FCRL1 Regulates B Cell Receptor-Induced ERK Activation through GRB2.

Regulation of BCR signaling has important consequences for generating effective Ab responses to pathogens and preventing production of autoreactive B cells during development. Currently defined functions of Fc receptor-like (FCRL) 1 include positive regulation of BCR-induced calcium flux, proliferation, and Ab production; however, the mechanistic basis of FCRL1 signaling and its contributions to B cell development remain undefined. Molecular characterization of FCRL1 signaling shows phosphotyrosine-dependent associations with GRB2, GRAP, SHIP-1, and SOS1, all of which can profoundly influence MAPK signaling. In contrast with previous characterizations of FCRL1 as a strictly activating receptor, we discover a role for FCRL1 in suppressing ERK activation under homeostatic and BCR-stimulated conditions in a GRB2-dependent manner. Our analysis of B cells in Fcrl1 -/- mice shows that ERK suppression by FCRL1 is associated with a restriction in the number of cells surviving splenic maturation in vivo. The capacity of FCRL1 to modulate ERK activation presents a potential for FCRL1 to be a regulator of peripheral B cell tolerance, homeostasis, and activation.

Characterization of GAB3 and its association with NNV resistance in the Asian seabass.

Understanding the functions of genes related to disease resistance and identifying polymorphisms in these genes are essential in molecular breeding for disease resistance. Viral nervous necrosis (VNN) is one of the major diseases in the Asian seabass, Lates calcarifer. Our previous works on QTL mapping, GWAS and cell-line transcriptome analysis of the Asian seabass after NNV challenge revealed that the gene GAB3 might be a candidate gene for VNN resistance. In this study, we cloned and characterized GAB3, and identified SNPs in the gene of the Asian seabass. The cDNA of the gene was 2165 bp, containing an ORF of 1674 bp encoding 557 amino acids. The gene consisted of 10 exons and nine introns. It was ubiquitously expressed in normal fish. An analysis of the association between two SNPs in the second intron and NNV resistance in 1035 fish descended from 43 families revealed that the two SNPs were significantly associated with VNN resistance. After NNV infection, the expression of GAB3 was significantly increased in the brain, spleen, muscle and gut, and was suppressed in the liver. The GAB3 protein was localized in the nucleus. Overexpression of GAB3 with specific GAB3-pcDNA was positively correlated to increased viral RNA and titer in NNV-infected Asian seabass cells. Our study provides new evidence to support that GAB3 may be an important gene related to NNV resistance. In addition, the SNPs provide DNA markers for the selection of candidate genes resistance to NNV at the juvenile stage of Asian seabass.

Murine eosinophil development and allergic lung eosinophilia are largely dependent on the signaling adaptor GRB2.

Eosinophils are innate effector cells associated with allergic inflammation. Their development and survival is largely dependent on IL-5 and the common beta chain (ßc ) of the IL-5 receptor that serves as docking site for several proteins that mediate down-stream signaling cascades including JAK/STAT, PI3 kinase, NFκB, and RAS-MAP kinase pathways. The relative contribution of these signaling pathways for eosinophil development and homeostasis in vivo are poorly understood. Here, we investigated the role of GRB2, an adaptor protein that binds to ßc and other proteins and elicits the RAS-MAP kinase pathway. By using GRB2 inhibitors and inducible deletion of the Grb2 gene in mouse eosinophils we demonstrate that GRB2 plays a critical role for development of eosinophils from bone marrow precursors. Furthermore, Aspergillus fumigatus-induced allergic lung eosinophilia was significantly reduced in mice with induced genetic deletion of Grb2. Our results indicate that GRB2 is important for eosinophil development in steady-state conditions and during allergic inflammation. Based on these findings pharmacologic GRB2 inhibitors may have the potential to dampen tissue eosinophilia in various eosinophil-associated diseases.

Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation.

