Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice

CD4+ T cells differentiate into subsets that promote immunity or minimize damage to the host. T helper 17 cells (Th17) are effector cells that function in inflammatory responses. T regulatory cells (Tregs) maintain tolerance and prevent autoimmunity by secreting immunosuppressive cytokines and expressing check point receptors. While the functions of Th17 and Treg cells are different, both cell fate trajectories require T cell receptor (TCR) and TGF-β receptor (TGF-βR) signals, and Th17 polarization requires an additional IL-6 receptor (IL-6R) signal. Utilizing high-resolution phosphoproteomics, we identified that both synergistic and additive interactions between TCR, TGF-βR, and IL-6R shape kinase signaling networks to differentially regulate key pathways during the early phase of Treg versus Th17 induction. Quantitative biochemical analysis revealed that CD4+ T cells integrate receptor signals via SMAD3, which is a mediator of TGF-βR signaling. Treg induction potentiates the formation of the canonical SMAD3/4 trimer to activate a negative feedback loop through kinases PKA and CSK to suppress TCR signaling, phosphatidylinositol metabolism, and mTOR signaling. IL-6R signaling activates STAT3 to bind SMAD3 and block formation of the SMAD3/4 trimer during the early phase of Th17 induction, which leads to elevated TCR and PI3K signaling. These data provide a biochemical mechanism by which CD4+ T cells integrate TCR, TGF-β, and IL-6 signals via generation of alternate SMAD3 complexes that control the development of early signaling networks to potentiate the choice of Treg versus Th17 cell fate.     

Mitogenic CD28 signals require the exchange factor Vav1 to enhance TCR signaling at the SLP-76-Vav-Itk signalosome

Almost all physiological T cell responses require costimulation-engagement of the clonotypic TCR with MHC/Ag and CD28 by its ligands CD80/86. Whether CD28 provides signals that are qualitatively unique or quantitatively amplify TCR signaling is poorly understood. In this study, we use superagonistic CD28 Abs, which induce T cell proliferation without TCR coligation, to determine how CD28 contributes to mitogenic responses. We show that mitogenic CD28 signals require but do not activate the proximal TCR components TCRzeta and Zap-70 kinase. In cell lines lacking proximal TCR signaling, an early defect in the CD28 pathway is in phosphorylation of the adaptor molecule SLP-76, which we show is essential for recruitment of the exchange factor Vav leading to Ca(2+) flux and IL-2 production. Point mutations in CD28 that result in diminished Vav phosphorylation also result in defective Ca(2+) flux, IL-2 production, and Tec-kinase phosphorylation. Using Vav1-deficient mice, we further demonstrate the importance of Vav1 for efficient proliferation, IL-2 production, and Ca(2+) flux. Our results indicate that CD28 signals feed into the TCR signaling pathway at the level of the SLP-76 signalosome.     

Galectin-1 induces partial TCR zeta-chain phosphorylation and antagonizes processive TCR signal transduction

Galectin-1 is an endogenous lectin with known T cell immunoregulatory activity, though the molecular basis by which galectin-1 influences Ag specific T cell responses has not been elucidated. Here, we characterize the ability of galectin-1 to modulate TCR signals and responses by T cells with well defined hierarchies of threshold requirements for signaling distinct functional responses. We demonstrate that galectin-1 antagonizes TCR responses known to require costimulation and processive protein tyrosine phosphorylation, such as IL-2 production, but is permissive for TCR responses that only require partial TCR signals, such as IFN-gamma production, CD69 up-regulation, and apoptosis. Galectin-1 binding alone or together with Ag stimulation induces partial phosphorylation of TCR-zeta and the generation of inhibitory pp21zeta. Galectin-1 antagonizes Ag induced signals and TCR/costimulator dependent lipid raft clustering at the TCR contact site. We propose that galectin-1 functions as a T cell "counterstimulator" to limit required protein segregation and lipid raft reorganization at the TCR contact site and, thus, processive and sustained TCR signal transduction. These findings support the concept that TCR antagonism can arise from the generation of an inhibitory pp21zeta-based TCR signaling complex. Moreover, they demonstrate that TCR antagonism can result from T cell interactions with a ligand other than peptide/MHC.     

