The expression and function of human CD300 receptors on blood circulating mononuclear cells are distinct in neonates and adults.

Neonates are more susceptible to infections than adults. This susceptibility is thought to reflect neonates' qualitative and quantitative defects in the adaptive and innate immune responses. Differential expression of cell surface receptors may result in altered thresholds of neonatal immune cell activation. We determined whether the expression and function of the lipid-binding CD300 family of receptors are different on neonatal immune cells compared to adult immune cells. A multiparametric flow cytometry analysis was performed to determine the expression of CD300 receptors on adult peripheral blood mononuclear cells and neonatal cord blood mononuclear cells. The expression of the CD300a inhibitory receptor was significantly reduced on cells from the newborn adaptive immune system, and neonatal antigen presenting cells exhibited a different CD300 receptors expression pattern. We also found differential LPS-mediated regulation of CD300 receptors expression on adult monocytes compared to cord blood monocytes, and that CD300c and CD300e-mediated activation was quantitatively different in neonatal monocytes. This is the first complete study examining the expression of CD300 receptors on human neonatal immune cells compared with adult immune cells. Significant differences in the expression and function of CD300 receptors may help to explain the peculiarities and distinctness of the neonatal immune responses.

CD300c is uniquely expressed on CD56 bright Natural Killer Cells and differs from CD300a upon ligand recognition.

Paired receptors on NK cells recognize similar ligands with varied strength of binding ability and perform different functions. The CD300 molecules are emerging as novel immune regulators in health and disease due to their interaction with their lipid-nature ligands. Particularly, the paired receptors CD300c and CD300a have been shown to elicit activating and inhibitory capabilities, respectively. In the current study, we seek to investigate the expression and function of CD300c on human NK cells. We demonstrate that IL-2 and IL-15 treatment significantly induce CD300c expression exclusively on CD56(bright) NK cells. CD300c up-regulation requires STAT5 and its expression is inhibited by IL-4. Consistently, IL-2 secreted from activated CD4(+) T cells specifically induces the expression of CD300c on CD56(bright) NK cells. Crosslinking CD300c with a specific antibody enhances the proficiency of CD56(bright) NK cells to degranulate and induce chemokine and cytokine secretion. We also show the differential binding of CD300a and CD300c to their ligands phosphatidylethanolamine (PE) and phosphatidylserine (PS) and their differential ability to affect CD56(bright) NK cell functions. Our results provide an insight into the novel set of paired receptors CD300a and CD300c that are distinctively expressed on CD56(bright) NK cells with varied effector functions.

The Biology and Disease Relevance of CD300a, an Inhibitory Receptor for Phosphatidylserine and Phosphatidylethanolamine.

The CD300a inhibitory receptor belongs to the CD300 family of cell surface molecules that regulate a diverse array of immune cell processes. The inhibitory signal of CD300a depends on the phosphorylation of tyrosine residues embedded in ITIMs of the cytoplasmic tail. CD300a is broadly expressed on myeloid and lymphoid cells, and its expression is differentially regulated depending on the cell type. The finding that CD300a recognizes phosphatidylserine and phosphatidylethanolamine, two aminophospholipids exposed on the outer leaflet of dead and activated cells, has shed new light on its role in the modulation of immune functions and in its participation in the host response to several diseases states, such as infectious diseases, cancer, allergy, and chronic inflammatory diseases. This review summarizes the literature on CD300a expression, regulation, signaling pathways, and ligand interaction, as well as its role in fine tuning immune cell functions and its clinical relevance.

A Human Anti-M2 Antibody Mediates Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Cytokine Secretion by Resting and Cytokine-Preactivated Natural Killer (NK) Cells.

The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.

CD300c is an activating receptor expressed on human monocytes.

Human CD300 molecules comprise a family of receptors that regulate many immune cell processes. They are mostly expressed on myeloid cells, although expression of two members, CD300a and CD300c, has also been described on lymphocytes. However, due to the lack of specific antibodies that distinguish between these two receptors, it has been difficult to determine the expression pattern and function of CD300a and CD300c in primary cells. Here, we have identified a specific monoclonal antibody, clone TX45, that recognizes only CD300c and show that within freshly isolated blood leukocytes, monocytes are the only cells that express CD300c on the cell surface. In vitro differentiation experiments revealed that CD300c is differentially expressed on different monocyte-derived cells, including macrophages and dendritic cells. Furthermore, TLR ligands LPS and flagellin dynamically regulate the expression of CD300c. Cross-linking of this receptor with clone TX45 monoclonal antibody induced calcium mobilization, upregulation of the costimulatory molecule CD86 and the production of inflammatory cytokines. Importantly, LPS-mediated production of inflammatory cytokines by monocytes was further enhanced if CD300c was simultaneously engaged by the agonist antibody. Altogether, our results show that human CD300c is an activating receptor expressed on monocytes and that it has a potential role in inflammatory responses.

Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity.

Natural killer (NK) cells are a major component of the anti-tumor immune response. NK cell dysfunctions have been reported in various hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here we investigated the role of tumor cell-released soluble and exosomal ligands for NK cell receptors that modulate NK cell activity. Soluble CLL plasma factors suppressed NK cell cytotoxicity and down-regulated the surface receptors CD16 and CD56 on NK cells of healthy donors. The inhibition of NK cell cytotoxicity was attributed to the soluble ligand BAG6/BAT3 that engages the activating receptor NKp30 expressed on NK cells. Soluble BAG6 was detectable in the plasma of CLL patients, with the highest levels at the advanced disease stages. In contrast, NK cells were activated when BAG6 was presented on the surface of exosomes. The latter form was induced in non-CLL cells by cellular stress via an nSmase2-dependent pathway. Such cells were eliminated by lymphocytes in a xenograft tumor model in vivo. Here, exosomal BAG6 was essential for tumor cell killing because BAG6-deficient cells evaded immune detection. Taken together, the findings show that the dysregulated balance of exosomal vs soluble BAG6 expression may cause immune evasion of CLL cells.

A novel role for reciprocal CD30-CD30L signaling in the cross-talk between natural killer and dendritic cells.

The interplay between dendritic cells (DCs) and natural killer (NK) cells directs adaptive immune responses. The molecular basis of the cross-talk is largely undefined. Here, we provide evidence for a contribution of CD30 (TNFRSF8) and its ligand CD30L (TNFSF8) expressed on NK cells and DCs, respectively. We demonstrate that CD30-mediated engagement of CD30L induced cytokine secretion from immature DCs via the mitogen-activated protein kinase pathway. Moreover, CD30L engagement promoted differentiation to mature DCs. On the contrary, the engagement of CD30 on NK cells resulted in an NF-κB-dependent release of TNF-α/IFN-γ. These data uncover a novel and unexpected role for CD30/CD30L that contributes to proinflammatory immune responses.

Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a.

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.

Human CD300a binds to phosphatidylethanolamine and phosphatidylserine, and modulates the phagocytosis of dead cells.

CD300a is an immunoreceptor tyrosine-based inhibitory motif (ITIM) containing molecule that belongs to the CD300 family of paired activating/inhibitory receptors. It has been shown that its ligation inhibits activation signals on cells of both myeloid and lymphoid lineages. The ligands for CD300a have not been identified. Here, we show that a CD300a-Ig fusion protein specifically binds to apoptotic cells that are evolutionary apart, such as human and insect cells, suggesting that the ligand has to be conserved. Using surface plasmon resonance, ultracentrifugation, ELISA, and reporter cell assays, we identified phosphatidylethanolamine (PE) and phosphatidylserine (PS), 2 phospholipids that translocate to the outer leaflet of the plasma membrane of dead cells, as the ligands for CD300a. Mutational and structural modeling studies identified residues that are involved in the binding of CD300a to PE and PS and that form a cavity where the hydrophilic heads of PE and PS, can penetrate. CD300a down-regulates the uptake of apoptotic cells by macrophages and its ectopic expression in CD300a-negative cell lines also decreased the engulfment of dead cells. Collectively, our results indicate that PE and PS are ligands for CD300a, and that this interaction plays an important role in regulating the removal of dead cells.

Differential expression of CD300a/c on human TH1 and TH17 cells.

BACKGROUND: Human memory CD4+ T cells can be either CD300a/c+ or CD300a/c- and subsequent analyses showed that CD4+ effector memory T (T(EM)) cells are mostly CD300a/c+, whereas CD4+ central memory T (T(CM)) cells have similar frequencies of CD300a/c+ and CD300a/c- cells. RESULTS: Extensive phenotypical and functional characterization showed that in both T(CM) and T(EM) 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 T(EM) 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.

Cutting edge: mouse CD300f (CMRF-35-like molecule-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, cosedimentation, 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.

CD300a is expressed on human B cells, modulates BCR-mediated signaling, and its expression is down-regulated in HIV infection.