The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT microcluster formation and vesicle delivery, using lattice light sheet microscopy to image a T cell from the earliest point of activation. A kinetic lag occurs between LAT microcluster formation and vesicular pool recruitment to the synapse. Correlative 3D light and electron microscopy show an absence of vesicles at microclusters at early times, but an abundance of vesicles as activation proceeds. Using TIRF-SIM to look at the activated T-cell surface with high resolution, we capture directed vesicle movement between microclusters on microtubules. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters.

An asthma-associated IL4R variant exacerbates airway inflammation by promoting conversion of regulatory T cells to TH17-like cells.

Mechanisms by which regulatory T (Treg) cells fail to control inflammation in asthma remain poorly understood. We show that a severe asthma-associated polymorphism in the gene encoding the interleukin (IL)-4 receptor alpha chain (Il4ra(R576)) promotes conversion of induced Treg (iTreg) cells toward a T helper 17 (TH17) cell fate. This skewing is mediated by the recruitment by IL-4Rα(R576) of the growth-factor-receptor-bound protein 2 (GRB2) adaptor protein, which drives IL-17 expression by activating a pathway that involves extracellular-signal-regulated kinase, IL-6 and the transcription factor STAT3. Treg cell-specific deletion of genes that regulate TH17 cell differentiation, including Il6ra and RAR-related orphan receptor gamma (Rorc), but not of Il4 or Il13, prevented exacerbated airway inflammation in mice expressing Il4ra(R576) (hereafter referred to as Il4ra(R576) mice). Furthermore, treatment of Il4ra(R576) mice with a neutralizing IL-6-specific antibody prevented iTreg cell reprogramming into TH17-like cells and protected against severe airway inflammation. These findings identify a previously unknown mechanism for the development of mixed TH2-TH17 cell inflammation in genetically prone individuals and point to interventions that stabilize iTreg cells as potentially effective therapeutic strategies.

IL-8 Alterations in HIV-1 Infected Children With Disease Progression.

Disease progression in HIV-1 infected children is faster than in adults. Less than 5% of the infected children maintain stable CD4 counts beyond 7 years of infection and are termed long-term nonprogressors (LTNPs). Delineating the host immune response in antiretroviral naïve (ART) and treated HIV-1 infected children at different disease stages will help in understanding the immunopathogenesis of the disease.A total of 79 asymptomatic, perinatally HIV-1 infected children (50 ART naïve and 29 ART treated) and 8 seronegative donors were recruited in this study. T- and B-cell activation PCR arrays were performed from the cDNA, using total RNA extracted from the peripheral blood mononuclear cells (PBMCs) of 14 HIV-1 infected children at different stages of the disease. The differentially expressed genes were identified. Quantitative RT-PCR was performed for the (interleukin-8) IL-8 gene and its transcriptional mediators, that is, SHP2, GRB2, and IL-8R (IL-8 receptor/CXCR1). Plasma levels of IL-8 were measured by flow cytometry.Gene array data revealed a higher expression of IL-8 in the ART naïve HIV-1 infected progressors and in ART nonresponders than LTNPs and ART responders, respectively. Quantitative RT-PCR analysis demonstrated a significant higher expression of IL-8 (P < 0.001), its receptor CXCR1 (P = 0.03) and the upstream signaling molecule SHP2 (P = 0.04) in the progressors versus LTNPs. Plasma levels of IL-8 were significantly higher in progressors versus LTNPs (P < 0.001), and ART nonresponders versus ART responders (P < 0.001). A significant negative correlation of plasma levels of IL-8 with CD4 counts (cells/µL) was observed in HIV-1 infected ART naïve subjects (r = -0.488; P < 0.001), while the IL-8 levels positively correlated with viral load in the ART treated children (r = 0.5494; P < 0.001). ART naïve progressors on follow up demonstrated a significant reduction in the mRNA expression (P = 0.05) and plasma levels of IL-8 (P = 0.05) post 6 months of ART initiation suggesting the beneficial role of ART therapy in reducing inflammation in infected children.Our data suggest that IL-8 may serve as a potential prognostic marker in adjunct with CD4 counts to monitor disease progression in the HIV-1 infected children and the efficacy of ART.

