iNKT reversed cGVHD through donor Treg expansion and prevented the onset of cGVHD. (A-C) cGVHD was established as per Figure 1, except that BM and T cells were harvested from B6.Foxp3.Luci.DTR-4 mice. Group 1 and 2 are BM only and cGVHD control, as described before. Groups 3 through 5 are cGVHD mice that received DT (group 3), iNKT infusion on day 28 and day 42 (group 4), or iNKT infusion and DT (group 5). (A) On day 43, mice were imaged by the Spectrum In Vivo Imaging system. (B) Quantification of the bioluminescent imaging signal shows depletion of Tregs by DT and expansion of Tregs by iNKT infusion. (C) PFTs were assessed as described in Figure 1. Treg depletion by DT injection completely abolishes iNKTs efficacy. (D) iNKT cells (white arrow) were identified by CD45.1⁺ in the GC area. (E) Mice were transplanted as per Figure 1. iNKT cells from wild-type, CXCR5^−/−, or IL-4^−/− mice were infused to transplanted mice on days 28 and 42. iNKT cells from CXCR5^−/− or IL-4^−/− mice lost the ability to reverse cGVHD. (F) cGVHD mice were infused with B6 iNKTs either on days 1 and 14 (prophylaxis) or on days 28 and 42 (therapy). Prophylactic iNKT infusion completely blocks cGVHD. (G) Prophylactic or therapeutic RGI2001 (2.5 μg/mouse) was given to transplanted mice. PFTs suggest RGI2001 prevents and reverses cGVHD. Five to 8 mice per group were analyzed for each assay. Significance: *P < .05; **P < .01; ***P < .001; ****P < .0001.