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1.  Mouse IgM Fc receptor, FCMR, promotes B cell development and modulates antigen-driven immune responses 
FCMR, a Fc receptor specific for pentameric IgM, is expressed at high levels by B cells. Although circulating IgM has profound effects on responses to pathogens, autoimmunity and B cell homeostasis, the biologic consequences of its binding to FCMR are poorly understood. We interrogated FCMR contributions to B cell function by studying mice lacking FCMR. FCMR transcripts are expressed at different levels by various B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic FO and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. Following immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are sensitive to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR is required for B cell differentiation and homeostasis, the prevention of autoreactive B cells and responsiveness to antigenic challenge.
doi:10.4049/jimmunol.1202227
PMCID: PMC3552009  PMID: 23267023
2.  Toso, a functional IgM receptor, is regulated by IL-2 in T and NK cells1 
We find that the cell surface receptor Toso is dramatically down-regulated by in vitro stimulation of human T and NK cells with IL-2 in a STAT5 dependent manner. The fact that IL-2 is known to prime NK and T cells for Fas/TNF-mediated activation induced cell death (AICD) fits nicely with the original and recent descriptions of Toso as an inhibitor of Fas/TNF-induced apoptosis. In support of this possibility, effector memory T cells express markedly lower levels of Toso than naïve T cells, indicating that activation in vivo correlates with the down-regulation of Toso. Moreover, in vitro activation of memory T cells through TCR dramatically down-regulates Toso expression compared to naïve CD4 T cells. However, over-expression of Toso in human NK cells and Jurkat T cells does not inhibit Fas-mediated apoptosis, and, in agreement with other recent reports, Toso clearly functions as an IgM receptor. Unlike CD16, Toso expression by NK cells does not convey cytotoxic potential, but its ligation does trigger intracellular signaling in NK cells. In summary, our data indicate that Toso is a functional IgM receptor that is capable of activating signaling molecules, is regulated by IL-2, and is not inherently an anti-apoptotic molecule.
doi:10.4049/jimmunol.1200840
PMCID: PMC3392415  PMID: 22675200
3.  Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) deficient mice have an altered immune cell phenotype 
Cross-linking of the collagen binding receptor LAIR-1 in vitro delivers an inhibitory signal that is able to down-regulate activation-mediated signals. To study the in vivo function of LAIR-1, we generated LAIR-1−/− mice. They are healthy and fertile, and have normal longevity; however, they show certain phenotypic characteristics distinct from wild-type mice, including increased numbers of splenic B, T regulatory and dendritic cells. As LAIR-1−/− mice age, the splenic T cell population shows a higher frequency of activated and memory T cells. Since LAIR-1+/+ and LAIR-1−/− T cells traffic with equal proficiency to peripheral lymphoid organs, this is not likely due to abnormal T lymphocyte trafficking. LAIR-1−/− mice have lower serum levels of IgG1 and, in response to T-dependent immunization with TNP-OVA, switch less efficiently to antigen specific IgG2a and IgG2b, while switching to IgG1 is not affected. Several mouse disease models, including EAE and colitis, were utilized to examine the effect of LAIR-1 deficiency and no differences in the responses of LAIR-1−/− and LAIR-1+/+ mice were observed. Taken together, these observations indicate that LAIR-1 plays a role in regulating immune cells and suggest that any adverse effects of its absence may be balanced in vivo by other inhibitory receptors.