DNAM-1 controls NK cell activation via an ITT-like motif.

DNAM-1 (CD226) is an activating receptor expressed on natural killer (NK) cells, CD8(+) T cells, and other immune cells. Upon recognition of its ligands, CD155 and CD112, DNAM-1 promotes NK cell-mediated elimination of transformed and virus-infected cells. It also has a key role in expansion and maintenance of virus-specific memory NK cells. Herein, the mechanism by which DNAM-1 controls NK cell-mediated cytotoxicity and cytokine production was elucidated. Cytotoxicity and cytokine production triggered by DNAM-1 were mediated via a conserved tyrosine- and asparagine-based motif in the cytoplasmic domain of DNAM-1. Upon phosphorylation by Src kinases, this motif enabled binding of DNAM-1 to adaptor Grb2, leading to activation of enzymes Vav-1, phosphatidylinositol 3' kinase, and phospholipase C-γ1. It also promoted activation of kinases Erk and Akt, and calcium fluxes. Although, as reported, DNAM-1 promoted adhesion, this function was signal-independent and insufficient to promote cytotoxicity. DNAM-1 signaling was also required to enhance cytotoxicity, by increasing actin polymerization and granule polarization. We propose that DNAM-1 promotes NK cell activation via an immunoreceptor tyrosine tail (ITT)-like motif coupling DNAM-1 to Grb2 and other downstream effectors.

Absence of both Sos-1 and Sos-2 in peripheral CD4(+) T cells leads to PI3K pathway activation and defects in migration.

Sos-1 and Sos-2 are ubiquitously expressed Ras-guanine exchange factors involved in Erk-MAP kinase pathway activation. Using mice lacking genes encoding Sos-1 and Sos-2, we evaluated the role of these proteins in peripheral T-cell signaling and function. Our results confirmed that TCR-mediated Erk activation in peripheral CD4(+) T cells does not depend on Sos-1 and Sos-2, although IL-2-mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos-1/2dKO CD4(+) T cells upon TCR and IL-2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL-2 stimulation in Sos-1/2dKO CD4(+) T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos-1/2dKO T cells and a subsequent impairment in T-cell migration.

Molecular cloning and characterization of growth factor receptor bound-protein in Clonorchis sinensis.

BACKGROUND: Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. CONCLUSION: Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths.

Paired immunoglobulin-like receptor A is an intrinsic, self-limiting suppressor of IL-5-induced eosinophil development.

Eosinophilia is a hallmark characteristic of T helper type 2 (TH2) cell-associated diseases and is critically regulated by the central eosinophil growth factor interleukin 5 (IL-5). Here we demonstrate that IL-5 activity in eosinophils was regulated by paired immunoglobulin-like receptors PIR-A and PIR-B. Upon self-recognition of ß2-microglobulin (ß2M) molecules, PIR-B served as a permissive checkpoint for IL-5-induced development of eosinophils by suppressing the proapoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway. PIR-B-deficient bone marrow eosinophils underwent compartmentalized apoptosis, resulting in decreased blood eosinophilia in naive mice and in mice challenged with IL-5. Subsequently, Pirb(-/-) mice displayed impaired aeroallergen-induced lung eosinophilia and induction of lung TH2 cell responses. Collectively, these data uncover an intrinsic, self-limiting pathway regulating IL-5-induced expansion of eosinophils, which has broad implications for eosinophil-associated diseases.

Activation of integrin ß1-focal adhesion kinase-RasGTP pathway plays a critical role in TGF beta1-induced podocyte injury.

