Donor alloreactive T cells in GVHD display a global increase in the phosphorylation of signal transduction proteins, including STAT-3 and ERK1/2. (A) A total of 107 CFSE-labeled purified B6 splenic T cells were infused into lethally irradiated B6 CD45.1 or C3FeB6F1 mice. Recipient spleens were analyzed at 4, 20, 44, and 67 hours for the proliferation of donor T cells and phosphorylation of selected intracellular signaling proteins. Bivariate plots demonstrating the proliferation with CFSE dilution versus the phosphorylation of STAT-3 (pS727) in both allogeneic and syngeneic settings are shown. Pink quadrant lines represent the limits of isotype control staining. These plots demonstrate an increase in the phosphorylation of STAT-3 with T-cell alloactivation (eg, at 44 hours) and proliferation, especially in the allogeneic setting. One of 3 independent experiments. (B) Lethally irradiated BALB/c mice (850 cGy) were transplanted with 5 × 106 B6 T cell–depleted bone marrow cells and 106 enriched B6 splenic T cells and harvested on day 14 after BMT. Donor T cells were analyzed by flow cytometry and levels of intracellular phosphorylated and total signal transduction proteins were compared with levels in the corresponding populations in nontransplanted B6 splenic T cells. All signal transduction proteins we examined demonstrated elevated levels of phosphorylation compared with levels in the corresponding naive B6 mouse. Naive donor T cells are not present at day 14 after BMT in T cell–replete transplants and were thus not analyzed. Populations were compared with corresponding populations in the normal B6 mouse. N = 8. One of 2 independent experiments.