Lck和Fyn对T细胞发育过程的影响

胸腺中T细胞的发育及次级淋巴组织中成熟T细胞的活化均需要细胞能够对环境信号分子做出适应性的反应。在共刺激分子及细胞因子受体介导的信号参与下通过TCR(T cell receptor)及其辅助受体CD4和CD8与MHC/抗原肽复合物相互作用,可以诱导TCR信号通路激活并最终导致T细胞免疫反应的发生。Src家族激酶Lck(Lymphocyte-specific protein tyrosine kinase)和Fyn(Proto-oncogene tyrosine-protein kinase)的激活是启动TCR信号通路的关键因素。在T细胞的发育、阳性选择、初始T细胞的外周存活及由淋巴细胞缺失诱导的细胞增殖中都起着关键性的作用。研究显示,虽然这两种信号分子紧密相关,但在某些条件下Lck发挥着比Fyn更重要的作用,并且Fyn仅可以补充Lck的部分功能。文章针对这两个激酶在T细胞发育的整个过程中的作用机制进行了论述。     

Lck和Fyn对T细胞发育过程的影响

胸腺中T细胞的发育及次级淋巴组织中成熟T细胞的活化均需要细胞能够对环境信号分子做出适应性的反应。在共刺激分子及细胞因子受体介导的信号参与下通过TCR(T cell receptor )及其辅助受体CD4和CD8与MHC/抗原肽复合物相互作用, 可以诱导TCR信号通路激活并最终导致T细胞免疫反应的发生。Src家族激酶Lck(Lymphocyte-specific protein tyrosine kinase)和Fyn (Proto-oncogene tyrosine-protein kinase)的激活是启动TCR信号通路的关键因素。在T细胞的发育、阳性选择、初始T细胞的外周存活及由淋巴细胞缺失诱导的细胞增殖中都起着关键性的作用。研究显示, 虽然这两种信号分子紧密相关, 但在某些条件下Lck发挥着比Fyn更重要的作用, 并且Fyn仅可以补充Lck的部分功能。文章针对这两个激酶在T细胞发育的整个过程中的作用机制进行了论述。     

Essential role of NF-kappa B-inducing kinase in T cell activation through the TCR/CD3 pathway

NF-kappa B-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-beta receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-kappa B activity in both mature and immature T cells; the impaired NF-kappa B activity in mature T cells was also associated with the failure of maintenance of activated NF-kappa B. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-kappa B activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.     

Orally tolerant CD4 T cells respond poorly to antigenic stimulation but strongly to direct stimulation of intracellular signaling pathways

The response of splenic CD4 T cells from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic mice after long-term feeding of a diet containing this antigen was examined. These CD4 T cells exhibited a decreased response to OVA peptide stimulation, in terms of proliferation, interleukin-2 secretion, and CD40 ligand expression, compared to those from mice fed a control diet lacking OVA, demonstrating that oral tolerance of T cells had been induced through oral intake of the antigen. We investigated the intracellular signaling pathways, which were Ca/CN cascade and Ras/MAPK cascade, of these tolerant CD4 T cells using phorbol-12-myristate-13-acetate (PMA) and ionomycin, which are known to directly stimulate these pathways. In contrast to the decreased response to TCR stimulation by OVA peptide, it was shown that the response of splenic CD4 T cells to these reagents in the state of oral tolerance was stronger. These results suggest that splenic CD4 T cells in the state of oral tolerance have an impairment in signaling, in which signals are not transmitted from the TCR to downstream signaling pathways, and have impairments in the vicinity of TCR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Proteomic characterization of plasma membrane-proximal T cell activation responses

Early downstream responses of T lymphocytes following T cell antigen receptor (TCR) activation are mediated by protein complexes that assemble in domains of the plasma membrane. Using stable isotope labeling with amino acids in cell culture and mass spectrometry, we quantitatively related the proteome of αCD3 immunoisolated native TCR signaling plasma membrane domains to that of control plasma membrane fragments not engaged in TCR signaling. Proteins were sorted according to their relative enrichment in isolated TCR signaling plasma membrane domains, identifying a complex protein network that is anchored in the vicinity of activated TCR. These networks harbor widespread mediators of plasma membrane-proximal T cell activities, including propagation, balancing, and attenuation of TCR signaling, immune synapse formation, as well as cytoskeletal arrangements relative to TCR activation clusters. These results highlight the unique potential of systematic characterizations of plasma membrane-proximal T cell activation proteome in the context of its native lipid bilayer platform.     

