CD4 up-regulation occurs following chronic stimulation in vitro. (A) Histograms represent CD4 fluorescence intensity following various stimulation periods. A distinct CD4^hi population starts to become evident at day 6 and is clearly evident at day 7 in data representative of experiments from 6 unique donors. (B) Up-regulation of activation markers occurs uniquely on CD4^hi T cells. Following 7 days of stimulation with an allogeneic PBMC pool, up-regulation of 10 activation markers (y-axes) was assessed with respect to CD4 fluorescence intensity (x-axis). In each case, activation-marker coexpression was restricted within the CD4⁺ T-cell subset to the CD4^hi population. Data from 1 donor shown are representative of experiments from 8 unique donors stimulated in the same fashion. (C) Proliferation of alloreactive T cells is restricted to the CD4^hi subset. A decrease in fluorescence intensity of CFSE, a dye used to label responder cells, is seen within the CD4⁺ T-cell population only in the CD4^hi subset following 7 days of stimulation. (D) Within the CD4⁺ T-cell population, CFSE^low cells are confined to the alloreactive CD4^hiCD38⁺ T-cell subset following allogeneic stimulation. The median number of divisions, calculated by the change in CFSE fluorescence intensity in the 2 peaks illustrated at right, is 4.1 ± 0.5, based on experiments from 5 unique donors. Assuming that nondividing cells did not die during the stimulation period, we estimate that the original precursor frequency of alloreactive cells was 6.9% ± 1.5% of the original CD4⁺ T-cell population.