Figure 1.
CD205-deficient mice demonstrate normal anatomical organization of thymic microenvironments.
Frozen tissue sections of adult CD205-deficient (Ly75−/−) and CD205-sufficient (Ly75+/−) littermate control mice were analyzed by confocal microscopy for: (A) organization of thymocytes, defined by expression of CD4 (blue) and CD8 (red), and dendritic cells by CD11c (green), final magnification ×100, (B) thymic epithelium defined by β5t (cortex, green) and ER-TR5 (medulla, red), final magnification ×250, and the presence of Aire+ (red) ER-TR5+ MTEC (green), final magnification ×400 (C). Quantitative analysis of total cortical and medullary areas of CD205-sufficient (white bars) and CD205-deficient (grey bars) adult thymi (D). Data are representative of two individual experiments, n.s. = not significant.
Figure 2.
Flow cytometric analysis of CD205-deficient thymi does not reveal defects in thymic epithelial compartments.
Thymi from adult Ly75+/− and Ly75−/− mice were enzymatically digested and analyzed by flow cytometry for: (A) CTEC (EpCAM+Ly51hi) and MTEC (EpCAM+Ly51low), cells gated on CD45−EpCAM+ thymic epithelium, (B) MHC class I and (C) MHC class II expression on CTEC and MTEC in Ly75+/− (grey solid line, open histogram) and Ly75−/− (black dashed line, open histogram) thymi, staining controls (filled histogram), and (D) Aire+ CD80+ MTEC, cells gated on CD45−EpCAM+Ly51low cells. Data are representative of 3 separate experiments.
Figure 3.
Efficient thymocyte development is unaffected in CD205-deficient thymus.
Thymocytes isolated from adult Ly75−/− and Ly75+/− 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− TCRβhi 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.
Figure 4.
Peripheral T cells generated in the absence of CD205 appear normal and do not display signs of activation.
Splenocytes isolated from Ly75−/− and Ly75+/− 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 CD62Llow phenotype (D, E) was assessed in 10-week-old Ly75−/− and Ly75+/− mice. Error bars show ± standard error for n = ≥4 mice, n.s. (not significant) using Mann Whitney U test.
Figure 5.
CD205-deficiency does not impact upon TCRVβ distribution in a polyclonal T cell repertoire.
Flow cytometric analysis of TCRVβ distribution in adult Ly75−/− and Ly75+/− 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 Ly75−/− and Ly75+/− mice.
Figure 6.
CD205 does not regulate positive selection of MHC class I and MHC class II restricted transgenic TCR specificities.
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 Ly75−/− or Ly75+/− 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).