The depletion of glomerular podocytes is the key mechanism of glomerulosclerosis and progressive renal failure. Transforming growth factor-ß (TGFß) is a central mediator of signaling networks that control a diverse set of cellular processes, such as cell proliferation, differentiation, and apoptosis. Though many key events in TGFß1 signaling have been documented at cellular and molecular level in podocytes, the complete effects of TGFß1 on podocyte integrity are still elusive. In this study, the function of adhesion protein integrin ß1, focal adhesion kinase (FAK), and a small GTPase Ras was explored in TGFß1-induced podocyte injury. In cultured mouse podocyte, caspase 3-positive cells were counted by flow cytometry to evaluate podocyte damage at different time points after TGFß1 treatment. Immunoblotting assay showed that integrin ß1, FAK, Src kinase, and an adaptor protein Grb2 were activated rapidly after TGFß1 stimulation. Active Ras Pull-Down assay revealed that the active Ras (GTP-bound Ras) level was upregulated in TGFß1-treated cell. Immunoprecipitation results displayed that TGFß1 enhanced the complex formation of integrin ß1, FAK and Src kinase, as well as FAK, Grb2 and Ras. The FAK inhibitor TAE226 and the specific knockdown of Grb2 remarkably alleviated TGFß1-induced podocyte apoptosis. The activation of p38MAPK and Erk1/2, and the nuclear translocation of NFκB(p65) were increased evidently in TGFß1-treated cell, which could be dramatically prohibited by the application of the p38MAPK inhibitor SB202190 and the Ras inhibitor FPT Inhibitor III. The Src kinase inhibitor PP2 obviously prevented the activation of FAK and Ras, as well as the translocation of NFκB(p65) from cytoplasm to nuclei. The PP2, FPT Inhibitor III, and SB202190 significantly decreased TGFß1-induced podocyte apoptosis. Taken together, these data demonstrated that the activation of integrin ß1/Src/FAK and Grb2/RasGTP should be responsible for TGFß1-induced podocyte damage through the p38MAPK and Erk1/2-mediated nuclear translocation of NFκB(p65).

A deficient translocation of CD3ζ, ZAP-70 and Grb2 to lipid raft, as a hallmark of defective adaptive immune response during chronic hepatitis B infection.

Hepatitis B is considered to be a worldwide public health problem. An immunosuppressor microenvironment has been proposed to contribute to viral persistence during chronic disease. Understanding the intracellular signaling cascade in T-cells from HBV-infected patients, will contribute to unravel the mechanisms that control the development of immune response during hepatitis B. We analyze lipid rafts formation and early activation signals in chronic HBV infected patients, compared to naturally immune subjects (NIS). Patients show: (1) diminished GM1 clustering, (2) A deficient lipid rafts recruitment of CD3ζ/ZAP-70/Grb2, and (3) these proteins do not merge with GM1 within the lipid rafts. Finally, immunoprecipitation assays proved that ZAP-70 does not associate to CD3ζ. These results show for the first time, defects regarding early key events in T-cell activation, in chronically infected HBV patients, which may contribute not only to understand HBV immune tolerance, but to reveal new potential therapeutic targets to control the infection.

Tespa1 is involved in late thymocyte development through the regulation of TCR-mediated signaling.

Signaling via the T cell antigen receptor (TCR) during the CD4(+)CD8(+) double-positive developmental stage determines thymocyte selection and lineage commitment. Here we describe a previously uncharacterized T cell-expressed protein, Tespa1, with critical functions during the positive selection of thymocytes. Tespa1(-/-) mice had fewer mature thymic CD4(+) and CD8(+) T cells, which reflected impaired thymocyte development. Tespa1 associated with the TCR signaling components PLC-γ1 and Grb2, and Tespa1 deficiency resulted in attenuated TCR signaling, as reflected by defective activation of the Erk-AP-1 and Ca(2+)-NFAT pathways. Our findings demonstrate that Tespa1 is a component of the TCR signalosome and is essential for T cell selection and maturation through the regulation of TCR signaling during T cell development.

B cell receptor-mediated antigen gathering requires ubiquitin ligase Cbl and adaptors Grb2 and Dok-3 to recruit dynein to the signaling microcluster.

