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Signaling in Lymphocyte Development
Summary: Ken Murphy's laboratory studies the receptors and signaling pathways that control the development and activation of lymphocytes.
The vertebrate immune system has innate and adaptive components, differing in the diversity of the molecular targets they recognize. Innate immunity is triggered by relatively few conserved molecular features derived from pathogens. Adaptive immunity is concerned with recognizing a greatly increased spectrum of molecules, through the generation of a highly diverse set of antigen receptors expressed on T and B lymphocytes. Our laboratory is interested in understanding the regulatory mechanisms controlling development and activation of these lymphocytes. We are particularly interested in identifying the receptors and signaling pathways that control T cell differentiation toward distinct effector subsets that mediate resistance against different classes of pathogens and control self-tolerance.
Our laboratory identified several of the critical pathways controlling peripheral CD4+ T cell differentiation during the immune response. Development of the T helper type 1 (Th1) response, necessary for effective resistance to many viral and bacterial pathogens, is regulated by several cytokines, including interferon-γ (IFN-γ) and interleukin-12 (IL-12), whose receptors signal through the JAK/STAT pathway. We have therefore focused much of our past and current efforts on characterizing the IL-12 signaling pathway. IL-12–induced Th1 development requires activation of the transcription factor STAT4. In studying IL-12 signaling, we have uncovered a previously unrecognized structural aspect of the regulation of STAT4 that we believe has also revealed an unrecognized general feature of the entire STAT family of transcription factors.
Cytokine receptors were thought to act on latent STAT monomers by phosphorylation of a conserved tyrosine residue, allowing formation of the active STAT dimer via reciprocal SH2-domain interactions. We recently discovered a functional role for a domain in STATs, the N-terminal protein interaction domain (N-domain), which is present in all STAT family members but whose function was unknown. We showed that the STAT4 N-domain forms a dimer and that the N-domain causes the association of two nonphosphorylated monomers, which takes place prior to any interaction with cytokine receptors. Furthermore, the nonphosphorylated dimer is the preferred substrate for interaction with and activation by the cytokine receptor. More generally, we found that N-domains of each of the other STAT family members also form such dimers. Importantly, this dimerization is homotypic, forming only between the N-domains of identical STAT monomers and not between heterologous STATs. This suggests a mechanism to enforce pair rules in STAT activation, thereby enhancing the specificity of STAT activation and signaling.
In analyzing the hierarchy of control between the transcription factors acting in Th1 and Th2 development, we found that IL-4 activates STAT6 to induce the transcription factor GATA-3 a threshold controlled by inhibitors such as FOG (friend of GATA)-1. When elevated beyond this threshold, transcriptional autoactivation leads to a positive-feedback cycle that maintains GATA-3 expression independently of IL-4 and STAT6. GATA-3, independently of STAT6, induces the defining properties of the Th2 phenotype: high IL-4 and low IFN-γ production, expression of c-Maf, and DNase I hypersensitivity within the
IL-4 locus. Since GATA-3 also inhibits expression of the IL-12 receptor–signaling subunit (IL-12Rβ2), this feedback also blocks subsequent activation of STAT4 through IL-12. In this way, GATA-3 autoactivation stabilizes Th2 development, providing a mechanism for Th2 phenotypic memory.
We have also attempted to identify new transcriptional targets of the signals that direct CD4+ T cell differentiation in the periphery. In some cases, these efforts have led us in unanticipated directions. Recently, we identified a novel cell surface immunoreceptor that is selectively expressed by Th1 clones and whose expression is exclusive to the immune system. This receptor, termed B and T lymphocyte attenuator (BTLA), is expressed by activated T and B cells and acts as an inhibitor of cellular activation. BTLA is a member of a larger class of immunoglobulin (Ig) family member cell surface receptors that regulate the strength of the immune responses, collectively called "costimulatory" receptors. BTLA joins the group of two other receptors, CTLA-4 (cytotoxic T lymphocyte–associated antigen 4) and PD-1 (programmed death 1), that are inhibitory rather than activating in nature. In mice, BTLA deficiency increases the strength of T and B cell–dependent immune responses, which can be manifested also by an increased susceptibility to autoimmunity and acquisition of autoantibodies. BTLA is polymorphic in both mice and in humans. We are attempting to determine whether human BTLA polymorphisms are related to naturally occurring autoimmune diseases.
Until recently, this class of Ig family costimulatory receptors, all members of the Ig superfamily, was thought to interact exclusively with ligands that are also members of the Ig superfamily. In cloning the ligand for BTLA, however, we were surprised to identify a member of the tumor necrosis factor (TNF) receptor (TNFR) superfamily known as herpesvirus entry mediator (HVEM). As a TNFR family member, HVEM binds members of the TNF family, specifically lymphotoxin-α (LT-α) and LIGHT. However, the Ig domain of BTLA also interacts with the most membrane distal CRD1 (cysteine-rich domain) region of HVEM, distinct from the regions interacting with LT-α and LIGHT. HVEM binding to BTLA induces the tyrosine phosphorylation of BTLA within its cytoplasmic domain and transmits an inhibitory signal in lymphocytes expressing BTLA. This interaction is unique structurally and provides a novel reverse signaling pathway employing the TNFR family. We are exploring whether the BTLA-HVEM interaction is one of a broader class of unrecognized interactions between the Ig and TNFR superfamily of receptors.
Ongoing studies also include identification of additional signaling and transcriptional pathways that regulate peripheral T cell differentiation. We have identified novel transcriptional participants in the regulation of the expression of cytokines in Th cells and have identified transcriptional targets of the Ets-related molecule ERM, a transcription factor induced by IL-12 in T cells. Additionally, ERM-knockout mice also manifest several phenotypes outside the immune system, including interruption of the spermatogonia stem cell niche in male testis, causing a Sertoli cell–only syndrome due to the failure of stem cell renewal that ensues at 4 weeks of age. We are characterizing this process in greater detail. Our finding that BTLA is highly expressed by anergic CD4+ T cells has motivated us to broaden our scope regarding peripheral T cell differentiation to include consideration of anergic/regulatory CD4+ T cell subsets, which are critical for maintaining self-tolerance. We are now generating models for the analysis and characterization of this state of differentiation.
Grants from the National Institutes of Health provided support for the work on Th1 development and IL-4 regulation.
Last updated: January 11, 2006
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