Intrathymic Medullary Accumulation of Vγ5⁺ DETC Progenitors occurs Independently of CCR4 and CCR7.

<p>(A) Expression of CCR4 and CCR7 on total Vγ5⁺ thymocytes and CD45RB^high/CD45RB^low Vγ5⁺ thymocyte subsets from E18 thymus. Black histograms show the levels of antibody staining using E18 thymocytes from the indicated chemokine receptor knockout mice as a control. Red histograms show the expression level of each chemokine receptor in WT E18 thymocytes. Numbers represent the mean fluorescent intensity of CCR4/7 KO then WT cells. (B-D) Representative confocal images of E18 WT (B), CCR4^−/− (C), CCR7^−/− (D) thymus. M denotes medulla, C denotes cortex. Scale bars in B-D represent 50 µm. (E) Confocal quantification of Vγ5⁺ thymocytes per in mm² medullary area in WT (n = 6), CCR4^−/− (n = 6) and CCR7^−/− (n = 6) E18 thymus. Error bars represent SEM.</p

Inhibition of G-Coupled Receptor Signaling Prevents Accumulation of Vγ5⁺ DETC Progenitors in the Thymic Medulla.

<p>(A) Experimental design. Freshly isolated E14 C57BL6 thymus lobes were treated with or without 250 ng/ml Pertussis toxin (PTX) for 30 minutes, cultured as FTOC for 48 hr, then harvested for flow cytometry or confocal analysis. (B) Vγ5⁺ thymocyte numbers from PTX treated or non-treated FTOC. Control; n = 8, PTX; n = 9. (C and D) Confocal analysis of Vγ5⁺ thymocyte distribution in control and PTX treated FTOC. Error bars represents SEM, and asterisks signify a significant difference, where <i>p</i><0.0001. Scale bars in D on upper panel represent 200 µm and on lower panel represent 50 µm.</p

Absence of CCR4 or CCR7 causes an Increase in Mature Vγ5⁺ T Cells in E18 Fetal Thymus.

<p>(A) Total number of thymocytes from both thymus lobes of individual E18 mouse embryos of the indicated strain. (B) Total cell number of Vγ5⁺ thymocytes from E18 thymus. (C) Shows the ratio of CD45RB^high and CD45RB^low subsets within total CD3⁺ Vγ5⁺ thymocytes. A minimum of 10 mice of each strain were analyzed. Error bars represent SEM, with asterisks signifying a significant difference, where <i>p</i><0.001.</p

Vγ5⁺ DETC are Selectively Reduced in the Epidermis of Adult, but not Neonatal, CCR4^−/− Mice.

<p>(A) Day 0 newborn epidermal sheets were digested and stained for Vγ5TCR and CD3 expression. The graph shows the percentage of T-cells within DAPI- cells of the indicated strains. WT; n = 12, CCR4^−/−; n = 8, CCR7^−/−; n = 9. (B) Percentages of Vγ5⁺ DETC within CD3⁺ T-cells in d0 newborn epidermis. (C) Adult ear epidermal sheets were digested with Collagenase D and stained for Vγ5TCR and CD3 expression. The graph shows the percentage of CD3⁺ T-cells within DAPI- cells. WT; n = 19, CCR4^−/−; n = 14, CCR7^−/−; n = 12. (D) Percentage of Vγ5⁺ DETC within CD3⁺ T-cells in adult epidermis. (E) Percentage of Annexin V⁺ cells in adult ear, after gating on Vγ5⁺ DETC. WT n = 4, CCR4^−/− n = 3. (F) shows the percentage of T-cells in the dermis of adult ear skin in WT and CCR4^−/− mice, while (G) shows the proportion of Vγ5⁺ cells within dermal T cells. WT; n = 4, CCR4^−/−; n = 4. Asterisks signify a significant difference, where ** <i>p</i><0.001, * <i>p</i><0.01.</p

Aire is not Required for Vγ5⁺ DETC Progenitor Clustering with mTEC.

<p>(A) Representative confocal analysis of E18 WT and Aire^−/− thymus sections. Vγ5⁺ cells are shown in green, CD8β⁺ cells are shown in red and EpCAM⁺ medullary areas are shown in blue. M denotes medulla, C denotes cortex. Scale bars in A represent 100 µm. (B) Quantification of Vγ5⁺ cells per mm² thymic medulla. WT; n = 6, Aire^−/−; n = 6. Error bars represents SEM. (C) Individual thymus lobes of E18 WT and Aire^−/− embryos were teased apart and stained for Vγ5, CD3, CD24 and CD45RB expression. Numbers shown on FACS plot are the mean percentages +/− SD. WT; n = 9, Aire^−/−; n = 4.</p