Figure 2.

Density of T cells, macrophages and putative dendritic cells (DCs) in the human rectum and colon. A: Representative 20x pseudo-color images in one individual of CD3 (T cells), CD68 (macrophages) and CD209 (DCs) in the rectum and colon. Tissue sections were stained as described in Methods for three-color immunofluorescence: anti-CD3, anti-CD68 or anti-CD209 in one fluorescence channel (pseudo-colored in white), mouse IgG isotype control for anti-CCR5 staining in a second fluorescence channel (red), and the nuclear counter-stain Sytox Orange in the third channel (blue). Note that these sections therefore also depict representative single isotype controls for the CCR5 staining data presented in Figures 3 and 4. The two control images in A are double isotype controls with no marker-specific primary antibodies. B: Dot plots corresponding to biopsies in panel A, but showing data from all cells in the entire tissue sections. Each dot corresponds to one cell, defined by its specific mean and maximum fluorescence intensity. Quadrant cutoffs were determined by isotype control stains. Only cells in the upper right quadrant were judged as positive. The numbers and percentages of cells in the upper right quadrants, and their corresponding cell densities, are specified in each plot. C: Summary of CD3⁺, CD68⁺ and CD209⁺ cell densities in all biopsies analyzed. The horizontal line in each box denotes the median, the ends of the box denote the 25th and 75th percentiles, and the whiskers extend to the extreme data points that are no more than 1.5 x the interquartile range. Paired samples, i.e., where the data were derived from a colon and rectum biopsy obtained from the same individual at the same time point, are connected by dotted lines. Unpaired (Mann-Whitney U test) and paired (Wilcoxon signed-rank test) p values for differences between rectum and colon are listed below the corresponding boxes. D: Median percentages of CD3⁺, CD68⁺ and CD209⁺ cells among all nucleated cells.