H4/H60-specific depletion abolishes in vivo T-cell responses but not GVHD. Splenocytes from female B6 mice were depleted with K^b-H4 and K^b-H60 tetramers, or mock depleted. Splenocytes (25 × 10⁶) together with 5 × 10⁶ T-cell–depleted BM cells were transferred into lethally irradiated male BALB.B recipients. Peripheral blood was obtained at indicated days after transfer and pooled, and the percentage of CD8⁺ T cells specific for the different antigens was determined by intracellular IFNγ staining upon a 5-hour peptide restimulation with the indicated MiHAg peptides, or with OVA peptide as a control. (A) Dot plots represent blood samples on day 9 after transfer obtained from mice that received H4/H60-specific T-cell–depleted splenocytes (n = 10; top), or mock depleted splenocytes (n = 9; bottom). Numbers in upper right quadrants reflect the percentage of IFNγ-producing CD8⁺ T cells of total CD8⁺ T cells. (B) Kinetics of antigen-specific T-cell responses against the different antigens, measured as described in panel A. (C) Clinical signs of GVHD scored daily starting from day 14 after transfer, when the mice had recovered from irradiation-induced weight loss. (D) Survival plot of male mice that received H4/H60-specific T-cell–depleted splenocytes (▪), mock-depleted splenocytes (□), or female mice that received a syngeneic transfer from female BALB.B donor mice (⊡; n = 4). (E) Histopathologic analysis of intestinal and liver sections obtained after the onset of clinical GVHD. Note that lymphocytic infiltrates are present throughout the intestinal wall and in the periportal area of BALB.B recipients receiving B6 splenocytes (top and middle rows) compared with sections from mice receiving a syngeneic transplant (bottom row). Magnifications: 10 × 1.25 (left panels); × 20 (right panels). L indicates lumen; LP, lamina propria; MM, muscularis mucosae; SM, submucosa; ME, muscularis externa; BD, bile duct; and PV, portal vein. (F) Incidence of histopathologic signs of GVHD in the classical target organs.