Impaired activation and cytokine response in alloreactive DNMAML CD4⁺ T cells. WT or DNMAML B6 CD4⁺ T cells were tracked after transplantation into lethally irradiated MHC-mismatched BALB/c mice (900 rads). (A) Decreased expression of CD25 (IL-2Rα) in DNMAML CFSE^low donor CD4⁺ T cells. Bar graphs show the percentage of cells expressing CD25 and the CD25 mean fluorescence intensity (MFI; mean ± SD, n = 3). (B) Intracellular staining for IFNγ, TNFα, and IL-2 after ex vivo restimulation with plate-bound anti-CD3 and anti-CD28 antibodies. Data are representative of 5 experiments. Bar graphs show mean ± SD (n = 3). (C) Cytokine production by 3 × 10⁴ donor-derived CD4⁺ T cells sort-purified 5 days after transplantation and cultured for 6 hours in the presence of anti-CD3/anti-CD28 antibodies (mean ± SD). (D) Intracellular staining for FoxP3 in donor WT and DNMAML CD4⁺ T cells on day 5. *P < .05, **P < .01 (2-tailed unpaired Student t test).