Thymus-infiltrating donor T cells proliferate in situ and express inflammatory cytokines during acute GVHD.

Acute or chronic GVHD was induced in unirradiated B6D2F₁mice. Cell cycling and cytokine mRNA expression of thymic cells were analyzed 2 weeks after transplantation. (A) Representative flow cytometric plots of thymocytes from a mouse with acute GVHD. Numbers depicted in each quadrant of the dot plots represent mean frequencies (%) of the respective populations. (B) For analysis of cytokine production, semiquantitative PCR of cDNA from frozen whole thymic tissue was used. PCR products were separated by gel electrophoresis, and the bands depict amplicons from 1 naive B6D2F₁ mouse and from 2 representative mice with acute or chronic disease on day 15 after transplantation. Bands were further analyzed by densitometry, and the intensities of each amplicon cytokine mRNA from mice with acute (▪) or chronic () GVHD were expressed as ratios compared with syngeneically transplanted controls (□). Expression levels for the different genes in the B6D2F₁ control were arbitrarily set at 1, and differences in initial cDNA input levels were corrected based on band intensities measured for GAPDH products. (C) Freshly isolated thymocytes from naive B6D2F₁ mice (F₁) and from transplanted mice with acute GVHD (day 15) were separated on a FACSvantage into donor- and host-derived CD4 SP (▪) and CD8 SP (▨) cell subsets based on their expression of Ly5.1. PCR amplification products (IFN-γ and TNF-α) were analyzed by densitometry, and optical densities of bands were compared. The graph represents pooled data from 5 independent experiments; 3 to 8 mice were analyzed for each group. *P < .001 versus mice with chronic GVHD and syngeneic controls, respectively. ^†P < .03 versus syngeneic controls (ANOVA).