The adaptor protein Crk controls activation and inhibition of natural killer cells

Natural killer (NK) cell inhibitory receptors recruit tyrosine phosphatases to prevent activation, induce phosphorylation and dissociation of the small adaptor Crk from cytoskeleton scaffold complexes, and maintain NK cells in a state of responsiveness to subsequent activation events. How Crk contributes to inhibition is unknown. We imaged primary NK cells over lipid bilayers carrying IgG1 Fc to stimulate CD16 and human leukocyte antigen (HLA)-E to inhibit through receptor CD94-NKG2A. HLA-E alone induced Crk phosphorylation in NKG2A(+) NK cells. At activating synapses with Fc alone, Crk was required for the movement of Fc microclusters and their ability to trigger activation signals. At inhibitory synapses, HLA-E promoted central accumulation of both Fc and phosphorylated Crk and blocked the Fc-induced buildup of F-actin. We propose a unified model for inhibitory receptor function: Crk phosphorylation prevents essential Crk-dependent activation signals and blocks F-actin network formation, thereby reducing constraints on subsequent engagement of activation receptors.     

The Role of NK Cells and NK Cell Receptors inAutoimmune Disease

Natural killer (NK) cells are the first line of defense against infection and transformation. Additionally, NK cells can play seemingly opposite roles in autoimmune disease. Here, we summarize the functions of NK cells as both regulators and inducers of autoimmune disease. The role NK cells play depends on which cells become targets for NK cell attack. The activity of NK cells is controlled by inhibitory receptors specific for MHC Class I molecules, and by activating receptors with diverse specificities. The ligands for both activating and inhibitory receptors are present on potential target cells. It is the balance in expression of these different ligands that determines NK cell activation and therefore whether the cell becomes a target for NK cell-mediated killing. We further discuss the roles of NK cell receptors and their ligands in autoimmune disease.     

The role of NK cells and NK cell receptors in autoimmune disease

Natural killer (NK) cells are the first line of defense against infection and transformation. Additionally, NK cells can play seemingly opposite roles in autoimmune disease. Here, we summarize the functions of NK cells as both regulators and inducers of autoimmune disease. The role NK cells play depends on which cells become targets for NK cell attack. The activity of NK cells is controlled by inhibitory receptors specific for MHC Class I molecules, and by activating receptors with diverse specificities. The ligands for both activating and inhibitory receptors are present on potential target cells. It is the balance in expression of these different ligands that determines NK cell activation and therefore whether the cell becomes a target for NK cell-mediated killing. We further discuss the roles of NK cell receptors and their ligands in autoimmune disease.     

Regulation of hematopoietic cell function by inhibitory immunoglobulin G receptors and their inositol lipid phosphatase effectors

Summary: Numerous autoimmune and inflammatory disorders stem from the dysregulation of hematopoietic cell activation. The activity of inositol lipid and protein tyrosine phosphatases, and the receptors that recruit them, is critical for prevention of these disorders. Balanced signaling by inhibitory and activating receptors is now recognized to be an important factor in tuning cell function and inflammatory potential. In this review, we provide an overview of current knowledge of membrane proximal events in signaling by inhibitory/regulatory receptors focusing on structural and functional characteristics of receptors and their effectors Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 and SH2 domain-containing inositol 5-phosphatase-1. We review use of new strategies to identify novel regulatory receptors and effectors. Finally, we discuss complementary actions of paired inhibitory and activating receptors, using FcγRIIA and FcγRIIB regulation human basophil activation as a prototype.     

BJ‐2266 ameliorates experimental autoimmune encephalomyelitis through down‐regulation of the JAK/STAT signaling pathway

CD4⁺ T cells differentiate into distinct effector subsets upon antigenic stimulation. Cytokines, and micro‐environmental factors present during T‐cell priming, direct differentiation of naïve CD4⁺ T cells into pro‐inflammatory Th1 and Th17 cells. From extensive screening of 2,4,5‐trimethylpyridin‐3‐ol derivatives with various functional groups at C(6)‐position, BJ‐2266, a 6‐thioureido‐derivative, showed potent inhibitory activity on in vitro T helper (Th)‐cell differentiation. This compound inhibited IFN‐γ and IL‐17 production from polyclonal CD4⁺ T cells and ovalbumin (OVA)‐specific CD4⁺ T cells that were activated by T‐cell receptor (TCR) engagement. We assessed the inhibitory effect of BJ‐2266 in experimental autoimmune encephalomyelitis (EAE). Our results suggest that BJ‐2266 treatment significantly suppresses EAE disease progression with reduced generation of Th1 and Th17 cells. Notably, Th‐cell differentiation was significantly suppressed by BJ‐2266 treatment with no effect on apoptosis, activation and proliferation of activated T cells. Furthermore, adoptive transfer of BJ‐2266 treated MOG‐reactive Th1 and Th17 cells led to a lower EAE disease score and better clinical recovery from EAE. The underlying mechanism of BJ‐2266 effect involved the inhibition of JAK/STAT phosphorylation that is critical for Th‐cell differentiation. We conclude that BJ‐2266 regulates the JAK/STAT pathway in response to cytokine signals and subsequently suppresses the differentiation of Th‐cell responses.     

