Progenitors of distinct lineages shape the diversity of mature type 2 conventional dendritic cells.

Conventional dendritic cells (cDC) are antigen-presenting cells comprising cDC1 and cDC2, responsible for priming naive CD8+ and CD4+ T cells, respectively. Recent studies have unveiled cDC2 heterogeneity and identified various cDC2 progenitors beyond the common DC progenitor (CDP), hinting at distinct cDC2 lineages. By generating Cd300ciCre-hCD2R26tdTomato reporter mice, we identified a bone marrow pro-cDC2 progenitor exclusively generating cDC2 in vitro and in vivo. Single-cell analyses and multiparametric flow cytometry demonstrated that pro-cDC2 encompasses myeloid-derived pre-cDC2 and lymphoid-derived plasmacytoid DC (pDC)-like precursors differentiating into a transcriptionally convergent cDC2 phenotype. Cd300c-traced cDC2 had distinct transcriptomic profiles, phenotypes, and tissue distributions compared with Ms4a3CreR26tdTomato lineage-traced DC3, a monocyte-DC progenitor (MDP)-derived subset that bypasses CDP. Mice with reduced Cd300c-traced cDC2 showed impaired humoral responses to T cell-dependent antigens. We conclude that progenitors of distinct lineages shape the diversity of mature cDC2 across tissues. Thus, ontogenesis may impact tissue immune responses.

Engagement of CD300c by a novel monoclonal antibody induces the differentiation of monocytes to M1 macrophages.

Human CD300c is expressed on various immune or cancer cells and is a novel B7 family member, functioning as an activity modulator on immune cells. To elucidate the function of CD300c, we developed CL7, a human CD300c-specific monoclonal antibody, and assessed its biological activity. The specific binding of CL7 monoclonal antibody against recombinant CD300c antigen was confirmed using enzyme-linked immunosorbent assay and surface plasmon resonance analysis. The binding affinity of CL7 was strong at the sub-nanomolar level. Furthermore, CL7 effectively bound to exogenously expressed CD300c on 293T cells. CL7 antibody differentiated monocytes to M1 macrophages, as evidenced by the upregulated expression of M1-specific cell surface markers and increased secretion of M1-specific cytokines in vitro in THP-1 cells and primary macrophages, as well as the increased population size of M1 macrophages in tumors grafted into mice. Additionally, CL7 treatment upregulated PD-L1 expression on THP-1 cells. We confirmed that the mechanism of M1 macrophage differentiation was through the mitogen-activated protein kinase and NF-κB signaling pathways. CD300c expression on various immune and cancer cells was similar to that of the well-known immune checkpoint PD-L1, suggesting the possibility of CD300c as a novel tumor biomarker. We also confirmed that the tumor size was substantially reduced by CL7 antibody treatment in the CT26 mouse model. Our study supports that CD300c is a potential therapeutic target in immuno-oncology. Overall, the CD300c-specific monoclonal antibody, CL7, is a promising immunotherapeutic agent, and it induces enhanced differentiation of M1 macrophages and/or their infiltration into the tumor microenvironment.

Recombinant CD300c-Ig fusion protein attenuates collagen-induced arthritis in mice.

OBJECTIVES: RA is a chronic autoimmune disease characterized by joint inflammation and tissue destruction. Immune responses mediated by T cells and autoantibodies are known to play critical roles in RA. Collagen type II (CII)-induced arthritis (CIA) is a commonly used animal model of human RA. We have previously reported the identification of a new T cell inhibitory molecule CD300c. Here we investigate the ability of recombinant CD300c-IgG2a Fc (CD300c-Ig) fusion protein to prevent and treat CIA. METHODS: Mice were induced to develop CIA by CII and injected with CD300c-Ig or control Ig protein before or after CIA symptoms occur. The mice were examined for CIA clinical and pathological scores, and analysed for the expression of proinflammatory cytokines, the percentage and activation of CD4 T cells and regulatory T cells, CII-specific T cell proliferation and cytokine production, and CII-specific autoantibody production. RESULTS: In a prevention model, CD300c-Ig significantly decreases CIA incidence, and reduces clinical and pathological arthritis scores. In the treatment model, CD300c-Ig ameliorates established CIA. The beneficial effects of CD300c-Ig are related to decreased expansion and activation of T cells in the spleen and reduced expression of proinflammatory cytokines in the joints. CD300c-Ig also inhibits CII-specific T cell proliferation and Th1 and Th17 cytokine production. In addition, CD300c-Ig treatment reduced the production of CII autoantibodies in the serum. Furthermore, CD300c-Ig inhibits the proliferation and activation of T cells from RA patients in vitro. CONCLUSION: CD300c-Ig protein has the potential to be used in the treatment of patients with RA.

T Cell Activation, Highly Armed Cytotoxic Cells and a Shift in Monocytes CD300 Receptors Expression Is Characteristic of Patients With Severe COVID-19.