Defective T cell receptor-mediated signal transduction in memory CD4 T lymphocytes exposed to superantigen or anti-T cell receptor antibodies

Lymphocytes must promote protective immune responses while still maintaining self-tolerance. Stimulation through the T cell receptor (TCR) can lead to distinct responses in naive and memory CD4 T cells. Whereas peptide antigen stimulates both naive and memory T cells, soluble anti-CD3 antibodies and bacterial superantigens stimulate only naive T cells to proliferate and secrete cytokines. Further, superantigens, like staphylococcal enterotoxin B (SEB), cause memory T cells to become anergic while soluble anti-CD3 does not. In the present report, we show that signal transduction through the TCR is impaired in memory cells exposed to either anti-CD3 or SEB. A block in signaling leads to impaired activation of the kinase ZAP-70 so that downstream signals and cell proliferation do not occur. We further show that the signaling defect is unique to each agent. In anti-CD3-treated memory T cells, the src kinase Lck is only transiently activated and does not phosphorylate and activate ZAP-70. In SEB-treated memory T cells, ZAP-70 does not interact with the TCR/CD3 complex to become accessible to Lck. Finally, we provide evidence that alternative signaling pathways are initiated in SEB-treated memory cells. Altered signaling, indicated by an elevation in activity of the src kinase Fyn, may be responsible for memory cell anergy caused by SEB. Thus, differentiation of naive T cells into memory cells is accompanied by alterations in TCR-mediated signaling that can promote heightened recall immunity or specific tolerance.     

IL-2 gene inducibility in T cells before T cell receptor expression. Changes in signaling pathways and gene expression requirements during intrathymic maturation

The ability to express the growth hormone IL-2 upon stimulation gives T lymphocytes one of their major effector functions in the immune system. IL-2 is apparently synthesized only by T cells, and only by a subset of T cells which constitutes a "helper" class. It remains unknown how and when the IL-2-producing lineage becomes distinct from other functional effector lineages. We have therefore examined immature T cell precursors to determine when IL-2 inducibility is acquired in relation to other maturation events, such as expression of an Ag-binding TCR, which is suspected to play an influential role in the determination of subclass commitment. In mature T cells, IL-2 is inducible via agonists of the phosphoinositide pathway, a network of signaling mediators shared by a wide variety of metazoan cell types. The universality of this activation pathway makes it seem less likely, a priori, to be a target of developmental change than the intrinsic susceptibility to induction of the IL-2 locus. However, our results presented here refute this expectation. In this report, we show that both TCR+ cells and pre-T cells too immature to express TCR can be induced to express IL-2 at high levels. The induction requirements for IL-2 expression, however, are different in TCR- and TCR+ cells. Even by using Ca2+ ionophore and phorbol ester to bypass the requirement for the TCR in cell activation, the TCR- cells also require the presence of the polypeptide hormone IL-1. By contrast, TCR+ mature cells not only can express IL-2 without IL-1, but also show no response to IL-1 when Ca2+ ionophore and phorbol ester are present. IL-1-dependent IL-2 producers appear in the thymus of repopulating radiation chimeras before "mature" (TCR+) T cells, whereas IL-1-independent IL-2 production is found only afterward. Thus, IL-2 inducibility per se apparently precedes TCR expression and all TCR-associated fate determination events. However, developmental alteration of signal transduction pathways may play a vital regulatory role in the later allocation of particular functional responses to appropriate lineages of T cells.     

The space and time frames of T cell activation at the immunological synapse

The selective recognition of antigenic peptides by T cells requires the spatio/temporal integration of a panoply of molecular triggers. The space frame of T cell antigen receptors (TCR) interaction with peptide/MHC complexes (pMHC) displayed by antigen presenting cells is delineated by the micrometer-scale area of the immunological synapse. The time frame of T cell stimulation is governed by a series of short TCR-pMHC interactions that are integrated into sustained signaling leading to productive activation. We discuss here how approaching antigen recognition from the time and space angles is key to the comprehension of the puzzling process of T cell activation.     

