Third-party CD4⁺ iNKT cells protect from lethal GVHD. BALB/c recipient mice were irradiated with 2 × 4 Gy, followed by transplantation of 5.0 × 10⁶ TCD-BM cells and 1.0 × 10⁶ Tcons from C57BL/6 donor mice. In addition, 5.0 × 10⁴ CD4⁺ iNKT cells from C57BL/6 donor or FVB/N third-party mice were transferred together with the graft. (A) Overall survival pooled from 2 independent experiments with 10 mice per group except irradiation control (n = 5). (B) Weight and (C) GVHD score from 1 of 2 independent experiments with 5 animals per group except irradiation control (n = 3). Error bars indicate standard error of the mean (SEM). (D) Photomicrographs of small intestines, large intestines, haired skin, and liver at day +25. Small intestinal histology is normal in BM mice with presence of regularly-shaped and -sized villi, regularly-spaced and -sized intestinal crypts, presence of a regular complement of enterocytes (including goblet cells), and absence of any significant inflammation in the lamina propria and submucosa. In Tcon mice, there are many apoptotic cells in the crypt epithelium (black arrows), which appear as shrunken, hypereosinophilic, round bodies with pyknotic nuclei. Mild-to-moderate lymphoplasmacytic inflammation (black asterisks) is also present in the lamina propria and/or submucosa of the same mice, causing mild separation of the intestinal crypts. Third-party CD4⁺ iNKT-cell–treated mice have essentially normal small intestines when compared with the BM control mice, except for the presence of mild inflammatory infiltrates in the lamina propria (black asterisks). H&E stain; original magnification ×400; bar = 50 µm. Large intestinal histology is normal in BM control and third-party CD4⁺ iNKT-cell–treated mice with presence of regularly-spaced and -sized intestinal glands, presence of a regular complement of enterocytes (including goblet cells), and absence of any significant inflammation in the lamina propria and submucosa. In Tcon mice, there is marked lymphoplasmacytic inflammation (black asterisks) in the lamina propria and/or submucosa associated with the loss of intestinal glands. The remaining intestinal glands are abnormal with loss of goblet cells and presence of many apoptotic cells (black arrow), which appear as shrunken, hypereosinophilic, round bodies with pyknotic nuclei. H&E stain; original magnification ×400; bar = 50 µm. The BM control and third-party CD4⁺ iNKT-cell–treated mice have normal histology of the skin with an intact, thin epidermis and a thick layer of subcutaneous adipose tissue (white asterisks). In the Tcon mice however, there is mild interface damage of the epidermis (as evidenced by apoptotic keratinocytes; black arrows) and mild-to-complete atrophy of the subcutaneous adipose tissue (black asterisks). A mild dermal inflammatory infiltrate is also noted within the Tcon mice. H&E stain; original magnification ×200 (inset magnification ×400); bar = 100 µm. The BM control and third-party CD4⁺ iNKT-cell–treated mice have normal liver histology, with portal triads containing a single portal vein (white asterisks), and one or two bile ductules (white arrows). The livers of Tcon mice also appear essentially normal, except for some minimal lymphoplasmacytic inflammation in periportal interstitial tissues (black arrows) and mild proliferation/hyperplasia of bile ductules. H&E stain; original magnification ×200; bar = 100 µm. BMT, bone marrow transplantation.