Betaglycan (TβRIII) Is Expressed in the Thymus and Regulates T Cell Development by Protecting Thymocytes from Apoptosis

<div><p>TGF-β type III receptor (TβRIII) is a coreceptor for TGFβ family members required for high-affinity binding of these ligands to their receptors, potentiating their cellular functions. TGF-β <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044217#pone.0044217-Massague1">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044217#pone.0044217-TenDijke1">[3]</a>, bone morphogenetic proteins (BMP2/4) and inhibins regulate different checkpoints during T cell differentiation. Although TβRIII is expressed on hematopoietic cells, the role of this receptor in the immune system remains elusive. Here, we provide the first evidence that TβRIII is developmentally expressed during T cell ontogeny, and plays a crucial role in thymocyte differentiation. Blocking of endogenous TβRIII in fetal thymic organ cultures led to a delay in DN-DP transition. In addition, <em>in vitro</em> development of TβRIII^−/− thymic lobes also showed a significant reduction in absolute thymocyte numbers, which correlated with increased thymocyte apoptosis, resembling the phenotype reported in Inhibin α ^−/− thymic lobes. These data suggest that Inhibins and TβRIII may function as a molecular pair regulating T cell development.</p> </div

TβRIII deficiency results in increased apoptosis of developing thymocytes.

<p>(A) Left panel, representative CD4 versus CD8 staining dot plots from TβRIII^+/+ and TβRIII^−/− fetal thymic lobes at day 7 of culture. Histograms show the expression of active caspase 3⁺ cells in each gated thymocyte subset. Right panel, graphs represent the percentage of active caspase 3⁺ cells and the levels of expression (MFI values) in each thymocyte subset. Data are representative of three independent experiments. (B) Left panel, representative histograms show the percentage of Annexin V⁺ cells in gated thymocyte subsets. Right panel, graph shows the analysis of the percentage of Annexin V⁺ cells in thymocytes from day 7 TβRIII^+/+ or TβRIII^−/− FTOCs. Data are representative of two independent experiments. Mean values ± SEM are shown (TβRIII^+/+ n = 3 and TβRIII^−/− n = 3). Asterisks indicate statistically significant differences (** p≤0.05).</p

The blocking of TβRIII in FTOCs alters T cell development.

<p>E14 thymic lobes were cultured in the presence of anti-TβRIII antibody or in the presence of pre-immune serum (control lobe). At day 3 and 7 of culture thymic lobes were disaggregated, counted and stained with antibodies to CD4, CD8. (A) Representative CD4 versus CD8 staining dot plots. (B). Comparative graphs represent the percentages of DN, DP, CD4SP and CD8SP thymocytes obtained after 3 and 7 days of culture between both treatments. (C) Analysis of cell numbers in non-treated and anti-TβRIII treated FTOCs at day 3 and 7. Data are representative of two independent experiments. Mean values ± SEM are shown (n  = 7 per group for day 3, and n  = 9 per group for day 7). Asterisks indicate *p≤0.05.</p

TβRIII is developmentally expressed during T cell ontogeny.

<p>Thymocytes from 4 to 6 week old C57BL/6 background mice were stained with antibodies to CD4, CD8, and TβRIII. Pre-immune serum was used as an internal background staining control. (A) Representative histograms showing the percentage of TβRIII⁺ cells in gated DN, DP, CD4⁺ SP and CD8⁺ SP subsets. Graphs show the percentage of TβRIII⁺ thymocytes and geometric MFI calculated after subtracting the background staining. (B) Representative histograms showing the percentage of TβRIII⁺ thymocytes in DN1, DN2, DN3 and DN4 immature subsets. Graph represents the analysis of TβRIII⁺ cells and geometric MFI in gated DN1, DN2, DN3 and DN4 immature subsets. Unstained (filled curve in gray), preimmune serum (gray line) and anti-TβRIII antiserum (black line). Data are representative of 4 independent experiments. (C) Left panel, graph shows the percentage of TβRIII⁺ cells and geometric MFI in gated CD69⁻ and CD69⁺ SP thymocytes as showed in histograms. Right panel, graph represents the analysis of TβRIII⁺ cells and geometric MFI in gated CD62L⁻, CD62L⁺ and CD62L^hi SP thymocytes as showed in upper panel. Mean values ± SEM are shown (n  = 5 per group). Asterisks indicate * p≤0.05, and **p≤0.01.</p