doi:10.4049/jimmunol.1102044
PMCID: PMC3286132  PMID: 22156345
4.  Cutting Edge: Mouse CD300f (CLM-1) recognizes outer-membrane exposed phosphatidylserine and can promote phagocytosis 
Reportedly, CD300f negatively regulates interactions between dendritic and T cells, and acts as an anti-inflammatory molecule in a multiple sclerosis mouse model. We found that a CD300f/Fc chimeric protein specifically binds to apoptotic/dead splenocytes and to apoptotic cells from starved or irradiated lymphocytic cell lines; an observation extended to insect cells. CD300f also binds PMA/ionomycin-activated splenocytes and Ag-stimulated T cells, an interaction inhibited by Annexin V. By ELISA, co-sedimentation and surface plasmon resonance using phospholipid containing liposomes, we show that CD300f preferentially binds phosphatidylserine and requires a metal ion. Exogenous expression of CD300f in cell lines results in enhanced phagocytosis of apoptotic cells. We conclude that expression of CD300f conveys additional capacity to recognize phosphatidylserine to myeloid cells. The result of this recognition may vary with the overall qualitative and quantitative receptor content, as well as signaling capacity of the expressing effector cell, but enhanced phagocytosis is one measurable outcome.
doi:10.4049/jimmunol.1101549
PMCID: PMC3178745  PMID: 21865548
5.  Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a 
BMC Immunology  2012;13:23.
Background
Activation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD300a, an ITIM bearing type I transmembrane protein, is expressed on many hematopoietic cells, including subsets of lymphocytes.
Results
We have taken two approaches to further define the mechanism by which CD300a acts as an inhibitor of immune cell receptor signaling. First, we have expressed in Jurkat T cells a chimeric receptor consisting of the extracellular domains of killer-cell immunoglobulin-like receptor (KIR)2DL2 fused to the transmembrane and cytoplasmic segments of CD300a (KIR-CD300a) to explore surrogate ligand-stimulated inhibition of superantigen stimulated T cell receptor (TCR) mediated cell signaling. We found that intact CD300a ITIMs were essential for inhibition and that the tyrosine phosphorylation of these ITIMs required the src tyrosine kinase Lck. Tyrosine phosphorylation of the CD300a ITIMs created docking sites for both src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Suppression of SHP-1 and SHP-2 expression in KIR-CD300a Jurkat T cells with siRNA and the use of DT40 chicken B cell lines expressing CD300a and deficient in several phosphatases revealed that SHP-1, but not SHP-2 or the src homology 2 domain containing inositol 5’ phosphatase SHIP, was utilized by CD300a for its inhibitory activity.
Conclusion
These studies provide new insights into the function of CD300a in tuning T and B cell responses.
doi:10.1186/1471-2172-13-23
PMCID: PMC3418551  PMID: 22537350
6.  Differential expression of CD300a/c on human TH1 and TH17 cells 
BMC Immunology  2011;12:62.
Background
Human memory CD4+ T cells can be either CD300a/c+ or CD300a/c- and subsequent analyses showed that CD4+ effector memory T (TEM) cells are mostly CD300a/c+, whereas CD4+ central memory T (TCM) cells have similar frequencies of CD300a/c+ and CD300a/c- cells.
Results
Extensive phenotypical and functional characterization showed that in both TCM and TEM cells, the CD300a/c+ subset contained a higher number of TH1 (IFN-γ producing) cells. Alternatively, TH17 (IL-17a producing) cells tend to be CD300a/c-, especially in the TEM subset. Further characterization of the IL-17a+ cells showed that cells that produce only this cytokine are mostly CD300a/c-, while cells that produce IL-17a in combination with other cytokines, especially IFN-γ, are mostly CD300a/c+, indicating that the expression of this receptor is associated with cells that produce IFN-γ. Co-ligation of the TCR and CD300a/c in CD4+ T cells inhibited Ca2+ mobilization evoked by TCR ligation alone and modulated IFN-γ production on TH1 polarized cells.
Conclusion
We conclude that the CD300a/c receptors are differentially expressed on human TH1 and TH17 cells and that their ligation is capable of modulating TCR mediated signals.
doi:10.1186/1471-2172-12-62
PMCID: PMC3219710  PMID: 22046970
7.  Endocytosis and Intracellular Trafficking of Human Natural Killer Cell Receptors 
Traffic (Copenhagen, Denmark)  2009;10(12):1735-1744.
