Thymic-specific regulation of TCR signaling by Tespa1

Double-positive(DP)thymocytes undergo positive selection to become mature single-positive CD4+and CD8+T cells in response to T cell receptor(TCR)signaling.Unlike mature T cells,DP cells must respond to low-affinity self-peptide-MHC ligands before full upregulation of their surface TCR expression can occur.Thus,DP thymocytes must be more sensitive to ligands than mature T cells.A number of molecules have been found that are able to enhance the strength of the TCR signal to facilitate positive selection.However,almost all of these molecules are also active in mature T cells.Themis(thymocyte expressed molecule involved in selection)and Tespa1(thymocyte expressed positive selection associated 1)are two recently discovered molecules essential for optimal TCR signaling and thymocyte development.A deficiency in both molecules leads to defects in positive selection.Here,we compared the relative contributions of Themis and Tespa1 to positive selection in thymocytes.We show that Tespa1 deficiency led to more limited and specific gene expression profile changes in cells undergoing positive selection.In mixed bone marrow transfer experiments,Tespa1^(−/−)cells showed more severe defects in thymocyte development than Themis^(−/−)cells.However,Tespa1^(−/−)cells showed a substantial degree of homeostatic expansion and became predominant in the peripheral lymphoid organs,suggesting that Tespa1 is a thymic-specific TCR signaling regulator.This hypothesis is further supported by our observations in Tespa1 conditional knockout mice,as Tespa1 deletion in peripheral T cells did not affect TCR signaling or cell proliferation.The different regulatory effects of Tespa1 and Themis are in accordance with their nonredundant roles in thymocyte selection,during which Tespa1 and Themis double knockouts showed additive defects.

Zebl represses TCR signaling, promotes the proliferation of T cell progenitors and is essential for NK1.1^(+) T cell development

T cell development proceeds un der the in fluence of a n etwork of transcription factors(TFs).The precise role of Zeb1,a member of this network,remains unclear.Here,we report that Zeb1 expression is induced early during T cell development in CD4^(-)CD8^(-)double-negative(DN)stage 2(DN2).Zeb1 expression was further increased in the CD4^(+)CD8^(+)double-positive(DP)stage before decreasing in more mature T cell subsets.We performed an exhaustive characterization of T cells in Cellophane mice that bear Zeb 7 hypomorphic mutations.The Zebl mutation profoundly affected all thymic subsets,especially DN2 and DP cells.Zeb1 promoted the survival and proliferation of both cell populations in a cell-intrinsic manner.In the periphery of Cellophane mice,the number of conventional T cells was near normal,but invariant NKT cells,NK1.1^(+)γδ T cells and Ly49^(+)CD8 T cells were virtually absent.This suggested that Zebl regulates the development of unconventional T cell types from DP progenitors.A transcriptomic analysis of WT and Cellophane DP cells revealed that Zebl regulated the expression of multiple genes involved in the cell cycle and TCR sign aling,which possibly occurred in cooperation with Tcf1 and Heb.In deed,Cellophane DP cells displayed stron ger signaling than WT DP cells upon TCR engagement in terms of the calcium respons巳phosphorylation events,and expression of early genes.Thus,Zebl is a key regulator of the cell cycle and TCR signaling during thymic T cell development.We propose that thymocyte selection is perturbed in Zeb7-mutated mice in a way that does not allow the survival of unconventional T cell subsets.

Novel function of perforin in negatively regulating CD4^＋ T cell activation by affecting calcium signaling

Perforin is a pore-forming protein engaged mainly in mediating target T cell death and is employed by cytotoxic T lymphocytes （CTLs） and natural killer cells. However, whether it also plays a role in conventional CD4^＋ T cell function remains unclear. Here we report that in perforin-deficient （PKO） mice, CD4^＋ T cells are hyperproliferative in response to T cell receptor （TCR） stimulation. This feature of hyperproliferation is accompanied by the enhancement both in cell division and in IL-2 secretion. It seems that the perforin deficiency does not influence T cell development in thymus spleen and lymph node. In vivo, perforin deficiency results in increased antigen-specific T cell proliferation and antibody production. Furthermore, PKO mice are more susceptible to experimental autoimmune uveitis. To address the molecular mechanism, we found that after TCR stimulation, CD4^＋ T cells from PKO mice display an increased intracellular calcium flux and subsequently enhance activation of transcription factor NFAT1. Our results indicate that perforin plays a negative role in regulating CD4^＋ T cell activation and immune response by affecting TCR-dependent Ca^2＋ signaling.

Trichosanthin inhibits T cell activation by interfering with the recruitment of ZAP-70 to CD3 ζchain

Plant protein Trichosanthin (Tk) has been shown in our previous experiments to suppress antigenic response of T cells. Here we explored its inhibitory mechanisms on the proliferation of human Jurkat leukemia T cell triggered by anti-CD3 McAb. By examination of tyrosine phosphorylation of cell lysate, we were able to show that Tk could interfere with the PTK-related activity in the TCR/CD3initiated signal transduction in addition to blocking the phosphorylation of PKC. As shown in our experiment,the expression intensity of ZAP-70, a kind of protein tyrosine kinase, was not changed but its phosphorylation could be inhibited. When physical link between CD3(chain and ZAP-70 was further examined by using coimmunoprecipitation after pluse-treatment of the cell line with Tk, the anti-CD3 McAb-induced recruitment of ZAP70 to CD3 ζ chain was observed to be blocked in some extent. This may account for, at least in part, how Trichosanthin was able to inhibit the TCR-triggered T cell proliferation.

Selective Function of PKC-θin T cells

T cell activation is a critical process in initiating adaptive immune response since only through this process the naive antigen specific T cells differentiate into armed effector T cells that mediate the actual immune response. During T cell activation, naive T cells undergo clonal expansion and acquire the capability to kill target cells infected with pathogens or produce cytokines essential for regulating immune response. Inappropriate activation or inactivation of T cells leads to autoimmunity or severe immunodeficiencies. PKC-θ is selectively expressed in T cells and required for mediating T cell activation process. Mice deficient in PKC-θ exhibit defects in T cell activation, survival and activation-induced cell death. PKC-θ selectively translocates to immunological synapse and mediates the signals required for activation of NF-κB, AP1 and NFAT that are essential for T cell activation. Furthermore, PKC-θ^-│- mice displayed multiple defects in the development of T cell-mediated immune responses in vivo. PKC-θ is thus a critical molecule that regulates T cell function at multiple stages in T cell-mediated immune responses in vivo.