The flow cytometric signature of primate GVHD. (A) Longitudinal analysis of the white blood cell (WBC) count, absolute neutrophil count (ANC), and absolute lymphocyte count (ALC) in R.1-R.3. (B) Longitudinal analysis of the absolute CD8⁺ T-cell count, the absolute CD4⁺ T-cell count, the absolute CD20⁺ B-cell count, and the absolute CD16⁺ natural killer (NK) cell count in R.1-R.3. (C) Forward-scatter (FSC-A) versus side-scatter (SSC-A) flow cytometric analysis of the lymphocyte blast phenotype (red circle) in GVHD. Example from R.1 before transplantation (top) and on day 7 (bottom), during rapid T-cell expansion. (D) Down-regulation of CD127 in expanding CD8⁺ T cells during GVHD. Panels on the left show pseudocolor dot plots and histogram analysis of CD127 expression on CD8⁺ T cells analyzed both before transplantation (top) and on day 7 (bottom). Panels on the right show longitudinal analysis of CD127 expression on CD8⁺ T cells in R.1-R.3, compared with pretransplantation expression levels of CD127. (E) Representative flow cytometric analysis of R.2, showing the phenotypic shift away from naive T cells that occurred in both CD4⁺ and CD8⁺ compartments during GVHD; before transplantation (top), day 6 after transplantation (bottom). (Third column from the left, top, and bottom) Example of CD4⁺ T cells shifting to a CD28⁺/CD95⁺ predominant phenotype after transplantation (compare top with bottom rows). (Fourth column from the left) Example of CD8⁺ T cells shifting to a CD28⁻/CD95⁺ predominant phenotype after transplantation (compare top with bottom rows). (F) Longitudinal analysis of CD4⁺ and CD8⁺ T-cell subsets during GVHD. R.1-R.3 all shift toward a predominant CD28⁻/CD95⁺ CD8⁺ phenotype (left) and a predominant CD28⁺/CD95⁺ CD4⁺ phenotype (right). (G) Representative CFSE (5,6-carboxyfluorescein diacetate, succinimidyl ester) MLR analysis of the proliferation of CD28⁺ and CD28⁻ CD8⁺ T cells. (Left) Representative flow cytometric analysis of CD3⁺/CD8⁺ T cells showing the CD28 and CD95 phenotypes. (Right) CFSE MLR analysis after a 5-day MLR culture. Minimal proliferation occurred in cultures incubated without any stimulators (“No stimulation”) or those incubated with autologous stimulator cells (“+ Auto-stimulation”). However, in those incubated with allogeneic PBMCs (“+ Allo-stimulation), proliferation occurred in both CD28⁺ and CD28⁻ subpopulations. The results shown are representative of ≥ 5 separate MLR assays with the use of distinct donor:recipient pairs. (H) CFSE MLR analysis of flow cytometrically sorted CD28⁺ and CD28⁻ populations. CD28⁺/CD95⁻, CD28⁺/CD95⁺, and CD28⁻/CD95⁺ CD8⁺ T cells were sorted with a FACSAria flow cytometer before CFSE MLR analysis either in the absence (left) or presence (right) of allogeneic stimulator cells. Shown are representative CFSE proliferation profiles from 1 of 3 replicate experiments. (I) Granzyme B expression is highly up-regulated during GVHD. Shown is a representative example of the mean fluorescence intensity of granzyme B in both CD28⁺/CD95⁺ and CD28⁻/CD95⁺ subpopulations for CD4⁺ and CD8⁺ T cells; granzyme B fluorescence before transplantation (black), granzyme B fluorescence at the time of necropsy in the setting of severe clinical GVHD (red). (J) BCl-2 and Ki-67 show significant shifts in expression during GVHD. (Top) Representative analysis from R.2, before transplantation. (Bottom) Day 6 after transplantation. (Left) T-cell subpopulations are first identified with CD28 and CD95 staining. The CD28⁺/CD95⁺ and CD28⁻/CD95⁺ cells were then queried for their expression of BCl-2 and Ki-67 (right). (K) CD4⁺/FoxP3⁺ cells expanded during GVHD in rhesus macaques. (Red) Longitudinal analysis of CD4⁺/FoxP3⁺ T-cell homeostasis in the untreated animals R.2, R.3, as well as a third MHC-disparate transplant recipient (R.1 could not be evaluated because of a technical problem with FoxP3 staining on the day of death). (Black) Longitudinal analysis of CD4⁺/FoxP3⁺ T-cell homeostasis in the CoBS-treated animals, R.4, R.5, and R.7 (R.6 could not be evaluated because of technical problems with CD3/CD4 staining for this animal). (L) FoxP3⁺/CD4⁺ T cells (gated population, and blue traces on the histograms) showed up-regulation of CD25, down-regulation of CD127, and up-regulation of CD27 compared with FoxP3⁻/CD4⁺ T cells (red traces).