The Role Of α-Dystroglycan In Thymocyte Development

The task of generating a T lymphocyte population that responds to foreignpeptides presented by MHC, but not to self peptides, is undertaken in thethymus. Thymocytes comprising the newly formed TCR repertoire are selectedfor their ability to recognize peptide in the context of MHC molecules. Positiveselection tests the ability of TCR to signal in response to self-peptide/MHC.Thymocytes that do not receive a TCR signal of sufficient strength die throughneglect. Negative selection eliminates thymocytes expressing TCRs thattransmit a strong signal in response to self-peptide/MHC. Current modelsemphasize the role of overall signal strength, reflecting the input fromcostimulatory and accessory molecules as well as TCR, in determining thedevelopmental outcome of TCR signaling. In mature T cells, TCR signaling isassociated with the formation of immunological synapse, at the T cell/APCinterface. Recent studies have begun to analyze immunological synapseformation during thymic selection using lipid bilayers containing peptide/MHCcomplexes, a negative selection system and a positive selection system.These studies provide direct evidence that thymocyte-epithelial cellinteractions leading to positive selection result in the redistribution of cellmembrane-associated signaling molecules to the thymocyte-epithelial cellinterface in a manner analogous to that seen in mature T cell-APC interactions.Dystroglycan consists ofαandβsubunits yielded by proteolysis cleavage of asingle precursor protein,α-Dystroglycan is a highly glycosylated extracellularprotein and non-covalently anchored to the transmembraneβ-dystroglycan.(α-Dystroglycan links to the extracellular matrix (ECM) via several ligands,whereasβ-dystroglycan linksα-dystroglycan to the actin cytoskeleton viadystrophin or utrophin. Accumulating evidence indicates that dystroglycan is involved in the formation of synapse in neural-muscle junction (NMJ) andcentral never system (CNS). Our previous studies have shown thatα-dystroglycan is expressed on lymphocytes and suggested thatα-dystroglycan might contribute to lymphocyte activation by participating insynapse formation. In this study we found thatα-dystroglycan was dynamicallyexpressed on various thymocytes especially on double positive thymocytesand showed that interruption ofα-dystroglycan led to increased apoptosis ofdouble positive thymocytes by affecting immune synapse formation, indicatingthatα-dystroglycan might be involved in thymocyte development byparticipating in synapse formation.1. Dynamic expression ofα-dystroglycan on fetal thymocytes in vivo andin fetal thymus organ culture (FTOC)(1) Dynamic expression ofα-dystroglycan in fetal thymuses fromdifferent gestation days mice: To investigate the possible roles ofα-dystroglycan in thymocyte development, the expression ofα-dystroglycan inthymus was first examined at mRNA level. The coding gene for dystroglycancould be specifically amplified from total RNA of thymocytes from 15-19gestation days (gd) fetal mice. And an expression peak appeared at 16 gd.The results suggested thatα-dystroglycan was dynamically expressed in fetalthymuses from different gd mice.(2) Dynamic expression ofα-dystroglycan on four subpopulation ofthymocytes in vivo: To examine which subpoplations of thymocytes expressα-dystroglycan in vivo, thymocytes from 15-19 gd fetal mice were stained withantibodies againstα-dystroglycan followed by FITC-labeled rabbit anti-mouseIgG along with anti-CD4-PerCP and anti-CD8-PE and analyzed by FACS. Theresults showed thatα-dystroglycan was dynamically expressed on foursubpopulation of thymocytes from different gd fetal mice, especially highlyexpressed on double positive (DP) cells and CD4+/CD8+ single positive (SP)cells. For example, the expression rates ofα-dystroglycan on total population,DN, DP, CD4+SP CD8+SP cells from 19 gd fetal mice were 92.66%, 8.30%,99.98%, 90.42% and 95.41% respectively; the expression rates of α-dystroglycan on DP cells from 15-19 gd fetal mice were 97.62%, 99.57%,99.59% and 99.98% respectively; the expression rates ofα-dystroglycan onCD4+SP cells from 15-19 gd fetal mice were 54.55%, 80.22%, 76.60% and90.42% respectively; the expression rates ofα-dystroglycan on CD8+SP cellsfrom 15-19 gd fetal mice were 52.34%, 50.35%, 62.71%, and 95.41%respectively. These results probably suggested a role ofα-dystroglycan inthymocyte development.(3) Dynamic expression ofα-dystroglycan on four subpopulation ofthymocytes in FTOC: To examine which subpoplations of thymocytesexpressα-dystroglycan in FTOC, thymocytes recovered from FTOC culturedfor 2-6 days were stained with antibodies againstα-dystroglycan followed byFITC-labeled rabbit anti-mouse IgG along with anti-CD4-PerCP andanti-CD8-PE and analyzed by FAGS. The results showed thatα-dystroglycanwas dynamically expressed on four subpopulation of thymocytes recoveredfrom FTOC cultured for 2-6 days, especially highly expressed on DP and SPcells. For example, the expression rates ofα-dystroglycan on total population,DN, DP, CD4+SP CD8+SP cells recovered from FTOC cultured for 6 dayswere 85.35%, 73.07%, 87.06%, 98.09% and 93.47% respectively and theexpression rates ofα-dystroglycan on the same subpopulation recovered fromFTOC cultured for 2-6 days also varied dynamically. These results probablysuggested a role ofα-dystroglycan in thymocyte development.2. Role ofα-dystroglycan in thymocyte development(1) Role ofα-dystroglycan in development of double negativethymocytesThe above results showed us that the expression ofα-dystroglycan on DPand SP cells was relatively higher, and the expression ofα-dystroglycan onDN cells was relatively lower, partly suggesting thatα-dystroglycan