Figure 2. CD45RA and CD31 expression on CD4⁺CD25^-, CD4⁺CD25^bright, and CD8⁺ T cell populations and the functional activity of sorted CD4⁺CD25^bright T cells.

(A) CD45RA status of patient CD4⁺CD25^bright (shaded area) and CD4⁺CD25^- (solid line) T cells is shown at various times after HSCT. (B) CD45RA expression on day +899 is shown for the CD4⁺CD25^bright (shaded area), CD4⁺CD25⁻ (solid line), and CD8⁺ (dotted line) T cell populations. The gating strategy excluded CD14⁺ monocytes and positively gated either CD4⁺ or CD8⁺ T cells. The CD4⁺CD25⁻ and CD4⁺CD25^bright populations were gated as shown. (C) The gating strategy and CD31 expression is depicted for the CD4⁺CD25^bright (shaded area), CD4⁺CD25⁻ (solid line), and CD8⁺ (dotted line) T cell populations on day +899. (D) CD14⁻CD4⁺CD25⁻ and CD14⁻CD4⁺CD25^bright T cells were sorted as described and immediately assayed for functional capability. Relative IL2 mRNA levels were measured after a 6-hour in vitro stimulation with anti-CD3, anti-CD28, and anti-CD2 conjugated beads either alone or in co-culture at the indicated ratios. ABL1 was used as the endogenous control, IL2 from CD14⁻CD4⁺CD25^bright cells as the calibrator, and CD14^-CD4⁺CD25⁻ as the positive control. Sufficient cells were available for stimulation cultures to be done in triplicate on day +899 and statistical significance by paired t-test analysis is shown.