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

Expression and immunolocalization of CA125 and CSC markers by immunofluorescence.

<p>Purified NAD and AD cells were evaluated by immunofluorescence using mouse monoclonal antibody (green) as described in the Methods and Materials. Cellular staining was visualized using the secondary Alexa 488 (green) fluorescent labeled antibody, and nuclei were detected by DAPI (blue) staining. Images are representative of three independent samples. Magnification was 200×; scale bar = 50 µm.</p

Tumorigenic properties of NAD and AD cells purified from the ascites of CR patients.

<p>(<b>A</b>) A phase contrast microscope image of NAD cells adhered to plastic before preparing the cell suspension for i.p. injection; (<b>B</b>) H and E staining of agarose embedded patient sample before injection; (<b>C</b>) image of solid tumor obtained from a mouse fourteen weeks after i.p. injection of NAD cells (5×10⁶); (<b>D</b>) H and E staining of mouse ascites NAD cells embedded on agarose; (<b>E</b>) Flow cytometric comparison of the expression of CA125, EpCAM and CD44 between the patient’s and mouse ascites cells. Results are representative of two independent samples. The filled histogram in each figure represents control IgG, black lines indicate protein expression in human cells, broken lines indicate the expression of the protein in mouse ascites cells.</p

Distribution of markers in isolated cells obtained from the ascites of ovarian cancer patients.

<p>The antigens have been scored as high expression (⁺⁺⁺), moderate expression (⁺⁺), low expression (⁺), and no expression (⁻).</p

mRNA expression of epithelial, mesenchymal and CSC markers in isolated ascites cells.

<p>qPCR was performed on purified NAD and AD populations as described in the Methods and Materials. Yields were converted to femtograms based on the standard curve for each PCR product, and the resultant mRNA levels were normalized to the 18S mRNA level per sample. The data were calculated from the results of eight independent samples assessed in triplicate. Significantly different in AD versus NAD cells *(p&lt;0.05) and **(p&lt;0.01).</p

mRNA expression of MMP2 and MMP9 in purified cells from CN and CR patients.

<p>qPCR was performed on isolated NAD and AD cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046858#pone-0046858-g009" target="_blank">Figure 9</a>. Results are expressed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046858#pone-0046858-g009" target="_blank">Figure 9</a>. Significantly different in CR versus CN samples, *(p&lt;0.05).</p

Morphological features, proliferation and cisplatin sensitivity of NAD and AD cells.

<p>(<b>A</b>) NAD spheroids and (<b>B</b>) AD cells were seeded on low attachment plates immediately after collection. Morphological features of (<b>C</b>) NAD spheroid and (<b>D</b>) AD cells on tissue culture plastic after 24 h following seeding. Images were assessed by phase contrast microscopy. Magnification was 100×, scale bar = 50 µm. The images are representative of (n = 25) samples. (<b>E</b>) [³H]-thymidine uptake in AD cells and in cells dispersed from spheroids was performed as described in Methods and Materials. The graph is a representation of one ascites sample performed in triplicate. (<b>F</b>) Effect of cisplatin on the proliferation {[³H]-thymidine uptake} of NAD and AD cells obtained from the ascites of ovarian cancer patients. The graph is a representation of three independent experiments, performed on three independent NAD and AD samples in triplicate. Significantly different between AD versus NAD cells, **p&lt;0.01.</p