CD205-deficiency does not impact upon TCRVβ distribution in a polyclonal T cell repertoire.

<p>Flow cytometric analysis of TCRVβ distribution in adult <i>Ly75</i>^−/− and <i>Ly75</i>^+/− littermates for (A) CD4⁺8⁻ (SP4) thymocytes, (B) CD4⁻8⁺ (SP8) thymocytes, (C) CD4⁺8⁻ splenocytes, and (D) CD4⁻8⁺ splenocytes. Error bars show ± standard error for 3 independent experiments. Statistical analysis performed using Mann Whitney U test, no significant (n.s.) differences were determined between TCRVβ usage in <i>Ly75</i>^−/− and <i>Ly75</i>^+/− mice.</p

Efficient thymocyte development is unaffected in CD205-deficient thymus.

<p>Thymocytes isolated from adult <i>Ly75</i>^−/− and <i>Ly75</i>^+/− littermate controls were analyzed by flow cytometry for: (A) CD4 and CD8 distribution, representative of ≥10mice, (B) Total thymus cellularity, n = ≥10, (C) CD4⁻8⁻ double negative (DN) thymocyte distribution, CD44⁺25⁻ (DN1), CD44⁺25⁺ (DN2), CD44⁻25⁺ (DN3), CD44⁻25⁻ (DN4), n = ≥4, (D) CD69⁺ CD4⁺8⁺ double positive (DP) thymocytes, n = ≥4, (E) Foxp3⁺ T regulatory CD4 thymocytes, gated on CD4⁺8⁻ TCRβ^hi CD25⁺ Foxp3⁺, n = ≥7, and (F) CD4⁺8⁻ SP4 and CD4⁻8⁺ SP ratios, n = ≥4. Statistical analysis performed using Mann Whitney U test. n.s. (not significant). Error bars show ± standard error for indicated number (n) of mice examined.</p

Flow cytometric analysis of CD205-deficient thymi does not reveal defects in thymic epithelial compartments.

<p>Thymi from adult <i>Ly75</i>^+/− and <i>Ly75</i>^−/− mice were enzymatically digested and analyzed by flow cytometry for: (A) CTEC (EpCAM⁺Ly51^hi) and MTEC (EpCAM⁺Ly51^low), cells gated on CD45⁻EpCAM⁺ thymic epithelium, (B) MHC class I and (C) MHC class II expression on CTEC and MTEC in <i>Ly75</i>^+/− (grey solid line, open histogram) and <i>Ly75</i>^−/− (black dashed line, open histogram) thymi, staining controls (filled histogram), and (D) Aire⁺ CD80⁺ MTEC, cells gated on CD45⁻EpCAM⁺Ly51^low cells. Data are representative of 3 separate experiments.</p

CD205 does not regulate positive selection of MHC class I and MHC class II restricted transgenic TCR specificities.

<p>Bone marrow (BM)-derived hematopoietic cells isolated from (A) OT-II and (B) SM1 MHC II-restricted, and (C) OT-I MHC I-restricted TCR-transgenic adult mice were transferred into lethally irradiated <i>Ly75</i>^−/− or <i>Ly75</i>^+/− littermate controls. Thymocytes were isolated 5 weeks after reconstitution and analyzed by flow cytometry for CD4 and CD8 (left hand panels). Histograms demonstrate TCR staining for transgenic-specific T cells, OT-II TCRVα2 (A), SM1 TCRVβ2 (B) and OT-I TCRVα2 (C) (open histograms), staining control (filled histogram). Right hand graphs demonstrate numbers of TCR transgenic T cells isolated from thymus and spleen, data points demonstrate total cell numbers for individual mice, gating as indicated (n = ≥3). Data analyzed using Mann Whitney U test. n.s. (not significant).</p

Peripheral T cells generated in the absence of CD205 appear normal and do not display signs of activation.

<p>Splenocytes isolated from <i>Ly75</i>^−/− and <i>Ly75</i>^+/− adult littermates were analyzed for: (A) total spleen cellularity, (B) CD4⁺8⁻ and CD4⁻8⁺ T cell ratios and (C) Foxp3⁺CD25⁺ CD4⁺8⁻ regulatory T cells. The occurrence of CD4⁺8⁻ splenocytes bearing an activated CD69⁺ and CD62L^low phenotype (D, E) was assessed in 10-week-old <i>Ly75</i>^−/− and <i>Ly75</i>^+/− mice. Error bars show ± standard error for n = ≥4 mice, n.s. (not significant) using Mann Whitney U test.</p

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

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

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

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