Flow cytometric analysis of CoBS-treated transplant recipients. (A) Longitudinal analysis of representative CD4⁺ (top) and CD8⁺ (bottom) T-cell subsets during CoBS treatment (R.4 and R.5 are shown). (B) CD127 expression on CD8⁺ T cells in CoBS-treated animals. (Left) Pseudocolor dot plots and histogram analysis are representative of CD127 expression on CD8⁺ T cells from R.5 analyzed both before transplantation (top) and on day 43 after transplantation (bottom). (Right) Longitudinal analysis is shown of CD127 expression on CD8⁺ T cells in R.4-R.7 normalized for pretransplantation expression levels of CD127. (C) Sirolimus levels measured longitudinally in animals R.4-R.7. The shaded area is the target trough range: 5-15 ng/mL. (D) BCl-2 and Ki-67 on CD4⁺ and CD8⁺ CD28⁺/CD95⁺ and CD28⁻/CD95⁺ T cells in a representative CoBS-treated animal (R.4). (E) CTLA4Ig-resistant alloproliferation of CD28⁻ T cells as measured by CFSE (5,6-carboxyfluorescein diacetate, succinimidyl ester) MLR. (Top) Representative dot plots showing T-cell alloproliferation in CD28⁺/CD95⁻, CD28⁺/CD95⁺, and CD28⁻/CD95⁺ T-cell populations in the absence of CTA4Ig treatment. (Bottom) Representative dot plots showing T-cell alloproliferation in CD28⁺/CD95⁻, CD28⁺/CD95⁺, and CD28-⁻CD95⁺ T-cell populations in the presence of 1.6μM CTLA4Ig, showing CTLA4Ig-resistant alloproliferation existing in the CD28⁻/CD95⁺ subpopulation. A dose-response curve of 0.8-12.8μM CTLA4Ig was performed (not shown), with similar results observed for all concentrations of CTLA4Ig tested. For each population, the percentage of cells that had undergone ≥ 1 cell division was measured with FlowJo flow cytometry analysis software (Tree Star) and is noted on the corresponding dot plots. (F) Correlation of the percentage of Ki-67^high CD28⁻/CD95⁺ CD8⁺ T cells with GVHD histopathology scores. The maximal percentage of CD28⁻/CD95⁺/CD8⁺ cells that up-regulated Ki-67 was compared with the total histopathology score (Table 4). The data fit a linear equation with an R² = 0.6557.