Analysis of phenotypic markers FoxP3, T-bet, and LAG-3. Heterologous CD4⁺ T cells (1 × 10⁶/mL), P8(1) clones, or peptide 72H-86L specific cell lines (both 1 × 10⁵/mL) were incubated in the presence or absence of anti-CD3 mAb for 24 hours at 37°C, 5% CO₂ before analysis of FoxP3 expression by flow cytometry (A). Top (resting cells) and bottom (activated cells) panels illustrate number of FoxP3⁺ cells in CD3⁺CD4⁺CD25^low (white histogram) and CD25^bright (gray) T-cell populations from T cells, clone, and line, respectively. Analysis of FoxP3 expression by Western blot (B). Representative Western blot of cell extracts analyzed by SDS-PAGE and stained for FoxP3 or the control protein actin. The extracts were obtained from Tr clone P8(1) either resting, stimulated by presentation of Rh protein autoantigen, or activated nonspecifically by anti-CD3 and anti-CD28 for 96 hours at 37°C, 5% CO₂. CD4⁺CD25⁺ peripheral blood cells isolated ex vivo are also included as a positive control for FoxP3 staining. Densitometric analysis normalized against actin levels confirmed increased FoxP3 expression by the Tr clone after specific Rh protein autoantigen stimulation (0.61) compared with nonstimulated control (0.43) or nonspecific stimulation (0.51) but similar to isolated CD4⁺CD25⁺ cells (0.59). Relative expression of the Th1 T-cell–associated transcription factor, T-bet (C). Cells were treated as described for panel A, and increases in number of T-bet⁺ clones or cell lines (gray histogram) at rest (top) or activated with anti-CD3 mAb (bottom) were compared with resting CD4⁺ T cells (white histogram). Comparison of LAG-3 analysis (D). CD4⁺ T cells shown in left panel (white and gray histograms are resting and activated cells, respectively) and clone, right panel, were costained with anti–LAG-3 antibody and compared for expression. Number of IL-10 positive cells are represented in upper right quadrant as a percentage of the total CD4⁺ T-cell population.