Natural killer (NK) cells play a vital role in the defense against viral infections and tumor development. NK cell function is primarily regulated by the sum of signals from a broad array of activation and inhibitory receptors. Key to generating the input level of either activating or inhibitory signals is the maintenance of receptor expression levels on the cell surface. Although the mechanisms of endocytosis and trafficking for some cell surface receptors, such as transferrin receptor, and certain immune receptors, are very well known, that is not the situation for receptors expressed by NK cells. Recent studies have uncovered that endocytosis and trafficking routes characteristic for specific activation and inhibitory receptors can regulate the functional responses of NK cells. In this review, we summarize the current knowledge of receptor endocytosis and trafficking, and integrate this with our current understanding of NK cell receptor trafficking.
doi:10.1111/j.1600-0854.2009.00973.x
PMCID: PMC2783697  PMID: 19719476
8.  Human Th1 Cells That Express CD300a Are Polyfunctional and After Stimulation Up-Regulate the T-Box Transcription Factor Eomesodermin 
PLoS ONE  2010;5(5):e10636.
Human naïve CD4 T cells express low levels of the immunomodulatory receptor CD300a, whereas effector/memory CD4 cells can be either CD300a+ or CD300a−. This suggested that CD300a expression could define a specific subset within the effector/memory CD4 T cell subpopulations. In fact, ex vivo analysis of the IFN-γ producing CD4 T cells showed that they are enriched in the CD300a+ subset. Moreover, stimulated CD4 T cells producing TNF-α and IL-2 besides IFN-γ (polyfunctional) are predominantly CD300a+. In addition to producing markedly higher levels of Th1-associated cytokines, the stimulated CD300a+ CD4 T cells are distinguished by a striking up-regulation of the T-box transcription factor eomesodermin (Eomes), whereas T-bet is up-regulated in both CD300a+ and CD300a− activated CD4 T cells to similar levels. The pleiotropic cytokine TGF-β1 has a determinant role in dictating the development of this Th1 subset, as its presence inhibits the expression of CD300a and down-regulates the expression of Eomes and IFN-γ. We conclude that CD300a+ human Th1 cells tend to be polyfunctional and after stimulation up-regulate Eomes.
doi:10.1371/journal.pone.0010636
PMCID: PMC2869357  PMID: 20498708
9.  A single residue, arginine 65, is critical for the functional interaction of leukocyte associated inhibitory receptor (LAIR)-1 with collagens1 
Immuno-receptor tyrosine based inhibitory motif (ITIM)-containing receptors play an essential role in modulating immune responses. Leukocyte associated inhibitory receptor (LAIR)-1, also known as CD305, is an ITIM-containing inhibitory receptor, expressed by all leukocytes, that binds collagens. In this report, we investigate the effect of a conservative R65K mutation on LAIR-1 ligand binding and function. Compared to LAIR-1 wild-type (wt) expressing cells, LAIR-1 R65K cells show markedly reduced binding to collagen, which correlates with a reduced level of LAIR-1 polarization to the site of interaction with collagens. Both LAIR-1 wt and R65K cells can generate intracellular signals when ligated by anti-LAIR-1 mAb, but only LAIR-1 wt cells respond to collagens or matrigel. In agreement, surface plasmon resonance (SPR) analyses showed that LAIR-1 R65K protein has markedly reduced avidity for collagen type I compared to LAIR-1 wt. Likewise, LAIR-1 R65K protein has decreased avidity for cells expressing transmembrane collagen XVII. Thus, a single residue, Arg 65, is critical for the interaction of LAIR-1 with collagens.
doi:10.4049/jimmunol.0804052
PMCID: PMC2714985  PMID: 19380792
Natural Killer Cells; Cell Surface Molecules; Signal Transduction; Cell Activation
10.  Endosomal Trafficking of the Ligated FcεRI Receptor 
Molecular immunology  2008;46(5):793-802.
