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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

Lck activation is required for RhoH transgene-induced DPs generation without TCRβ-gene rearrangement.(A) FACS analysis of the in vitro differentiation of thymocytes to the DP stage. DN3 cells from Rag2-/-, Rag2-/-RhoHtg, and Rag2+/+ C57B6 mice were differentiated for 14 days with the stromal cell line TSt-4/Dll-1 in the presence of IL-7. Representative two parameter plots show CD4 versus CD8 staining on cultured thymocytes at the indicated days. Results shown are from one out of three independent experiments. (B) DN3 cells were cultured with vehicle alone or a pharmacological inhibitor of Lck at the indicated concentration. Bar graphs represent the percentages of DP thymocytes compared with the vehicle control group. Data are representative of more than three independent experiments. ***P<0.001.
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pone.0131047.g004: Lck activation is required for RhoH transgene-induced DPs generation without TCRβ-gene rearrangement.(A) FACS analysis of the in vitro differentiation of thymocytes to the DP stage. DN3 cells from Rag2-/-, Rag2-/-RhoHtg, and Rag2+/+ C57B6 mice were differentiated for 14 days with the stromal cell line TSt-4/Dll-1 in the presence of IL-7. Representative two parameter plots show CD4 versus CD8 staining on cultured thymocytes at the indicated days. Results shown are from one out of three independent experiments. (B) DN3 cells were cultured with vehicle alone or a pharmacological inhibitor of Lck at the indicated concentration. Bar graphs represent the percentages of DP thymocytes compared with the vehicle control group. Data are representative of more than three independent experiments. ***P<0.001.

Mentions: To address whether generation of DPs in Rag-/-RhoHtg mice was thymocyte intrinsic, we next tested using an in vitro differentiation culture system. Sorted DN3 cells from wild-type mice were able to differentiate into DPs in vitro in a week on monolayers of Notch ligand-expressing stromal cells (TSt-4/Dll-1), while DN3 cells from Rag2-/- mice could not (Fig 4A). We found that sorted DN3 cells from Rag2-/-RhoHtg mice could successfully differentiate into DP cells (Fig 4A), demonstrating that these DPs were truly differentiated from DN3 cells, and that this transition was thymocyte intrinsic. Furthermore, this in vitro DPs generation was strongly inhibited by the addition of Lck inhibitor (Fig 4B, S6 Fig), indicating that RhoH-transgene induced generation of DPs was dependent on Lck activity.


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

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

Lck activation is required for RhoH transgene-induced DPs generation without TCRβ-gene rearrangement.(A) FACS analysis of the in vitro differentiation of thymocytes to the DP stage. DN3 cells from Rag2-/-, Rag2-/-RhoHtg, and Rag2+/+ C57B6 mice were differentiated for 14 days with the stromal cell line TSt-4/Dll-1 in the presence of IL-7. Representative two parameter plots show CD4 versus CD8 staining on cultured thymocytes at the indicated days. Results shown are from one out of three independent experiments. (B) DN3 cells were cultured with vehicle alone or a pharmacological inhibitor of Lck at the indicated concentration. Bar graphs represent the percentages of DP thymocytes compared with the vehicle control group. Data are representative of more than three independent experiments. ***P<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482576&req=5

pone.0131047.g004: Lck activation is required for RhoH transgene-induced DPs generation without TCRβ-gene rearrangement.(A) FACS analysis of the in vitro differentiation of thymocytes to the DP stage. DN3 cells from Rag2-/-, Rag2-/-RhoHtg, and Rag2+/+ C57B6 mice were differentiated for 14 days with the stromal cell line TSt-4/Dll-1 in the presence of IL-7. Representative two parameter plots show CD4 versus CD8 staining on cultured thymocytes at the indicated days. Results shown are from one out of three independent experiments. (B) DN3 cells were cultured with vehicle alone or a pharmacological inhibitor of Lck at the indicated concentration. Bar graphs represent the percentages of DP thymocytes compared with the vehicle control group. Data are representative of more than three independent experiments. ***P<0.001.
Mentions: To address whether generation of DPs in Rag-/-RhoHtg mice was thymocyte intrinsic, we next tested using an in vitro differentiation culture system. Sorted DN3 cells from wild-type mice were able to differentiate into DPs in vitro in a week on monolayers of Notch ligand-expressing stromal cells (TSt-4/Dll-1), while DN3 cells from Rag2-/- mice could not (Fig 4A). We found that sorted DN3 cells from Rag2-/-RhoHtg mice could successfully differentiate into DP cells (Fig 4A), demonstrating that these DPs were truly differentiated from DN3 cells, and that this transition was thymocyte intrinsic. Furthermore, this in vitro DPs generation was strongly inhibited by the addition of Lck inhibitor (Fig 4B, S6 Fig), indicating that RhoH-transgene induced generation of DPs was dependent on Lck activity.

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