CCL21 protein is presented by thymic mesenchyme at sites of thymic exit. (A) Confocal images of thymus sections from postnatal day–10 (P10) WT mice stained with antibodies to the endothelial marker CD31 (white), mTEC marker ERTR5 (green), and CCL21 protein (red). Blue dotted line indicates the CMJ, and blue arrows indicate vessels investigated at higher magnification in panel C. (B) Image of a thymus section from a Ccl21a knockout P10 mouse stained with anti-CCL21 (red), anti-CD31 (white), and ERTR5 (green). Note the absence of CCL21 staining. Scale bars in panels A and B denote 50 μm. (C) High-power images of CCL21⁻ (upper panels) and CCL21⁺ (lower panels) vessels identified by blue arrowheads in panel A. Images show individual channels for ERTR5 (green), CD31 (white), and CCL21 protein (red), as well as a combined image showing all markers simultaneously. Scale bars denote 25 μm. Data are representative of 4 mice from 2 separate experiments. (D) Schematic diagram and flow cytometric analysis of thymic mesenchymal populations associated with thymic blood vessels. Schematic is based on findings of Sitnik et al³⁵  and demonstrates CD34⁻integrin α7⁺ pericytes and CD34⁺integrin α7⁻ adventitial mesenchymal cells that surround thymic blood vessels. Flow cytometric analysis shows identification of these populations in P10 WT thymus. (E) Flow cytometric analysis of presentation of CCL21-mRFP by indicated thymic stromal populations in plt/plt P10 thymus suspensions. Gray histograms represent control staining seen in the absence of CCL21-mRFP. Bar chart indicates percentages of CCL21-mRFP⁺ cells within each stromal subset. (F) Flow cytometric analysis of stromal cell presentation of full-length CCL21-mRFP (red), full-length CCL19-mRFP (blue), or tCCL21-mRFP (green) by CD34⁻integrin α7⁺ pericytes and CD34⁺integrin α7⁻ adventitial mesenchymal cells. Gray filled histograms represent staining levels observed when no chemokines were added. Bar chart shows mRFP mean fluorescence intensity (MFI) for each fluorescent chemokine and indicated stromal cell type. For analysis of data in panels E and F, multiple comparison analysis was achieved by a 1-way analysis of variance (ANOVA) followed by Tukey’s post-test (E) or 2-way ANOVA followed by Sidak’s posttest (F) in GraphPad Prism to determine statistical differences. All data shown representative of 3 independent experiments, with a total of 7 to 11 mice for each analysis. Error bars represent mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001.