In addition to initiating signaling cascades leading to mast cell mediator release, aggregation of the high affinity IgE receptor (FcεRI) leads to rapid internalization of the cross-linked receptor. However, little is known about the trafficking of the internalized FcεRI. Here we demonstrate that in RBL-2H3 cells, aggregated FcεRI appears in the early endosomal antigen 1 (EEA1+) domains of the early endosomes within 15 minutes after ligation. Minimal co-localization of FcεRI with Rab5 was observed by 30 minutes, followed by its appearance in the Rab7+ late endosomes and lysosomes at later time points. During endosomal sorting, FcεRI α and γ subunits remain associated. In Syk-deficient RBL-2H3 cells, the rate of transport to lysosomes is markedly increased. Taken together, our data demonstrate time-dependent sorting of aggregated FcεRI within the endosomal-lysosomal network, and that Syk may play an essential role in regulating the trafficking and retention of FcεRI in endosomes.
doi:10.1016/j.molimm.2008.09.002
PMCID: PMC2668543  PMID: 18945491
FcεRI; endosomes; co-localization; Syk
11.  Endocytosis as a mechanism of regulating natural killer cell function: unique endocytic and trafficking pathway for CD94/NKG2A 
Immunologic research  2009;43(1-3):210-222.
Natural killer (NK) cells are lymphocytes generally recognized as sentinels of the innate immune system due to their inherent capacity to deal with diseased (stressed) cells, including malignant and infected. This ability to recognize many potentially pathogenic situations is due to the expression of a diverse panel of activation receptors. Because NK cell activation triggers an aggressive inflammatory response, it is important to have a means of throttling this response. Hence, NK cells also express a panel of inhibitory receptors that recognize ligands expressed by “normal” cells. Little or nothing is known about the endocytosis and trafficking of NK cell receptors, which are of great relevance to understanding how NK cells maintain the appropriate balance of activating and inhibitory receptors on their cell surface. In this review, we focus on the ITIM-containing inhibitory receptor CD94/NKG2A showing that it is endocytosed by a previously undescribed macropinocytic-like process that may be related to the maintenance of its surface expression.
doi:10.1007/s12026-008-8072-7
PMCID: PMC2752144  PMID: 18979076
NK cells; Inhibitory/activating receptors; Endocytosis; Trafficking; CD94/NKG2A
12.  Short communication 
Molecular immunology  2007;45(1):253-258.
To achieve an adequate response, cells of the immune system must be tightly regulated to avoid hypo- or hyper-responsiveness. One of the mechanisms used by the immune system to avoid excessive inflammation is the modulation of the response through inhibitory receptors containing immunoreceptor tyrosine based inhibitory motifs (ITIM). Here, we show that human neutrophils from peripheral blood express the ITIM containing CD300a (also known as IRp60 and CMRF-35H) receptor. By using the HL-60 differentiation model, we show that the expression of CD300a receptor is developmentally regulated. Stimulation of human neutrophils with LPS and GM-CSF increased the cell surface expression of CD300a as a result of the rapid translocation of an intracellular pool of the receptor to the cell surface. Co-ligation of CD300a with the immunoreceptor tyrosine based activating motif (ITAM) containing CD32a (FcγRIIa) activation receptor inhibited CD32a mediated signaling, whereas it did not inhibit Toll like receptor (TLR)-4 mediated reactive oxygen species (ROS) production. Therefore, at least for human neutrophils, the inhibitory signals mediated by the CD300a receptor may be selective in their action.
doi:10.1016/j.molimm.2007.05.006
PMCID: PMC2000843  PMID: 17588661
neutrophils; CD300a; inhibitory receptor; reactive oxygen species; ITAM; ITIM
13.  Exclusion of Lipid Rafts and Decreased Mobility of CD94/NKG2A Receptors at the Inhibitory NK Cell SynapseV⃞ 
Molecular Biology of the Cell  2004;15(7):3210-3223.
