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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

RhoH transgene expression enables transition from DN to DP stages in Rag2-/- mice.FACS analysis of thymocyte differentiation in Rag2-/- (A) or MHC-/- (B) background. Representative two parameter plots show CD4 versus CD8 staining on thymocytes and CD25 versus CD44 staining on DN cells. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from five mice per group. **P<0.01, ***P<0.001.
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pone.0131047.g003: RhoH transgene expression enables transition from DN to DP stages in Rag2-/- mice.FACS analysis of thymocyte differentiation in Rag2-/- (A) or MHC-/- (B) background. Representative two parameter plots show CD4 versus CD8 staining on thymocytes and CD25 versus CD44 staining on DN cells. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from five mice per group. **P<0.01, ***P<0.001.

Mentions: Rag2 deficient mice are unable to complete gene rearrangement of the TCR locus, therefore T cell development stops at the β-selection checkpoint, resulting in complete arrest at the DN3 stage. To our surprise, when we crossed RhoHtg mice with Rag2-/- mice, DP thymocytes emerged in the thymus (Fig 3A). The generation of DP cells associated with increased gene expression of RhoH was in a dose-dependent manner (Fig 3A), because transgene homozygous tg/tg mice generated more DP than transgene heterozygous tg/- mice. Introduction of RhoH transgene did not induce gene rearrangement in the TCRβ locus, because TCRβ protein was not observed in these DP cells (S5A Fig). Overexpression of RhoH could successfully bypass β-selection to generate DPs, however it could not bypass positive selection because differentiation of mature CD4SP and CD8SP cells was not observed in the Rag2-/-RhoHtg mice (Fig 3A). This was further confirmed by the results from MHC-/-RhoHtg mice, which contained no single positive cells (Fig 3B), proving that simple overexpression of RhoH is not sufficient for bypassing positive selection. Taken together, overexpression of RhoH enabled bypass of β-selection without TCRβ-gene rearrangement, however it was not sufficient to permit bypass of positive selection.


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

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

RhoH transgene expression enables transition from DN to DP stages in Rag2-/- mice.FACS analysis of thymocyte differentiation in Rag2-/- (A) or MHC-/- (B) background. Representative two parameter plots show CD4 versus CD8 staining on thymocytes and CD25 versus CD44 staining on DN cells. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from five mice per group. **P<0.01, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482576&req=5

pone.0131047.g003: RhoH transgene expression enables transition from DN to DP stages in Rag2-/- mice.FACS analysis of thymocyte differentiation in Rag2-/- (A) or MHC-/- (B) background. Representative two parameter plots show CD4 versus CD8 staining on thymocytes and CD25 versus CD44 staining on DN cells. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from five mice per group. **P<0.01, ***P<0.001.
Mentions: Rag2 deficient mice are unable to complete gene rearrangement of the TCR locus, therefore T cell development stops at the β-selection checkpoint, resulting in complete arrest at the DN3 stage. To our surprise, when we crossed RhoHtg mice with Rag2-/- mice, DP thymocytes emerged in the thymus (Fig 3A). The generation of DP cells associated with increased gene expression of RhoH was in a dose-dependent manner (Fig 3A), because transgene homozygous tg/tg mice generated more DP than transgene heterozygous tg/- mice. Introduction of RhoH transgene did not induce gene rearrangement in the TCRβ locus, because TCRβ protein was not observed in these DP cells (S5A Fig). Overexpression of RhoH could successfully bypass β-selection to generate DPs, however it could not bypass positive selection because differentiation of mature CD4SP and CD8SP cells was not observed in the Rag2-/-RhoHtg mice (Fig 3A). This was further confirmed by the results from MHC-/-RhoHtg mice, which contained no single positive cells (Fig 3B), proving that simple overexpression of RhoH is not sufficient for bypassing positive selection. Taken together, overexpression of RhoH enabled bypass of β-selection without TCRβ-gene rearrangement, however it was not sufficient to permit bypass of positive selection.

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