPN, spleen; THY, thymus.