The B cell receptor (BCR) mediates B cell antigen gathering and acquisition for presentation to T cells. Although the amount of antigen presentation to T cells determines the extent of B cell activation, the molecular mechanisms underlying antigen gathering remain unexplored. Here, through a combination of high-resolution imaging, genetics and quantitative mass spectrometry, we demonstrate that adaptors Grb2 and Dok-3, and ubiquitin ligase Cbl in signaling BCR microclusters mediate association with the microtubule motor dynein. Furthermore, we visualize the localization and movement of these microclusters on the underlying microtubule network. Importantly, disruption of this network or diminished dynein recruitment in Grb2-, Dok-3-, or Cbl-deficient B cells, does not influence microcluster formation or actin-dependent spreading, but abrogates directed movement of microclusters and antigen accumulation. Thus we identify a surprising but pivotal role for dynein and the microtubule network alongside Grb2, Dok-3, and Cbl in antigen gathering during B cell activation.

Growth-factor receptor-bound protein-2 (Grb2) signaling in B cells controls lymphoid follicle organization and germinal center reaction.

Grb2 (growth-factor receptor-bound protein-2) is a signaling adaptor that interacts with numerous receptors and intracellular signaling molecules. However, its role in B-cell development and function remains unknown. Here we show that ablation of Grb2 in B cells results in enhanced B-cell receptor signaling; however, mutant B cells do not form germinal centers in the spleen after antigen stimulation. Furthermore, mutant mice exhibit defects in splenic architecture resembling that observed in B-cell-specific lymphotoxin-ß-deficient mice, including disruption of marginal zone and follicular dendritic cell networks. We find that grb2(-/-) B cells are defective in lymphotoxin-ß expression. Although lymphotoxin can be up-regulated by chemokine CXCL13 and CD40 ligand stimulation in wild-type B cells, elevation of lymphotoxin expression in grb2(-/-) B cells is only induced by anti-CD40 but not by CXCL13. Our results thus define Grb2 as a nonredundant regulator that controls lymphoid follicle organization and germinal center reaction. Loss of Grb2 has no effect on B-cell chemotaxis to CXCL13, indicating that Grb2 executes this function by connecting the CXCR5 signaling pathway to lymphotoxin expression but not to chemotaxis.

The B-lymphoid Grb2 interaction code.

The growth factor receptor-bound protein 2 (Grb2) is a ubiquitously expressed and evolutionary conserved adapter protein possessing a plethora of described interaction partners for the regulation of signal transduction. In B lymphocytes, the Grb2-mediated scaffolding function controls the assembly and subcellular targeting of activating as well as inhibitory signalosomes in response to ligation of the antigen receptor. Also, integration of simultaneous signals from B-cell coreceptors that amplify or attenuate antigen receptor signal output relies on Grb2. Hence, Grb2 is an essential signal integrator. The key question remains, however, of how pathway specificity can be maintained during signal homeostasis critically required for the balance between immune cell activation and tolerance induction. Here, we summarize the molecular network of Grb2 in B cells and introduce a proteomic approach to elucidate the interactome of Grb2 in vivo.

Grb2, a simple adapter with complex roles in lymphocyte development, function, and signaling.

Lymphocyte development, activation, and tolerance depend on antigen receptor signaling transduced via multiple intracellular signalosomes. These signalosomes are assembled by different adapters. Given that signaling molecules can be either positive or negative regulators for a biochemical target, the complex of a target with different regulator may dictate the final signaling outcome. Grb2 is a simple adapter known to be involved in a variety of growth factor receptor signaling. However, its role in antigen receptor signaling as well as lymphocyte development and function has emerged only recently. Despite its simple molecular structure, recent experiments show that Grb2 may play a complex role in T and B-cell antigen receptor signaling. In this article, we review recent findings about the physiological role of Grb2 in T and B-cell development and activation and summarize the current mechanistic understanding of how Grb2 exerts its function following T and B-cell antigen receptor stimulation.