Ets-1 deficiency leads to altered B cell differentiation, hyperresponsiveness to TLR9 and autoimmune disease

It has been shown that mice with a targeted mutation in the Ets-1 gene exhibit increased B cell terminal differentiation to IgM-secreting plasma cells. Here, we show that mice, formerly described to lack Ets-1 protein, actually express low levels of an internally deleted Ets-1 protein. Mice harboring this Ets-1 hypomorphic allele possess very few marginal zone B cells and have increased expression of activation markers on follicular B cells. Adoptive transfer experiments indicate that this activated phenotype can be reversed upon transfer of Ets-1-deficient B cells to a wild-type host, suggesting a role for B cell-extrinsic factors in regulating the activated state. Supporting this observation, the reverse transfer experiment of wild-type B cells into an Ets-1-deficient host resulted in increased expression of activation markers on the transferred B cells. However, there are also cell-intrinsic changes in Ets-1-deficient B cells as demonstrated by their increased differentiation to plasma cells in vitro in response to stimulation with cytosine-phosphate-guanine DNA sequence-containing oligodeoxynucleotide [CpG DNA, a Toll-like receptor (TLR) 9 ligand]. Consistent with the activated phenotype and increased terminal differentiation of Ets-1-deficient B cells, Ets-1 mutant mice develop autoimmune disease. Hence, our studies establish Ets-1 as an important regulator of peripheral B cell differentiation and B cell responses to TLR9 activation.     

Signaling through human killer cell activating receptors triggers tyrosine phosphorylation of an associated protein complex

Our understanding of the biology of human natural killer (NK) cells has significantly advanced in recent years upon identification of a family of NK cell-expressed genes that encode killer cell inhibitory receptors (KIR). Individual KIR can selectively bind various HLA class I allotypes and consequently transduce inhibitory signals that block NK cell lysis of ligand-bearing target cells. A distinct subset of related and linked genes express truncated versions of KIR that are otherwise highly homologous in amino acid sequence. Interestingly, these receptors appear to transmit stimulatory signals into NK cells and have been termed killer cell activating receptors (KAR). In this report, we demonstrate that recognition of HLA-Cw3 by the p50 KAR, NKAT8, can potentiate the cytotoxic response of appropriate NK cell clones. Specific cross-linking of this KAR with a monoclonal antibody resulted in intracellular calcium mobilization, protein tyrosine phosphorylation, and phosphorylation of the MAP kinases, ERK1 and ERK2. In addition, we identified a KAR-associated disulfide-linked dimer of a 13-kDa protein that was absent in the Jurkat T cell line and is predicted to participate in these activation signaling events. Upon treatment of NK cells with pervanadate, the disulfide-linked p13 and additional proteins of 25, 30, 37 and 50 – 95 kDa were identified as KAR-associated tyrosine phosphoproteins. Importantly, p13 was inducibly tyrosine phosphorylated upon cross-linking of NKAT8, which strongly suggests that the associated p13 provides KAR with appropriate cytoplasmic structure to couple with tyrosine kinase-mediated signaling effectors.     

The adaptor 3BP2 activates CD244-mediated cytotoxicity in PKC- and SAP-dependent mechanisms

Natural killer (NK) cell cytotoxicity requires triggering of activation receptors over inhibitory receptors. CD244, a member of CD150 receptor family, positively regulates NK-mediated lyses by activating an intracellular multiproteic signaling network that involves the adaptors X-linked lymphoproliferative gene product SAP and 3BP2. However, the exact mechanisms used by 3BP2 to enhance CD244-mediated cytotoxicity are still not fully understood. Here using the human NK cell line YT-overexpressing 3BP2, we found that the adaptor increases CD244, PI3K, and Vav phosphorylation upon CD244 engagement. The use of enzymatic inhibitors revealed that 3BP2-dependent cytolysis enhancement was PKC-dependent and PI3K-ERK independent. Furthermore, 3BP2 overexpression enhanced PKC delta phosphorylation. SAP knockdown expression inhibited PKC delta activation, indicating that the activating role played by 3BP2 depends upon the presence of SAP. In conclusion, our data show that 3BP2 acts downstream of SAP, increases CD244 phosphorylation and links the receptor with PI3K, Vav, PLC gamma, and PKC downstream events in order to achieve maximum NK killing function.     

