Absence of PKCα and θ in donor T cells more comprehensively abrogates GVHD lethality and pathology than deficiency of PKCθ alone. Recipient BALB/c mice were lethally irradiated (800 cGy) and transplanted with 5 × 10⁶ genotype-matched TCD-BM cells alone (n = 2 for each genotype) or in addition to 1 × 10⁶ T cells (CD4⁺, CD8⁺, and CD25^–) from littermate WT C57BL/6 or PKCα^−/−, PKCθ^−/−, or PKCα^−/−/θ^−/− mice (n = 11, 10, 15, and 15 recipients, respectively). Recipient mice were monitored throughout the experimental period for survival (A) and weight change (B), and pooled data from 3 separate experiments are represented. In separate experiments, recipients (n = 4 recipients per group per experiment) were euthanized 2 weeks posttransplant, and samples of skin, liver, lung, small intestine, and large intestine were collected in formalin for routine hematoxylin and eosin and scored for microscopic GVHD severity by a pathologist blinded to the treatment groups. Photomicrographs depicting the average disease score morphology from 1 representative experiment out of 3 separate experiments (C) and average scores for GVHD target organs across 3 separate experiments (D) are depicted. Recipients surviving to 120 days post-BMT in experiments depicted in panels A-B were euthanized, and their spleens were subjected to total splenocyte count (E) and flow cytometric staining and analysis for H2kb, CD4, CD8, and B220 surface expression (F) and plated with 1 ug/mL anti-CD3 or 5 ug/mL LPS for 72 hours followed by overnight tritium thymidine incorporation to observe activation of T cells or B cells, respectively (G). T- and B-cell activities are depicted as per cell activity normalized to the original spleen count. *P < .05; **P < .01; ***P < .001 (compared with WT [brackets indicate statistical significance for comparisons between PKCα^−/−/θ^−/− and PKCθ^−/− treatment groups]). All error bars indicate standard error of the mean (SEM).