A role for TCR affinity in regulating naive T cell homeostasis

Homeostatic signals that control the overall size and composition of the naive T cell pool have recently been identified to arise from contact with self-MHC/peptide ligands and a cytokine, IL-7. IL-7 presumably serves as a survival factor to keep a finite number of naive cells alive by preventing the onset of apoptosis, but how TCR signaling from contact with self-MHC/peptide ligands regulates homeostasis is unknown. To address this issue, murine polyclonal and TCR-transgenic CD8+ cells expressing TCR with different affinities for self-MHC/peptide ligands, as depicted by the CD5 expression level, were analyzed for their ability to respond to and compete for homeostatic factors under normal and lymphopenic conditions. The results suggest that the strength of the TCR affinity determines the relative "fitness" of naive T cells to compete for factors that support cell survival and homeostatic proliferation.     

Strength of TCR-peptide/MHC interactions and in vivo T cell responses

The TCR can detect subtle differences in the strength of interaction with peptide/MHC ligand and transmit this information to influence downstream events in T cell responses. Manipulation of the factor commonly referred to as TCR signal strength can be achieved by changing the amount or quality of peptide/MHC ligand. Recent work has enhanced our understanding of the many variables that contribute to the apparent cumulative strength of TCR stimulation during immunogenic and tolerogenic T cell responses. In this review, we consider data from in vitro studies in the context of in vivo immune responses and discuss in vivo consequences of manipulation of strength of TCR stimulation, including influences on T cell-APC interactions, the magnitude and quality of the T cell response, and the types of fate decisions made by peripheral T cells.     

CD8+ T cell activation is governed by TCR-peptide/MHC affinity, not dissociation rate

Binding of peptide/MHC (pMHC) complexes by TCR initiates T cell activation. Despite long interest, the exact relationship between the biochemistry of TCR/pMHC interaction (particularly TCR affinity or ligand off-rate) and T cell responses remains unresolved, because the number of complexes examined in each independent system has been too small to draw a definitive conclusion. To test the current models of T cell activation, we have analyzed the interactions between the mouse P14 TCR and a set of altered peptides based on the lymphocytic choriomeningitis virus epitope gp33-41 sequence bound to mouse class I MHC D(b). pMHC binding, TCR-binding characteristics, CD8+ T cell cytotoxicity, and IFN-gamma production were measured for the peptides. We found affinity correlated well with both cytotoxicity and IFN-gamma production. In contrast, no correlation was observed between any kinetic parameter of TCR-pMHC interaction and cytotoxicity or IFN-gamma production. This study strongly argues for an affinity threshold model of T cell activation.     

Quantitative analysis of phosphotyrosine signaling networks triggered by CD3 and CD28 costimulation in Jurkat cells

The mechanism by which stimulation of coreceptors such as CD28 contributes to full activation of TCR signaling pathways has been intensively studied, yet quantitative measurement of costimulation effects on functional TCR signaling networks has been lacking. In this study, phosphotyrosine networks triggered by CD3, CD28, or CD3 and CD28 costimulation were analyzed by site-specific quantitative phosphoproteomics, resulting in identification of 101 tyrosine and 3 threonine phosphorylation sites and quantification of 87 sites across four cell states. As expected, CD3 stimulation induced phosphorylation of CD3 chains and upstream components of TCR pathways such as Zap70, while CD28 stimulation induced phosphorylation of CD28, Vav-1, and other adaptor proteins including downstream of tyrosine kinase 1, Grb2-associated protein 2 (Grap2), and Wiskott-Aldrich syndrome protein. CD3 and CD28 costimulation induced a complex response including decreased threonine phosphorylation in the ERK1 and ERK2 activation loops and increased phosphorylation of selected tyrosine sites on ERK1/2, p38, phospholipase C-gamma, Src homology 2 domain-containing transforming protein 1, Grap2, and Vav-1, potentiating T cell activation. Hierarchical clustering and self-organizing maps were used to identify modules of coregulated phosphorylation sites within the network. Quantitative information in our study suggests quantitative and qualitative contribution by costimulation of CD28 on CD3-stimulated TCR signaling networks via enhanced phosphorylation of phospholipase C-gamma/Src homology 2 domain-containing transforming protein 1/Grap2/Vav-1 and their effects on downstream components including MAPKs.