COVID-19 manifests with a wide diversity of clinical phenotypes characterized by dysfunctional and exaggerated host immune responses. Many results have been described on the status of the immune system of patients infected with SARS-CoV-2, but there are still aspects that have not been fully characterized or understood. In this study, we have analyzed a cohort of patients with mild, moderate and severe disease. We performed flow cytometric studies and correlated the data with the clinical characteristics and clinical laboratory values of the patients. Both conventional and unsupervised data analyses concluded that patients with severe disease are characterized, among others, by a higher state of activation in all T cell subsets (CD4, CD8, double negative and T follicular helper cells), higher expression of perforin and granzyme B in cytotoxic cells, expansion of adaptive NK cells and the accumulation of activated and immature dysfunctional monocytes which are identified by a low expression of HLA-DR and an intriguing shift in the expression pattern of CD300 receptors. More importantly, correlation analysis showed a strong association between the alterations in the immune cells and the clinical signs of severity. These results indicate that patients with severe COVID-19 have a broad perturbation of their immune system, and they will help to understand the immunopathogenesis of COVID-19.

CD300c costimulates IgE-mediated basophil activation, and its expression is increased in patients with cow's milk allergy.

BACKGROUND: Basophils express high-affinity IgE receptors (FcεRI), which play an essential role in allergic diseases. It is important to characterize new cell-surface receptors that modulate IgE-mediated basophil activation threshold to design promising immunomodulatory therapies. OBJECTIVES: We sought to analyze the expression of CD300 receptors on human basophils and their implication in IgE-mediated basophil activation processes. METHODS: Blood samples from healthy subjects and patients with cow's milk allergy were collected through the Basque Biobank under an institutional review board-approved protocol. PBMCs were obtained by means of density centrifugation, basophils were purified with a specific isolation kit, and phenotypic and functional studies were performed by using flow cytometry. RESULTS: We demonstrate that basophils express the activating receptor CD300c, which is specifically upregulated in response to IL-3. CD300c works as a costimulatory molecule during IgE-mediated basophil activation, as shown by a significant increase in degranulation and cytokine production when basophils are activated in the presence of CD300c cross-linking compared with activation through the IgE/FcεRI axis alone. Coligation of FcεRI and CD300c increased intracellular calcium mobilization and phosphorylation of signaling intermediates evoked only by FcεRI ligation. We show that the natural ligands of CD300c, phosphatidylserine and phosphatidylethanolamine, modulate IgE-mediated basophil activation. Furthermore, we have observed that CD300c expression in children with cow's milk allergy is increased compared with that in healthy control subjects and that the intensity of expression correlates with the severity of the hypersensitivity symptoms. CONCLUSION: CD300c could be considered a biomarker and therapeutic target in patients with IgE-mediated allergic diseases because it seems to be involved in the modulation of IgE-mediated basophil activation.

Monocytes Phenotype and Cytokine Production in Human Immunodeficiency Virus-1 Infected Patients Receiving a Modified Vaccinia Ankara-Based HIV-1 Vaccine: Relationship to CD300 Molecules Expression.

A modified vaccinia Ankara-based HIV-1 vaccine clade B (MVA-B) has been tested for safety and immunogenicity in low-risk human immunodeficiency virus (HIV)-uninfected individuals and as a therapeutic vaccine in HIV-1-infected individuals on combined antiretroviral therapy (cART). As a therapeutic vaccine, MVA-B was safe and broadly immunogenic; however, patients still showed a viral rebound upon treatment interruption. Monocytes are an important part of the viral reservoir and several studies suggest that they are partly responsible for the chronic inflammation observed in cART-treated HIV-infected people. The CD300 family of receptors has an important role in several diseases, including viral infections. Monocytes express CD300a, c, e, and f molecules and lipopolysaccharide (LPS) and other stimuli regulate their expression. However, the expression and function of CD300 receptors on monocytes in HIV infection is still unknown. In this work, we investigated for the first time the expression of CD300 molecules and the cytokine production in response to LPS on monocytes from HIV-1-infected patients before and after vaccination with MVA-B. Our results showed that CD300 receptors expression on monocytes from HIV-1-infected patients correlates with markers of HIV infection progression and immune inflammation. Specifically, we observed a positive correlation between the expression of CD300e and CD300f receptors on monocytes with the number of CD4+ T cells of HIV-1-infected patients before vaccination. We also saw a positive correlation between the expression of the inhibitory receptor CD300f and the expression of CD163 on monocytes from HIV-1-infected individuals before and after vaccination. In addition, monocytes exhibited a higher cytokine production in response to LPS after vaccination, almost at the same levels of monocytes from healthy donors. Furthermore, we also described a correlation in the expression of CD300e and CD300f receptors with TNF-α production in response to LPS, only in monocytes of HIV-1-infected patients before vaccination. Altogether, our results describe the impact of HIV-1 and of the MVA-B vaccine in cytokine production and monocytes phenotype.