Preserved proliferation and in vivo expansion of DNMAML alloreactive CD4⁺ T cells. (A) Identification of donor-derived CD4⁺ T cells in the spleen on day 5 after transplantation. DNMAML T cells expressed the DNMAML-GFP fusion protein. Data are representative of 5 experiments. (B) Absolute number of donor-derived CD4⁺ T cells in the spleen at day 5 (mean ± SD, n = 4). (C) Tracking of CFSE-labeled donor CD4⁺ T cells at day 5. Both WT and DNMAML CD4⁺ T cells had undergone extensive proliferation, as shown by the high percentage of CFSE^low cells. Fluorescence levels appear higher in the DNMAML-expressing cells due to fluorescence of the covalently fused GFP protein. This effect is absent in Rbpj knockout mice (see supplemental Figures 3-4). (D) Tracking of eFluor670-labeled donor WT and DNMAML CD4⁺ T cells at day 5, showing extensive proliferation in both (% eFluor670^low cells). (E) BrdU uptake by donor WT and DNMAML CD4⁺ T cells at day 5. Mice were pulsed with BrdU 6 hours before harvest (n = 5 per group). (F) Absolute number of CD45.2⁺ donor-derived WT and DNMAML CD4⁺ T cells in spleen, liver, BM, and thymus on day 14 (WT, n = 12; DNMAML, n = 11) and 21 (WT, n = 7; DNMAML, n = 5) posttransplantation (pool of 2 independent experiments). (G) Decreased number of donor-derived DNMAML compared with WT CD4⁺ T cells in the lamina propria of the small intestine on days 11 (WT, n = 5; DNMAML, n = 5) and 14 after transplantation (WT, n = 6; DNMAML, n = 4). *P < .05, **P < .01 (2-tailed unpaired Student t test).