Fig. 5: Transgenic TCR expression impacts CXCR4 chemokine receptor expression.

A E-cadherin expression on thymic epithelial cells that were isolated by protease digestion of thymus tissues and further identified by the absence of CD45 but high-level expression of EpCAM. Results are representative of 3 independent experiments. B UMAP plots of single-cell clusters generated by reanalyzing public mTEC scRNA-Seq data that identify 8 distinct populations. Relative expression of the indicated genes (i.e., Cdh1, Aire, Ccl21a, and Trpm5) are indicated for each cluster. C Bar graph shows the protein abundance of CXCR4 (MFI) on CXCR4⁺CD24⁺CCR7^–CD69^– immature DP thymocytes of WT (n = 4), OT-II (n = 4), AND (n = 3), and 2D2 (n = 3) transgenic mice. Results are a summary of 3 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. *, P < 0.05; **, P < 0.01; NS, not significant. One-Way Anova P = 0.001. Data are presented as mean values ± SD. D Histograms show the CXCR4 expression on CCR7^neg pre-selection thymocytes of WT, 2D2, AND, and OT-II transgenic mice in the presence or absence of CD103^Tgβ7^Tg. Results are representative of 6 independent experiments. E Bar graph shows the protein abundance of CXCR4 (MFI) on CCR7^neg thymocytes of WT (n = 7), 2D2 (n = 7), and 2D2.CD103^Tgβ7^Tg (n = 8) mice. Results are a summary of 5 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. **, P < 0.01. One-Way Anova P < 0.0001. Data are presented as mean values ± SD. F, G Histograms show intracellular staining for RORγt in DP thymocytes of WT, OT-II, and AND transgenic mice (F) and of WT, 2D2, and 2D2.CD103^Tgβ7^Tg mice (G). Results are representative of 3 independent experiments with at least 3 mice per group. Ctrl Ab: control antibody. Bar graph shows the protein abundance of RORγt expression (ΔMFI) on DP thymocytes of WT (n = 9), 2D2 (n = 8), and 2D2.CD103^Tgβ7^Tg (n = 11) mice. Results are a summary of 5 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. *, P < 0.05; ***, P < 0.001; NS, not significant. One-Way Anova P = 0.0026. Data are presented as mean values ± SD. H Histogram (left) is representative and bar graph (right) shows the summary of Helios expression (ΔMFI) in DP thymocytes of WT (n = 4), 2D2 (n = 10), and 2D2.CD103^Tgβ7^Tg (n = 4) mice. Results are a summary of 3 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. ***, P < 0.001. One-Way Anova P < 0.0001. Data are presented as mean values ± SD. I Histogram (left) is representative and bar graph (right) shows the summary of Ikaros expression (ΔMFI) in DP thymocytes of WT (n = 2), 2D2 (n = 6), and 2D2.CD103^Tgβ7^Tg (n = 4) mice. Results are a summary of 2 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. **, P < 0.01; NS, not significant. One-Way Anova P = 0.0084. Data are presented as mean values ± SD. J Histogram (left) is representative and bar graph (right) shows the summary of CXCR4 expression (MFI) on immature DP thymocytes of Ikfz1^WT and Ikfz1^ΔT mice. Results are a summary of 2 independent experiments with 2 Ikfz1^WT and 3 Ikfz1^ΔT mice. Data are presented as mean values ± SD. K, L Histogram is representative (left) and bar graph (right) shows a summary of TCRβ expression (MFI ratio) on DP thymocytes of WT (n = 5), OT-II (n = 4), AND (n = 4), and 2D2 (n = 4) transgenic mice (K). The line graph shows the inverse correlation between TCRβ expression (MFI) and CXCR4 (MFI) in DP thymocytes of the indicated mice (L). Results are the summary of 4 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. *, P < 0.05; ***, P < 0.001. One-Way Anova P < 0.0001. Data are presented as mean values ± SD. M Bar graph shows surface CXCR4 expression (ΔMFI) on freshly isolated or overnight (O/N) medium-cultured DP thymocytes of WT (n = 6), 2D2 (n = 5), and 2D2.CD103^Tgβ7^Tg (n = 8) mice. Results are the summary of 3 independent experiments. Paired two-tailed Student’s t-test was used to calculate P values. **, P < 0.01; ***, P < 0.001. Data are presented as mean values ± SD.