Lopmental progression through the DN stages, resulting in fewer DP (Fig. 2C). In contrast, 3KO mice showed a modest increase in DP/ DN, which may represent more efficient development of DP due to predominantly RasGRP1 driven signaling during development. No statistical difference in DP/DN ratio was observed between 1KO and DKO mice. Therefore, it appears that in addition to regulating positive selection, RasGRP1 also regulates the generation of DP. Since RasGRP1 deficiency results in inefficient DN to DP development, we next examined the DN compartment of wildtype and RasGRP1/3 deficient thymi using the CD44/CD25 profile of DN (CD42CD82Thy1.2+CD3lo) thymocytes. We found that 1KO and DKO thymi showed significantly increased frequencies and numbers of DN3 thymocytes (CD442CD25+) relative to B6 (Fig. 3A,B), suggesting defects in b-selection. One important result of b-selection is differentiation of DN3 thymocytes into DN4 thymocytes (CD442CD252). To evaluate this critical differentiation step in the 18325633 thymocyte developmental program, we examined the ratio of frequencies of DN3 to DN4 thymocytes. 1KO and DKO mice showed significant increases in DN3/DN4, providing further evidence of impaired b-selection in the absence of RasGRPs (Fig. 3B). The DN3/DN4 seemed to be modestlyRasGRP1 Is Required for b-SelectionThy1.2+ cells from B6 (n = 9), 1KO (n = 6), 3KO (n = 9) and DKO thymi (n = 13). B. Numbers of mature Thy1.2+CD3+CD442 CD4 and CD8 SP thymocytes. C. CD3 by CD1d Tetramer (PBS57) profiles of bulk thymocytes from B6 (n = 5), 1KO (n = 6), 3KO (n = 6) and DKO (n = 7) thymi. D. Frequencies and numbers of mature CD3+CD1d(PBS57) Tetramer+ iNKT cells. *p,0.05, **p,0.01 and ***p,0.001. doi:10.1371/journal.pone.0053300.gFigure 1. RasGRP1, but not RasGRP3, is required for thymocyte positive selection and iNKT selection. A. CD4 by CD8 profiles ofhigher in DKO thymi compared to 1KO, although this was not statistically significant. Loss of RasGRP3 alone did not appear to influence the development of DN3 into DN4 and the contribution of RasGRP1 to b-selection appears to be dominant to RasGRP3. The bifurcation of ab and cd T cell development is thought to occur at the DN3 stage. Since we saw a significant increase in DN3 numbers in RasGRP1/3 deficient thymi and DN3 can give rise to both ab and cd T cells, we wanted to address possible alterations in cd T cell development due to RasGRP1/3 deficiency. To examine thymic cd development we looked for the presence of mature cdTCR+CD3+ thymocytes in B6 and RasGRP1/3 deficient thymi. We found 1KO, 3KO and DKO thymi showed Licochalcone-A similar numbers and frequencies of mature cd thymocytes as B6 (Fig. 4 A,B). These results suggested that the increased DN3/DN4 seen in RasGRP1/3 deficient thymi was likely due to defects in ab development and not cd development. Therefore, we next focused on ab development from DN3 in RasGRP1/3 deficient mice. Following b-selection, DN4 GW0742 manufacturer upregulate surface expression of TCRb as they mature into DP thymocytes. To gain insight into progression through b-selection, we examined DN4 for surface expression of TCRb. 1KO and DKO thymi showed statistically significant reductions in frequencies of surface TCRb+ DN4 compared to B6, while 3KO thymi showed similar frequencies of surface TCRb+ DN4 as B6 (Fig. 4C). Since cd T cells also contribute to the DN4 pool, we examined DN4 for surface expression of cdTCR. 1KO and DKO thymi showed statistically significant increases in frequencies of cdTCR+ DN4 compared to.Lopmental progression through the DN stages, resulting in fewer DP (Fig. 2C). In contrast, 3KO mice showed a modest increase in DP/ DN, which may represent more efficient development of DP due to predominantly RasGRP1 driven signaling during development. No statistical difference in DP/DN ratio was observed between 1KO and DKO mice. Therefore, it appears that in addition to regulating positive selection, RasGRP1 also regulates the generation of DP. Since RasGRP1 deficiency results in inefficient DN to DP development, we next examined the DN compartment of wildtype and RasGRP1/3 deficient thymi using the CD44/CD25 profile of DN (CD42CD82Thy1.2+CD3lo) thymocytes. We found that 1KO and DKO thymi showed significantly increased frequencies and numbers of DN3 thymocytes (CD442CD25+) relative to B6 (Fig. 3A,B), suggesting defects in b-selection. One important result of b-selection is differentiation of DN3 thymocytes into DN4 thymocytes (CD442CD252). To evaluate this critical differentiation step in the 18325633 thymocyte developmental program, we examined the ratio of frequencies of DN3 to DN4 thymocytes. 1KO and DKO mice showed significant increases in DN3/DN4, providing further evidence of impaired b-selection in the absence of RasGRPs (Fig. 3B). The DN3/DN4 seemed to be modestlyRasGRP1 Is Required for b-SelectionThy1.2+ cells from B6 (n = 9), 1KO (n = 6), 3KO (n = 9) and DKO thymi (n = 13). B. Numbers of mature Thy1.2+CD3+CD442 CD4 and CD8 SP thymocytes. C. CD3 by CD1d Tetramer (PBS57) profiles of bulk thymocytes from B6 (n = 5), 1KO (n = 6), 3KO (n = 6) and DKO (n = 7) thymi. D. Frequencies and numbers of mature CD3+CD1d(PBS57) Tetramer+ iNKT cells. *p,0.05, **p,0.01 and ***p,0.001. doi:10.1371/journal.pone.0053300.gFigure 1. RasGRP1, but not RasGRP3, is required for thymocyte positive selection and iNKT selection. A. CD4 by CD8 profiles ofhigher in DKO thymi compared to 1KO, although this was not statistically significant. Loss of RasGRP3 alone did not appear to influence the development of DN3 into DN4 and the contribution of RasGRP1 to b-selection appears to be dominant to RasGRP3. The bifurcation of ab and cd T cell development is thought to occur at the DN3 stage. Since we saw a significant increase in DN3 numbers in RasGRP1/3 deficient thymi and DN3 can give rise to both ab and cd T cells, we wanted to address possible alterations in cd T cell development due to RasGRP1/3 deficiency. To examine thymic cd development we looked for the presence of mature cdTCR+CD3+ thymocytes in B6 and RasGRP1/3 deficient thymi. We found 1KO, 3KO and DKO thymi showed similar numbers and frequencies of mature cd thymocytes as B6 (Fig. 4 A,B). These results suggested that the increased DN3/DN4 seen in RasGRP1/3 deficient thymi was likely due to defects in ab development and not cd development. Therefore, we next focused on ab development from DN3 in RasGRP1/3 deficient mice. Following b-selection, DN4 upregulate surface expression of TCRb as they mature into DP thymocytes. To gain insight into progression through b-selection, we examined DN4 for surface expression of TCRb. 1KO and DKO thymi showed statistically significant reductions in frequencies of surface TCRb+ DN4 compared to B6, while 3KO thymi showed similar frequencies of surface TCRb+ DN4 as B6 (Fig. 4C). Since cd T cells also contribute to the DN4 pool, we examined DN4 for surface expression of cdTCR. 1KO and DKO thymi showed statistically significant increases in frequencies of cdTCR+ DN4 compared to.
