Distribution of alloantigen-specific CD154⁺CD4⁺ cells over naive and memory CD4⁺ T cells. A typical example of the distribution of CD154-expressing CD4⁺ T cells within the different T-cell subsets after a 24-hour allogeneic MLR (A). Briefly, viable lymphocytes are selected based on forward scatter (FSC)/side scatter (SSC) characteristics and 7-amino-actinomycin D expression. These lymphocytes are then plotted in a CD3 versus CD4 dot plot to select the CD3⁺CD4⁺ T (Th) lymphocytes. CD154 expression is than visualized using a dot plot of CD4 versus CD154 (i). Next, both CD154⁻ (ii) and CD154⁺ (iii) CD4⁺ lymphocytes are displayed in a dot plot with CD45RO on the x-axis and CCR7 on the y-axis, allowing a dissection of the allogeneic response into the different CD4⁺ T-lymphocyte subsets. Naive CD4⁺ lymphocytes are defined as CD45RO⁻CCR7⁺; memory CD4⁺ lymphocytes, as CD45RO⁺CCR7⁺ (central memory cells) or CCR7⁻ (effector memory cells). Subsequently, CD154⁺ expression for total, naive, and memory CD4⁺ lymphocytes at 24 hours is plotted against the number of MMs on HLA-DR where □, , and ■ represent 0 (n = 6), 1 (n = 12), and 2 (n = 6) MMs on HLA-DR, respectively (B). *P < .05. Error bars represent SD.