The immunomodulatory receptor CD300a is expressed on human B cells. Naive B cells express very low levels of this receptor, whereas memory B cells and plasmablasts/cells express variable levels of CD300a. Germinal center B cells are negative for CD300a expression. Stimulation of naive B cells via B-cell receptor (BCR) and Toll-like receptor 9, along with T-cell help, failed to up-regulate CD300a cell surface expression despite the increased expression of the memory marker CD27 and the down-regulation of CD305. However, Toll-like receptor 9 stimulation alone significantly increased CD300a expression on memory B cells, whereas interleukin-4 and transforming growth factor-ß1 act as negative regulators of CD300a expression on memory B cells. Coligation of BCR and CD300a inhibits Ca(2+) mobilization and nuclear factor of activated T cell transcriptional activity evoked by BCR ligation alone. Suppression of CD300a expression in primary B cells with siRNA resulted in increased BCR-mediated proliferation, thereby confirming the inhibitory capacity of CD300a. Finally, we show that CD300a expression levels are significantly down-regulated in the circulating B cells of HIV-infected patients. Altogether, these data demonstrate a novel mechanism for suppressing the activity of B cells and suggest a potential role for CD300a in the B-cell dysfunction observed in HIV-induced immunodeficiency.

Human Th1 cells that express CD300a are polyfunctional and after stimulation up-regulate the T-box transcription factor eomesodermin.

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-gamma producing CD4 T cells showed that they are enriched in the CD300a(+) subset. Moreover, stimulated CD4 T cells producing TNF-alpha and IL-2 besides IFN-gamma (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-beta1 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-gamma. We conclude that CD300a(+) human Th1 cells tend to be polyfunctional and after stimulation up-regulate Eomes.

Heat shock protein 90 inhibitor BIIB021 (CNF2024) depletes NF-kappaB and sensitizes Hodgkin's lymphoma cells for natural killer cell-mediated cytotoxicity.

PURPOSE: In Hodgkin's lymphoma, constitutive activation of NF-kappaB promotes tumor cell survival and proliferation. The molecular chaperone heat shock protein 90 (HSP90) has immune regulatory activity and supports the activation of NF-kappaB in Hodgkin's lymphoma cells. EXPERIMENTAL DESIGN: We analyzed the effect of HSP90 inhibition on viability and NF-kappaB activity in Hodgkin's lymphoma cells and the consequences for their recognition and killing through natural killer (NK) cells. RESULTS: The novel orally administrable HSP90 inhibitor BIIB021 (CNF2024) inhibited Hodgkin's lymphoma cell viability at low nanomolar concentrations in synergy with doxorubicin and gemcitabine. Annexin V/7-aminoactinomycin D binding assay revealed that BIIB021 selectively induced cell death in Hodgkin's lymphoma cells but not in lymphocytes from healthy individuals. We observed that BIIB021 inhibited the constitutive activity of NF-kappaB and this was independent of IkappaB mutations. Furthermore, we analyzed the effect of HSP90 inhibition on NK cell-mediated cytotoxicity. BIIB021 induced the expression of ligands for the activating NK cell receptor NKG2D on Hodgkin's lymphoma cells resulting in an increased susceptibility to NK cell-mediated killing. In a xenograft model of Hodgkin's lymphoma, HSP90 inhibition significantly delayed tumor growth. CONCLUSIONS: HSP90 inhibition has direct antitumor activity in Hodgkin's lymphoma in vitro and in vivo. Moreover, HSP90 inhibition may sensitize Hodgkin's lymphoma cells for NK cell-mediated killing via up-regulation of ligands engaging activating NK cell receptors.

A novel bispecific protein (ULBP2-BB4) targeting the NKG2D receptor on natural killer (NK) cells and CD138 activates NK cells and has potent antitumor activity against human multiple myeloma in vitro and in vivo.

The inability of the immune system to recognize and kill malignant plasma cells in patients with multiple myeloma (MM) has been attributed in part to the ineffective activation of natural killer (NK) cells. In order to activate and target NK cells to the malignant cells in MM we designed a novel recombinant bispecific protein (ULBP2-BB4). While ULBP2 binds the activating NK receptor NKG2D, the BB4 moiety binds to CD138, which is overexpressed on a variety of malignancies, including MM. ULBP2-BB4 strongly activated primary NK cells as demonstrated by a significant increase in interferon-gamma (IFN-gamma) secretion. In vitro, ULBP2-BB4 enhanced the NK-mediated lysis of 2 CD138+ human MM cell lines, U-266 and RPMI-8226, and of primary malignant plasma cells in the allogenic and autologous setting. Moreover, in a nude mouse model with subcutaneously growing RPMI-8226 cells, the cotherapy with ULBP-BB4 and human peripheral blood lymphocytes abrogated the tumor growth. These data suggest potential clinical use of this novel construct in patients with MM. The use of recombinant NK receptor ligands that target NK cells to tumor cells might offer new approaches for other malignancies provided a tumor antigen-specific antibody is available.