cGVHD induces T-cell infiltration and a proinflammatory cytokine profile in the brain. Messenger RNA expression of selected proinflammatory cytokines detected by quantitative real-time polymerase chain reaction in the thick coronal section (A) and hippocampus (B) of GVHD and TCD mice at days 35 and 70 after transplant (n = 6-15 mice per group; data pooled from 2 independent experiments). Expression calculated relative to the Hprt gene and reported as a fold change of the mean of the TCD group. (C) Schematic of transplantation regime. Lethally irradiated B6D2F1 recipient mice received 5 × 10⁶ BM from C57Bl/6 donors ubiquitously expressing GFP and 0.5 × 10⁶ sort-purified CD90.2⁺ T cells from C57Bl/6 donors ubiquitously expressing RFP. (D) Representative dot plots indicating gating strategy for identifying live CD90.2⁺ CD4 and CD8 T-cell subsets within the CD45^hiCD11b⁻ population, gated on forward and side scatter. Cells were isolated from digested coronal brain segments of transplant recipients at days 14, 35, and 70 after transplant. (E-I) Quantification of lymphocyte populations in dissociated GVHD brains at days 14, 35, and 70 after transplant. (E) CD90.2⁺ proportion of the CD45^hiCD11b⁻ population. (day 14: n = 6; data pooled from 2 independent experiments; day 35: n = 3; data from 1 independent experiment; day 70: n = 17; data pooled from 4 independent experiments). (F) Absolute number of CD90.2⁺ T cells within the CD45^hiCD11b⁻ population (day 14: n = 8; data pooled from 3 independent experiments; day 35: n = 6; data pooled from 2 independent experiments; day 70: n = 16; data pooled from 4 independent experiments). (G) Proportions of CD4⁺ and CD8⁺ T cells within the CD90.2⁺ population (day 14: n = 5; data pooled from 2 independent experiments; day 35: n = 7; data pooled from 2 independent experiments; day 70: n = 16; data pooled from 4 independent experiments). (H) Comparison of the proportions of CD8⁺ T cells derived from the T-cell (red) and BM (green) grafts (days 14 and 35: n = 3 mice per time point; day 70: n = 7; data pooled from 3 independent experiments). (I) Comparison of the proportions of CD4⁺ T cells derived from the T-cell (red) and BM (green) grafts (days 14 and 35: n = 3 mice per time point; day 70: n = 7; data pooled from 3 independent experiments). Data are presented as mean ± standard error of the mean. Significant differences calculated using unpaired Student t test (A-B) or 1-way analysis of variance with Tukey multiple comparisons test (E-H). *P < .05, **P < .01, ***P < .001, ****P < .0001.