Figure 3
Antigen-specific memory CD4
+ T cell proliferation at the site of the MT.
(A) Double immunofluorescence staining of representative biopsies from days 0, 3, 7, and 14 following MT induction. Green indicates Ki67; red indicates CD4. Original magnification, ×400. (B) Double immunofluorescence staining of representative biopsies (days 0, 3, 7, and 14; n = 5 per time point) shows CD4+ (green) and Foxp3+ (red) cells in a perivascular lymphocytic infiltrate (original magnification, ×400). The 5 largest perivascular infiltrates present in the upper and middle dermis were selected for analysis. Cell numbers were expressed as the mean absolute number of cells counted within the frame. (C) The proportion of Ki67+CD4+ cells was also determined by flow cytometry using blister cells (n = 3–5 per time point). Representative dot plots for day 7 PPD blister cells and PBMCs are shown. Numbers denote the percentage of cells expressing Ki67 relative to control gate set with an irrelevant antibody. (D) Number of total CD4+ cells (filled squares) and number of Ki67+CD4+ cells (open squares) in perivascular infiltrates following PPD injection. (E) Percentage of CD4+ cells expressing Ki67 found per perivascular infiltrate in each donor. Each circle represents an average of 5 perivascular infiltrates counted for each individual (n = 5–7 per time point; horizontal lines indicate the mean). (F) Number of total CD4+ cells (filled squares) and CD4+Foxp3+ cells (open squares) in perivascular infiltrates following PPD injection. (G) Percentage of CD4+ cells expressing Foxp3 per perivascular infiltrate counted. Each symbol represents an average of 5 perivascular infiltrates counted for each individual (n = 5–7 per time point). Data are mean ± SEM.
