Host NKT cells facilitate the early proliferation of chimeric donor CD4⁺Foxp3⁺ Tregs. (A) Representative FACS analyses of CD4 versus intracellular Foxp3 staining at day 6 of gated H-2K^b+CD4⁺ splenocytes from normal untreated (UNT) C57BL/6 control mice and from the following hosts: 800 cGy TBI/ATS wild-type BALB/c hosts or TLI/ATS wild-type BALB/c hosts given untreated transplants, TLI/ATS wild-type BALB/c hosts given CD25 T-cell–depleted transplants, TLI/ATS Jα18^−/− BALB/c hosts given untreated transplants, TLI/ATS Jα18^−/− BALB/c hosts given 0.5 × 10⁶ sorted wild-type BALB/c NKT cells with untreated donor cell transplants, and TLI/ATS Jα18^−/− BALB/c hosts given 0.5 × 10⁶ sorted BALB/c NKT cells with CD25⁺ T-cell–depleted donor cell transplants. Percentage of CD4⁺Foxp3⁺ cells among gated CD4⁺ donor cells is shown enclosed in boxes. (B) Mean (± SE; × 10⁵) absolute number of H-2K^b+CD4⁺CD25⁺Foxp3⁺ cells isolated from spleen at day 6 in groups of transplanted BALB/c hosts from panel A. n = 5-10 per group. (C) Representative FACS plots showing CD4 versus intracellular Foxp3 staining in gated H-2K^b+CD4⁺ cells from MLN and colon of wild-type BALB/c hosts conditioned with either TBI/ATS or TLI/ATS at day 6 after injection of untreated donor cell transplants. Percentage of Foxp3⁺ cells is shown. (D) Representative histograms showing CFSE-staining intensity versus cell number for gated CD45.1⁺CD4⁺TCRαβ⁺Foxp3⁺ cells and CD45.1⁺CD8⁺TCRαβ⁺ cells from the pretransplant donor cell inoculum at day 0 (DONOR C57BL/6 [PRE]) and cells isolated from spleen of transplanted TLI/ATS-conditioned hosts at day 6. SYN, Wild-type CD45.2 C57BL/6 TLI/ATS-conditioned hosts given C57BL/6 CD45.1⁺ splenocytes and CD45.2⁺ marrow cells; ALLO, wild-type CD45.2 BALB/c TLI/ATS-conditioned hosts given C57BL/6 CD45.1⁺ splenocytes and CD45.2⁺ marrow cells. n = 2-3 pooled animals per group. Percentage of cells with 2 or more divisions is shown. (E) Mean (± SE; %) gated CD45.1⁺CD4⁺TCRαβ⁺Foxp3⁺ cells and CD45.1⁺CD8⁺TCRαβ⁺ CFSE^low cells in the groups shown in panel D. Mean of 2 to 3 experiments. n = 2-3 pooled animals per group. Determination of CFSE^low cells is detailed in “Methods.”