Adoptively transferred donor-type NKT cells protect against GVHD. (A) Doses of NKT cells (DX5⁺TCRβ⁺CD4⁺) from C57BL6 donors (H-2^b) were adoptively transferred into lethally irradiated BALB/c mice (H-2^d) that had TCD-BM and 5.0 × 10⁵ Tcons (to induce GVHD). Significant improvement in survival was observed with the transfer of either 1.0 × 10⁴ or 2.5 × 10⁴ NKT cells (P < .01). Data shown in graph were pooled from 4 independent experiments. Groups: irradiation control (no BMT; ■; n = 25); TCD-BM only (●; n = 25); 5 × 10⁵ Tcons (▾; n = 25); Tcons + 1 × 10⁵ NKT (▵; n = 20); Tcons + 5 × 10⁴ NKT (◇; n = 18); Tcons + 2.5 × 10⁴ NKT (○; n = 23); and Tcons + 1 × 10⁴ NKT (□; n = 19). (B) Further sorting of NKT cells into PBS-57 CD1d–PBS-57 tetramer-positive and tetramer-negative subpopulations indicates that both invariant and noninvariant NKT cells provide protection against GVHD (P < .05). Graph contains data pooled from 4 independent experiments (n = 25). Groups: irradiation control (no BMT; ■); TCD-BM only (●); 5 × 10⁵ Tcons (▾); Tcons + 1 × 10⁴ PBS-57 CD1d⁻ NKT (▵); and Tcons + 1 × 10⁴ PBS-57 CD1d⁺ NKT (▴). (C) Mice were euthanized at day 39 after transplant. Samples of haired skin, small intestines, large intestines, liver, thymus, spleen, and mesenteric lymph nodes were collected and fixed in formalin for 48 hours before being routinely processed for histologic analysis of stained sections. Evidence for acute GVHD pathology in the haired skin as follows: reduced number of follicles (white arrows), increased inflammation in the panniculus (white asterisk), increased epidermal thickness because of hyperplasia (white measurement bar), increased density of dermal collagen (black asterisk), and mild vacuolar degeneration of the stratum basale with presence of apoptotic bodies (black arrowheads). GVHD-associated colitis was noted with increased numbers of lymphocytes and plasma cells in the lamina propria (black arrows) Increased numbers of intraepithelial lymphocytes are also present. A decreased number of goblet cells is observed, along with evidence of gland regeneration (hyperbasophilia of gland enterocytes, along with increased mitoses and nuclear crowding). Apoptotic bodies (black arrowhead) are present within the glands as well. Severe atrophy (lymphoid depletion) of the lymphoid follicles of splenic white pulp areas (white arrowhead) was noted in the mice receiving Tcon cells only. 100× magnification bar = 200 microns; 200× magnification bar = 100 microns; 600× magnification bar = 25 microns.