Therapeutic iNKT cell infusion reversed established cGVHD. B10.BR mice were conditioned by 2 doses of cyclophosphamide (120 mg/kg body weight, intraperitoneally) on day 3 and day 2 of transplantation. On day 1, B10.BR mice were irradiated (830 Gy by X-ray) and then infused with T-cell–depleted BM only or with 75 000 purified T cells to induce cGVHD. (A-B) On day 28 after transplantation, splenocytes were harvested from BM-only mice and cGVHD mice and naive mice of donor and recipient strains. Cells were stained with fluorochrome conjugated PBS57-CD1D tetramer, anti-CD4, anti-TCR-β, and viability dye. iNKT cells were identified by CD4⁺ TCR-β⁺PBS57-CD1D⁺ live cells. (A) Gating of iNKT cells. Cells were gated on live CD4⁺ T cells. (B) Frequency of iNKT is significantly reduced in cGVHD mice. (C-D) On days 28 and 42 posttransplantation, fluorescence-activated cell sorter sorted CD45.1 B6 iNKTs were infused to some cGVHD mice at a lower (50 000) or higher (100 000) dose. (C) Pulmonary function tests (PFTs), including resistance, elastance, and compliance, were performed on day 56 posttransplantation. iNKT infusion significantly improves the pulmonary function. (D) Hydroxyproline was measured in the lungs of mice from (C). iNKT infusion at the 100 000 cell dose significantly reduces hydroxyproline. (E) Trichrome staining that identifies collagen was performed on cryosections of lungs and imaged at 200×. (F) Collagen deposition was quantified by measuring the blue area by Fiji software. iNTK infusion significantly reduce collagen deposition in the lung. (G) cGVHD was established as previously described. Mice received Balb/c iNKT cells on days 28 and 42. Balb/c iNKT cells reverse cGVHD. (H) Cryosections of spleen (day 56) were stained with fluorochrome conjugated anti-CD4 , anti-Foxp3 (eFluor660), and peanut-agglutinin (PNA) (Rhodamine) and imaged by Olympus FV1000 system at 400×. Dotted lines delineate GC areas by PNA staining. (I) Follicular Tregs were identified as CD4⁺Foxp3⁺ cells within the GC areas. (J) Average GC size is decreased by iNKT. (K) Follicular Treg density is increased by iNKT. (L-M) Splenic GC B cells and follicular Tregs frequencies were determined by flow cytometry on day 55 post-transplantation. iNKT infusion decreases GC B and increases follicular Treg frequencies. Unpaired student t-test was used when comparing 2 groups. Data shown are representative of 2 to 4 independent experiments with 5 to 8 mice per group, except (G) with 1 experiment with 5 to 8 mice. *P < .05; **P < .01; ***P < .001; ****P < .0001.