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Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint.

Tamehiro N, Oda H, Shirai M, Suzuki H - PLoS ONE (2015)

Bottom Line: Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells.Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells.This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, Chiba, Japan.

ABSTRACT
RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+ CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

No MeSH data available.


Related in: MedlinePlus

Analysis of T cell development in RhoH overexpressing mice.Analysis by flow cytometry of thymocytes and splenocytes from RhoH+/+RhoHTg (red) and RhoH+/+ (blue) mice. Representative two parameter plots show CD4 versus CD8 staining on thymocytes (A, n = 8) and splenocytes (E, n = 13). (B) Representative single-parameter histogram plots show intracellular staining of Phospho-src (pY416) gated on CD4-CD8-CD44-CD25+ (DN3) cells (n = 5). Representative single-parameter histogram plots show cell surface staining of CD2 and CD5 antigens on DP cells from RhoH+/+RhoHTg and RhoH+/+ mice in either MHC+/+ (C, n = 6) or MHC-/- (D, n = 5) background. Solid line and dashed line represent RhoH+/+ and RhoH+/+RhoHTg, respectively. (F, G) Flow cytometric analysis of CD44 versus CD62L expression profile on splenic CD4+ T (F) or CD8+ T (G) cells gated on TCRβ+ T cells (n = 10). Data are shown as mean +SD and samples were from more than four independent experiments. **P<0.01, ***P<0.001, ****P<0.0001.
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pone.0131047.g002: Analysis of T cell development in RhoH overexpressing mice.Analysis by flow cytometry of thymocytes and splenocytes from RhoH+/+RhoHTg (red) and RhoH+/+ (blue) mice. Representative two parameter plots show CD4 versus CD8 staining on thymocytes (A, n = 8) and splenocytes (E, n = 13). (B) Representative single-parameter histogram plots show intracellular staining of Phospho-src (pY416) gated on CD4-CD8-CD44-CD25+ (DN3) cells (n = 5). Representative single-parameter histogram plots show cell surface staining of CD2 and CD5 antigens on DP cells from RhoH+/+RhoHTg and RhoH+/+ mice in either MHC+/+ (C, n = 6) or MHC-/- (D, n = 5) background. Solid line and dashed line represent RhoH+/+ and RhoH+/+RhoHTg, respectively. (F, G) Flow cytometric analysis of CD44 versus CD62L expression profile on splenic CD4+ T (F) or CD8+ T (G) cells gated on TCRβ+ T cells (n = 10). Data are shown as mean +SD and samples were from more than four independent experiments. **P<0.01, ***P<0.001, ****P<0.0001.

Mentions: To determine gain-of-function effects of RhoH on T cell development, we examined HA-tagged RhoHtg mice in C57BL/6 background. Although lack of RhoH caused strong inhibition of T cell development [4, 5] overexpression of RhoH did not affect overall differentiation of T cells, as the number and proportion of each subset in the thymus were unchanged (Fig 2A and S2A Fig). GFP expression on DP to CD4SP cells from RhoHtg Nur77-GFP mice, which were able to monitor the strength of TCR signaling in vivo during T cell developments [23], was also the same as the wild-type controls (S3A Fig). We finally observed RhoH transgene made small increase of the phosphorylation of Src family protein which act as the key kinases on T cell development in thymus (Fig 2B), but subpopulations of DN thymocytes were all unaltered in RhoHtg mice (S2C Fig), indicating that β-selection was not affected. Although prominent differences were not observed in the thymus, we noticed a slight but consistent up-regulation of CD2 and CD5 expression in DP cells from RhoHtg mice (Fig 2C). These changes were still observed in MHC-/- (I-Aβ and β2m DKO) background (Fig 2D), which is defective in positive and negative selection, indicating that the increase of CD2 and CD5 was independent of any events later than positive selection. Since expression levels of CD2 and CD5 correlate well to the strength of TCR signal [24, 25], these results may indicate augmented pre-TCR signaling in RhoH-overexpressing thymocytes. In the periphery, the percentage and numbers of splenic CD4+ and CD8+ T cells from RhoHtg mice were similar to that of wild-type mice (Fig 2E and S2B Fig). However, frequency and numbers of naïve CD4 and CD8 T cells (CD44lowCD62Lhi) were significantly reduced, and activated/memory T cells were concomitantly increased in RhoHtg mice (Fig 2F and 2G). Collectively, compared to the severe phenotypes of RhoH deficient mice, overexpression of RhoH had little effect on T cells, apart from the increased phosphorylation of Src family kinases in DN3, increased expression of CD2 and CD5 on DP, decreased number of naive T cells, and increased activated/memory T cells in the periphery. Other than conventional TCRαβ lineage T cells, development of unconventional T cells such as pTregs, NKT cells, and TCRγδ T cells was not changed in the RhoHtg mice (S4A–S4C Fig).


Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint.