CD94/NKG2A is an inhibitory receptor expressed by most human natural killer (NK) cells and a subset of T cells that recognizes human leukocyte antigen E (HLA-E) on potential target cells. To elucidate the cell surface dynamics of CD94/NKG2A receptors, we have expressed CD94/NKG2A-EGFP receptors in the rat basophilic leukemia (RBL) cell line. Photobleaching experiments revealed that CD94/NKG2A-EGFP receptors move freely within the plasma membrane and accumulate at the site of contact with ligand. The enriched CD94/NKG2A-EGFP is markedly less mobile than the nonligated receptor. We observed that not only are lipid rafts not required for receptor polarization, they are excluded from the site of receptor contact with the ligand. Furthermore, the lipid raft patches normally observed at the sites where FcεR1 activation receptors are cross-linked were not observed when CD94/NKG2A was coengaged along with the activation receptor. These results suggest that immobilization of the CD94/NKG2A receptors at ligation sites not only promote sustenance of the inhibitory signal, but by lipid rafts exclusion prevent formation of activation signaling complexes.
doi:10.1091/mbc.E03-11-0779
PMCID: PMC452577  PMID: 15133125
14.  Recognition of Human Histocompatibility Leukocyte Antigen (HLA)-E Complexed with HLA Class I Signal Sequence–derived Peptides by CD94/NKG2 Confers Protection from Natural Killer Cell–mediated Lysis  
Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical HLA class I molecule, the gene for which is transcribed in most tissues. It has recently been reported that this molecule binds peptides derived from the signal sequence of HLA class I proteins; however, no function for HLA-E has yet been described. We show that natural killer (NK) cells can recognize target cells expressing HLA-E molecules on the cell surface and this interaction results in inhibition of the lytic process. Furthermore, HLA-E recognition is mediated primarily through the CD94/NKG2-A heterodimer, as CD94-specific, but not killer cell inhibitory receptor (KIR)–specific mAbs block HLA-E–mediated protection of target cells. Cell surface HLA-E could be increased by incubation with synthetic peptides corresponding to residues 3–11 from the signal sequences of a number of HLA class I molecules; however, only peptides which contained a Met at position 2 were capable of conferring resistance to NK-mediated lysis, whereas those having Thr at position 2 had no effect. Interestingly, HLA class I molecules previously correlated with CD94/NKG2 recognition all have Met at residue 4 of the signal sequence (position 2 of the HLA-E binding peptide), whereas those which have been reported not to interact with CD94/NKG2 have Thr at this position. Thus, these data show a function for HLA-E and suggest an alternative explanation for the apparent broad reactivity of CD94/NKG2 with HLA class I molecules; that CD94/NKG2 interacts with HLA-E complexed with signal sequence peptides derived from “protective” HLA class I alleles rather than directly interacting with classical HLA class I proteins.
PMCID: PMC2212178  PMID: 9480992
15.  NKG2A Complexed with CD94 Defines a Novel Inhibitory Natural Killer Cell Receptor 
CD94 is a C-type lectin expressed by natural killer (NK) cells and a subset of T cells. Blocking studies using anti-CD94 mAbs have suggested that it is a receptor for human leukocyte antigen class I molecules. CD94 has recently been shown to be a 26-kD protein covalently associated with an unidentified 43-kD protein(s). This report shows that NKG2A, a 43-kD protein, is covalently associated with CD94 on the surface of NK cells. Cell surface expression of NKG2A is dependent on the association with CD94 as glycosylation patterns characteristic of mature proteins are found only in NKG2A that is associated with CD94. Analysis of NK cell clones showed that NKG2A was expressed in all NK cell clones whose CD16-dependent killing was inhibited by cross-linking CD94. The induction of an inhibitory signal is consistent with the presence of two immunoreceptor tyrosine-based inhibitory motifs (V/LXYXXL) on the cytoplasmic domain of NKG2A. Similar motifs are found on Ly49 and killer cell inhibitory receptors, which also transmit negative signals to NK cells.
PMCID: PMC2196137  PMID: 9034158

Results 1-15 (15)