Purified and adoptively transferred CD4⁺ iNKT cells protect from GVHD in a dose-dependent manner. (A) Magnetic-activated cell sorting (MACS) followed by fluorescence-activated cell sorting (FACS) of CD4⁺ iNKT cells. Shaded histogram curves depict CD4 isotype control. (B) Survival, (C) weight, and (D) GVHD score of BALB/c mice co-injected with 5.0 × 10⁶ TCD-BM cells, 1.0 × 10⁶ Tcons, and increasing doses of CD4⁺ iNKT cells (▲ 1.0 × 10⁴; ● 2.5 × 10⁴; ♦ 5.0 × 10⁴; ▪ 1.0 × 10⁵ per mouse) isolated from C57BL/6 donor mice. Shown are 5 animals per group from 1 of 2 independent experiments. (E) Representative photomicrographs of hematoxylin and eosin–stained sections of haired skin (×200 magnification; scale bar = 100 μ; inset ×400 magnification) and large intestines (×400 magnification; scale bar = 50 μ). The haired skin in all mice on day +10 appears essentially normal except for atrophy of subcutaneous adipose tissues (solid asterisk) in the Tcons and CD4⁺ iNKT groups (compared with normal adipose tissues in the bone marrow control group [open asterisk]). By day +35, the haired skin remained normal (bone marrow group) or reverted to normal (CD4⁺ iNKT group), but there were significant lesions in the Tcon group, with the presence of marked epidermal hyperplasia, continued atrophy of subcutaneous adipose tissues (solid asterisk), loss of hair follicles, and presence of moderate numbers of observable apoptotic keratinocytes in the stratum basale (arrows). On higher magnification (inset), these apoptotic keratinocytes appear as shrunken, hypereosinophilic (deep red) round bodies with pyknotic nuclei. The large intestines in both the bone marrow and CD4⁺ iNKT group appear essentially normal on days +10 and +35, except for the presence of rare apoptotic enterocytes (arrows), which appear as shrunken, hypereosinophilic (deep red) round bodies with pyknotic nuclei. In contrast, in the Tcon group, there was already development of mild lymphocytic colitis on day +10 characterized by small numbers of lymphocytes in the lamina propria (solid asterisks) that caused mild separation of intestinal glands, which themselves displayed mild loss of goblet cells with replacement by undifferentiated, proliferative basophilic (deep blue-purple) crypt enterocytes. Note the presence of small numbers of apoptotic enterocytes (arrows) as well. By day +35, the large intestinal lesions had worsened in the Tcon mice, with larger numbers of lymphocytes in the lamina propria (solid asterisks), larger separation of intestinal glands, marked loss of goblet cells with replacement undifferentiated crypt enterocytes, and larger numbers of apoptotic enterocytes (arrows). Histopathologic findings in recipient livers were mild, and only minor differences between groups were observed (not shown). †Indicates all animals from the respective group died or needed to be euthanized. Error bars indicate standard error of the mean. BM, bone marrow (control group); BMT, bone marrow transplantation.