Tamehiro N, Oda H, Shirai M, Suzuki H - PLoS ONE (2015)

Analysis of T cell development in RhoH overexpressing mice.Analysis by flow cytometry of thymocytes and splenocytes from RhoH+/+RhoHTg (red) and RhoH+/+ (blue) mice. Representative two parameter plots show CD4 versus CD8 staining on thymocytes (A, n = 8) and splenocytes (E, n = 13). (B) Representative single-parameter histogram plots show intracellular staining of Phospho-src (pY416) gated on CD4-CD8-CD44-CD25+ (DN3) cells (n = 5). Representative single-parameter histogram plots show cell surface staining of CD2 and CD5 antigens on DP cells from RhoH+/+RhoHTg and RhoH+/+ mice in either MHC+/+ (C, n = 6) or MHC-/- (D, n = 5) background. Solid line and dashed line represent RhoH+/+ and RhoH+/+RhoHTg, respectively. (F, G) Flow cytometric analysis of CD44 versus CD62L expression profile on splenic CD4+ T (F) or CD8+ T (G) cells gated on TCRβ+ T cells (n = 10). Data are shown as mean +SD and samples were from more than four independent experiments. **P<0.01, ***P<0.001, ****P<0.0001.
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Related In: Results  -  Collection

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pone.0131047.g002: Analysis of T cell development in RhoH overexpressing mice.Analysis by flow cytometry of thymocytes and splenocytes from RhoH+/+RhoHTg (red) and RhoH+/+ (blue) mice. Representative two parameter plots show CD4 versus CD8 staining on thymocytes (A, n = 8) and splenocytes (E, n = 13). (B) Representative single-parameter histogram plots show intracellular staining of Phospho-src (pY416) gated on CD4-CD8-CD44-CD25+ (DN3) cells (n = 5). Representative single-parameter histogram plots show cell surface staining of CD2 and CD5 antigens on DP cells from RhoH+/+RhoHTg and RhoH+/+ mice in either MHC+/+ (C, n = 6) or MHC-/- (D, n = 5) background. Solid line and dashed line represent RhoH+/+ and RhoH+/+RhoHTg, respectively. (F, G) Flow cytometric analysis of CD44 versus CD62L expression profile on splenic CD4+ T (F) or CD8+ T (G) cells gated on TCRβ+ T cells (n = 10). Data are shown as mean +SD and samples were from more than four independent experiments. **P<0.01, ***P<0.001, ****P<0.0001.
Mentions: To determine gain-of-function effects of RhoH on T cell development, we examined HA-tagged RhoHtg mice in C57BL/6 background. Although lack of RhoH caused strong inhibition of T cell development [4, 5] overexpression of RhoH did not affect overall differentiation of T cells, as the number and proportion of each subset in the thymus were unchanged (Fig 2A and S2A Fig). GFP expression on DP to CD4SP cells from RhoHtg Nur77-GFP mice, which were able to monitor the strength of TCR signaling in vivo during T cell developments [23], was also the same as the wild-type controls (S3A Fig). We finally observed RhoH transgene made small increase of the phosphorylation of Src family protein which act as the key kinases on T cell development in thymus (Fig 2B), but subpopulations of DN thymocytes were all unaltered in RhoHtg mice (S2C Fig), indicating that β-selection was not affected. Although prominent differences were not observed in the thymus, we noticed a slight but consistent up-regulation of CD2 and CD5 expression in DP cells from RhoHtg mice (Fig 2C). These changes were still observed in MHC-/- (I-Aβ and β2m DKO) background (Fig 2D), which is defective in positive and negative selection, indicating that the increase of CD2 and CD5 was independent of any events later than positive selection. Since expression levels of CD2 and CD5 correlate well to the strength of TCR signal [24, 25], these results may indicate augmented pre-TCR signaling in RhoH-overexpressing thymocytes. In the periphery, the percentage and numbers of splenic CD4+ and CD8+ T cells from RhoHtg mice were similar to that of wild-type mice (Fig 2E and S2B Fig). However, frequency and numbers of naïve CD4 and CD8 T cells (CD44lowCD62Lhi) were significantly reduced, and activated/memory T cells were concomitantly increased in RhoHtg mice (Fig 2F and 2G). Collectively, compared to the severe phenotypes of RhoH deficient mice, overexpression of RhoH had little effect on T cells, apart from the increased phosphorylation of Src family kinases in DN3, increased expression of CD2 and CD5 on DP, decreased number of naive T cells, and increased activated/memory T cells in the periphery. Other than conventional TCRαβ lineage T cells, development of unconventional T cells such as pTregs, NKT cells, and TCRγδ T cells was not changed in the RhoHtg mice (S4A–S4C Fig).

Bottom Line: Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells.Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells.This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, Chiba, Japan.

ABSTRACT
RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+ CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

No MeSH data available.


Related in: MedlinePlus