Immunohistochemical analysis of primate brain GVHD. Rhesus macaques used in this study were housed at the Yerkes National Primate Research Center and treated in accordance with Emory University Institutional Animal Care and Use Committee regulations. The following experimental groups were assessed: (1) healthy untransplanted controls (n = 4); (2) autologous transplanted controls (n = 3); (3) recipients of MHC-mismatched allogeneic HCT that did not receive GVHD prophylaxis (n = 5, mean survival time (MST) = 7 days); (4) allogeneic HCT recipients treated with rapamycin monotherapy (n = 6, MST = 17 days); and (5) allogeneic HCT recipients treated with tacrolimus/methotrexate (n = 3, MST = 47 days). For pretransplant preparation, all recipients received myeloablative irradiation (either 8 Gy [single fraction] or a dose equivalent [9.6 Gy] given in 2 fractions). Recipients received PBSC transplants (mean total nucleated cell dose of 6.2 ± 0.98 × 10⁸ cells/kg, mean CD3⁺ T-cell dose of 1.2 ± 0.21 × 10⁸ cells/kg). At necropsy, brains were preserved in 10% neutral buffered formalin, and immunoperoxidase staining for various antibodies was performed on 4-µm-thick formalin-fixed paraffin-embedded sections with the use of the standard avidin-biotin peroxidase complex technique (Dako, Carperinteria, CA). No evidence for infectious etiology was found, including no viral cytopathic effect, no cytomegalovirus viremia (data not shown), and no antigenic evidence for RhLCV (the rhesus macaque Epstein-Barr virus homolog) or SV40 in any of the sections examined (data not shown). Antibodies shown in this figure include CD3 (clone PC; Dako), CD4 (clone 1F6; Vector), CD8 (clone 1A5; Vector), CD163 (clone 10D6; Thermo Scientific), Ki-67 (clone MIB-1; Dako), Granzyme B (clone GRB-7; Chemicon), and LFA-1 (clone 345913; R&D). In preparation for analysis, tissue sections were washed and developed with DAB Chromagen (Dako) and counterstained with Mayer’s hematoxylin. Isotype matched relevant controls were tested on all tissues. Four to 5areas were scanned at 10×, and immunopositive cells were counted from 1 representative section to provide scores ranging from 0 to 11 according to the following strategy: no positive cells, score of 0; 1 to 5 cells, score of 1; 6 to 10 cells, score of 2; 11 to 15 cells, score of 3; 16 to 20 cells, score of 4; 21 to 25 cells, score of 5; 26 to 30 cells, score of 6; 31 to 35 cells, score of 7; 36 to 40 cells, score of 8; 41 to 45 cells, score of 9; 46 to 50 cells, score of 10; >51 cells, score of 11. The cell scores for individual markers were then analyzed statistically for significance by analysis of variance (ANOVA) and Mann-Whitney t test using Graph Pad Prism (La Jolla, CA). Representative photomicrographs were photographed using a ×20 objective. A 50 μM standard is included in each micrograph. (A) Representative IHC for each of the treatment groups and each of the antibodies of interest. (B) Corresponding statistical analysis of the IHC scoring for all animals. The ANOVA statistic is indicated for each comparison. Red bars indicate individual comparisons for which P ≤ .05. (C) Representative CD3 IHC from (left) a representative normal control and (right) a representative recipient of allogeneic HCT that did not receive GVHD prophylaxis for (upper) the perivasculature and (lower) the parenchyma. (D) Representative spectral analysis from a recipient of allogeneic HCT that did not receive GVHD prophylaxis for (top) CD3/CD8 and (bottom) CD8/Ki-67, performed with an Olympus Vanox S microscope and tunable filter-based camera. Cri-Nuance software (Woburn, MA) software was used to measure the colocalization between (top) CD3 and CD8 and (bottom) CD8 and Ki67. For this analysis, CD3 and Ki-67 were assigned a green pseudo-color and CD8 was assigned a red pseudo-color. Double-stained cells are assigned a yellow pseudo-color.