Complement regulatory protein Crry/p65-mediated signaling in T lymphocytes: role of its cytoplasmic domain and partitioning into lipid rafts

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I-related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR-dependent activation signals, including p56lck-, zeta-associated protein-70 (ZAP-70), Vav-1, Akt, and extracellular signal-regulated kinase (ERK) phosphorylation but also costimulation-dependent mitogen-activated protein kinases (MAPK), such as the stress-activated c-Jun N-terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent-insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3epsilon, and p59-60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol-3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin-4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP-70, Akt, Vav-1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity.     

The lectin jacalin plus costimulation with anti-CD28 antibody induces phosphorylation of p38 MAPK and IL-4 synthesis-I

Costimulatory signals play an important role in the development of T helper cell type 1 (Th1) or Th2 type. Little is known about jacalin plus CD28-mediated signaling and cytokine secretion. In the present study, we analyzed the intracellular signaling events following stimulation of CD4+ T cells with jacalin plus CD28 cross-linking (CD28XL) with anti-CD28 antibody. Our results indicate enhanced phosphorylation of Tec and linker for activation of T cells when compared with stimulation with jacalin alone or CD28XL alone. Stimulation with jacalin or CD28XL appears to be insufficient to induce interleukin (IL)-4 secretion; however, CD28XL followed by stimulation with jacalin resulted in enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and increased secretion of IL-4. However, compared with stimulation with phorbol 12-myristate 13-acetate plus ionomycin, jacalin plus CD28XL resulted in decreased levels of tumor necrosis factor alpha secretion. Addition of p38 inhibitor, SB203580, inhibited p38 phosphorylation and IL-4 secretion. These data suggest that jacalin stimulation alone appears to be insufficient for Th2 development, and addition of CD28 costimulation induced Th2 generation. We propose that jacalin plus CD28XL induces Th2 differentiation via activation of p38 MAPK.     

In vivo expression of recombinant pregnancy-specific glycoprotein 1a induces alternative activation of monocytes and enhances Th2-type immune response

It has been proposed that pregnancy-specific factors could be responsible for shift the balance of cytokine profiles during maternal immune response from Th1-type reactivity into a "less-damaging" Th2-type reactivity. In the present work, we investigated the in vivo function of human pregnancy-specific glycoprotein (PSG)1a, the major variant of PSG polypeptides released into the circulation during pregnancy, on the modulation of the innate and adaptive immune response. For this, BALB/c mice were injected with a vaccinia virus-based vector harboring the human PSG1a cDNA (Vac-PSG1a) 4 days before immunization with ovalbumin (OVA) in complete Freund's adjuvant, and the early specific T cell response against OVA was evaluated 8 days post-immunization. We also studied the activation status of spleen and peritoneal monocytes/macrophages (Mo) populations from Vac-PSG1a-treated mice, and explored whether PSG1a-targeted Mo could affect the Th-type commitment by investigating their impact on the differentiation of naive T cells. Our data show that the treatment with Vac-PSG1a is able to induce a state of alternative activation on Mo. Furthermore, the generation of the immune response in the context of these alternatively activated antigen-presenting cells may shift T cell differentiation to Th2-type immunity which is more compatible with a successful pregnancy.     

The activation of Csk by CD4 interferes with TCR-mediated activatory signaling

CD4-Lck recruitment to TCR/CD3, as well as Lck activation is essential for T cell activation. Indeed, the blockage of CD4-Lck recruitment to TCR during antigen recognition exerts a drastic inhibitory effect on T cell activation by interfering with both early and late phases of T cell signaling. In the present work, we report a novel inhibitory mechanism by which CD4 can shut down proximal T cell-activating signals. Indeed, we show that upon ligation of CD4 by antibodies the inhibitory kinase, p50(csk), is strongly induced and prolonged during the time. In contrast, p50(csk) was not activated when TCR and CD4 were properly engaged by their ligands. We also demonstrate that anti-CD4 treatment stimulated Csk kinase associated to the membrane adapter, PAG/Cbp, without affecting the total amount of Csk bound to PAG/Cbp. As a consequence, early tyrosine phosphorylation events as well as downstream signaling pathways leading to IL-2 gene expression induced by TCR were inhibited in anti-CD4 pretreated cells. We suggest a new model to explain the activation of negative signals by CD4 molecule.