Vav and Rac activation in B cell antigen receptor endocytosis involves Vav recruitment to the adapter protein LAB.

The signal transduction events supporting B cell antigen receptor (BCR) endocytosis are not well understood. We have identified a pathway supporting BCR internalization that begins with tyrosine phosphorylation of the adapter protein LAB. Phosphorylated LAB recruits a complex of Grb2-dynamin and the guanine nucleotide exchange factor Vav. Vav is required for activation of the small GTPases Rac1 and Rac2. All these proteins contribute to (and dynamin, Vav, and Rac1/2 are required for) BCR endocytosis and presentation of antigen to T cells. This is the first description of a sequential signal transduction pathway from BCR to internalization and antigen presentation.

CD72, a coreceptor with both positive and negative effects on B lymphocyte development and function.

INTRODUCTION: B lymphocytes remain in a resting state until activated by antigenic stimuli through interaction with the B cell receptor (BCR). Coreceptors on B cells can modulate the thresholds for signaling through the BCR for growth and differentiation. CD72 is a B cell coreceptor that has been shown to interact with CD100, a semaphorin, and to enhance BCR signaling. DISCUSSION: CD72 ligation induces a variety of early signaling events such as activation of the Src kinases Blk and Lyn and the non-src kinase Btk leading to activation of the mitogen-activated protein (MAP) kinases, events usually associated with positive signaling. CD72 signals can enable Btk-deficient B cells to overcome their unresponsiveness to BCR signaling. On the other hand, BCR-mediated signals are enhanced in CD72-deficient cells but are reduced in CD100 null cells. The dual effects of CD72 on B cells can be explained by its association with positive and negative signaling molecules. Thus, CD72 interacts with SHP-1, an SH2-domain containing protein tyrosine phosphatase, a negative regulator of signaling, and Grb2, an adaptor protein associated with the Ras/MAPK pathway. Ligation of CD72 also triggered its association with CD19, a positive modulator of B cell receptor signaling. We propose a dual signaling hypothesis to explain the growth and differentiation promoting properties of CD72. Deficiency in either CD72 or CD100 leads to autoimmunity in mouse models. CD72 expression and polymorphisms exhibit some association with autoimmune diseases such as lupus, Sjogren's syndrome, and type 1 diabetes.

CD28 stimulation triggers NF-kappaB activation through the CARMA1-PKCtheta-Grb2/Gads axis.

CD28 stimulation contributes to activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including nuclear factor kappaB (NF-kappaB)/Rel family members. However, the signal-transducing cascades linking the CD28 molecule and activation of NF-kappaB remain unclear. Protein kinase C (PKC) , CARMA1 and Bcl10 have recently been reported to integrate TCR-mediated NF-kappaB activation. However, since the data in these studies were drawn from experiments in which T cells were usually stimulated with both TCR and CD28, the relative contributions of TCR- and CD28-mediated signals to initiation of the NF-kappaB pathway remain elusive. To examine the role of these molecules in NF-kappaB activation through CD28-mediated stimulation, Bcl10 was over-expressed in Jurkat cells and their NF-kappaB activation by CD28- or TCR-cross-linking was evaluated. We found that CD28 stimulation alone can induce NF-kappaB activation in Bcl10-over-expressing Jurkat cells, whereas TCR stimulation alone has only little effect. In addition, we found that Bcl10-induced NF-kappaB activation through CD28-mediated stimulation could be blocked by the dominant-negative form of PKC or CARMA1. Furthermore, genetic studies revealed that Grb2/Gads binding, but not phosphatidylinositol 3-kinase binding, is important in CD28-mediated NF-kappaB activation. These findings indicate that the PKC-CARMA1-Bcl10 signaling pathway participates in the CD28 co-stimulatory signal independently of the TCR-signaling pathway, which leads us to propose that the activation of the NF-kappaB-signaling pathway via PKC-CARMA1-Bcl10 may be markedly dependent on CD28 stimulation rather than TCR stimulation.