might playa role in development of DP and SP cells, while the possibility thatα-dystroglycan might play a role in development of DN cells was much lower.To explore whetherα-dystroglycan plays a role in development of DN cells, wetreated FTOC with anti-α-dystroglycan blocking antibody IIH6C4 and found that blockage ofα-dystroglycan had no effect on the proportion and number ofDN cells compared with non-treated or isotype antibody-treated FTOC. Theresults indicated thatα-dystroglycan might not play a role in development ofDN cells.To further confirm the above results from Ab blocking experiment, weestablished reaggregate thymus organ culture (RTOC) with virus-producingcells (VPCs) which produced viruses carrying anti-sense cDNA ofα-dystroglycan and found that viruses carrying anti-sense cDNA ofα-dystroglycan had no effect on the proportion and number of DN cellscompared with no viruses and control viruses. The results further indicated thatα-dystroglycan might not play a role in development of DN cells.(2) Role ofα-dystroglycan in development of double positive thymocytesThe results thatα-dystroglycan was highly expressed on DP partlysuggested thatα-dystroglycan might play a role in development of DP cells. Toexplore whetherα-dystroglycan plays a role in development of DP cells, wetreated FTOC with anti-α-dystroglycan blocking antibody IIH6C4 and foundthat blockage ofα-dystroglycan decreased the proportion and number of DPcells compared with non-treated or isotype antibody-treated FTOC. The resultsindicated thatα-dystroglycan might play a role in development of DP cells.To further confirm the above results from Ab blocking experiment, weestablished reaggregate thymus organ culture (RTOC) with virus-producingcells (VPCs) which produced viruses carrying anti-sense cDNA ofα-dystroglycan and found that downregulation ofα-dystroglycan decreasedthe proportion and number of DP cells compared with no viruses and controlVPCs. The results further indicated thatα-dystroglycan might play a role indevelopment of DP cells.(3) Role ofα-dystroglycan in development of single positive thymocytesThe results thatα-dystroglycan was moderately expressed on SP partlysuggested thatα-dystroglycan might play a role in development of SP cells. Toexplore whetherα-dystroglycan plays a role in development of SP cells, wetreated FTOC with anti-α-dystroglycan blocking antibody IIH6C4 and foundthat blockage ofα-dystroglycan decreased the proportion and number of SP cells to a less extent than that of DP cells compared with non-treated orisotype antibody-treated FTOC.To further confirm the above results from Ab blocking experiment, weestablished reaggregate thymus organ culture (RTOC) with virus-producingcells (VPCs) which produced viruses carrying anti-sense cDNA ofα-dystroglycan and found that downregulation ofα-dystroglycan decreasedthe proportion and number of SP cells to a less extent than that of DP cellscompared with no viruses and control VPCs. These results were similar withthe results from Ab blocking experiments. Interruption ofα-dystroglycaninhibited development of both DP cells and SP cells, indicating thatα-dystroglycan might play a role in development of both DP cells and SP cellsor that the effects ofα-dystroglycan on development of SP cells were thefollowing outcome of the effects ofα-dystroglycan on development of DP cells.3. Mechanisms for the role ofα-dystroglycan in thymocyte development(1) The effect ofα-dystroglycan on apoptosis of thymocytes: Toinvestigate whether apoptosis was the reason that caused the numberdecrease of thymocytes, we examined the apoptosis status of thymocytes inIIH6C4-treated lobes. Enhanced apoptosis was observed in DP cells and to aless extent in SP cells in IIH6C4-treated lobes. The results suggested thatα-dystroglycan might be related with anti-apoptosis and/or survival signals ofthymocytes especially that of DP cells.(2) The role ofα-dystroglycan in synapse formation between thymocytesand thymic epithelial cells: To explore the mechanisms for the role ofα-dystroglycan in development of DP cells, we then investigated whetherα-dystroglycan played a role in synapse formation between DP cells andthymic epithelial cells. Using a thymocyte-epithelial cell conjugate model wefound thatα-dystroglycan blockage reduced conjugate formation and actinrearrangement between DP cells and thymic epithelial cells. These resultsindicated thatα-dystroglycan was involved in synapse formation between DPcells and thymic epithelial cells. (3) The pathways involved in the effect ofα-dystroglycan on apoptosis ofthymocytes: Then we examined the possible pathways that might be involvedin the effect ofα-dystroglycan on apoptosis of thymotyes. FAGS analysisdemonstrated that in IIH6C4-treated lobes, the expression of CD95 andCD95L was preferentially increased in DP cells and to a less extent in SP cellsbut not in DN cells compared to that of non-treated or isotype antibody-treatedlobes. Real-time RT-PCR showed that in IIH6C4-treated lobes, the expressionof Bcl-2 was decreased compared to that of non-treated or isotypeantibody-treated lobes. The results indicated that both CD95/CD95L and Bcl-2pathways were probably involved in the increased apoptosis of thymocytesespecially that of DP cells induced byα-dystroglycan blockage. FAGS analysisalso showed that blockage ofα-Dystroglycan downregulated ERKphosphorylation in DP cells, indicating that ERK pathway might be related toα-Dystroglycan signaling.In summary, our results suggested that interruption ofα-dystroglycan led toincreased apoptosis of DP thymocytes by affecting immunological synapseformation, indicating thatα-dystroglycan might be involved in thymocytedevelopment by participating in synapse formation.