Human IgMhiCD300a+ B Cells Are Circulating Marginal Zone Memory B Cells That Respond to Pneumococcal Polysaccharides and Their Frequency Is Decreased in People Living with HIV.

CD300a is differentially expressed among B cell subsets, although its expression in immunoglobulin (Ig)M+ B cells is not well known. We identified a B cell subset expressing CD300a and high levels of IgM (IgMhiCD300a+). The results showed that IgMhiCD300a+ B cells were CD10-CD27+CD25+IgDloCD21hiCD23-CD38loCD1chi, suggesting that they are circulating marginal zone (MZ) IgM memory B cells. Regarding the immunoglobulin repertoire, IgMhiCD300a+ B cells exhibited a higher mutation rate and usage of the IgH-VDJ genes than the IgM+CD300a- counterpart. Moreover, the shorter complementarity-determining region 3 (CDR3) amino acid (AA) length from IgMhiCD300a+ B cells together with the predicted antigen experience repertoire indicates that this B cell subset has a memory phenotype. IgM memory B cells are important in T cell-independent responses. Accordingly, we demonstrate that this particular subset secretes higher amounts of IgM after stimulation with pneumococcal polysaccharides or a toll-like receptor 9 (TLR9) agonist than IgM+CD300a- cells. Finally, the frequency of IgMhiCD300a+ B cells was lower in people living with HIV-1 (PLWH) and it was inversely correlated with the years with HIV infection. Altogether, these data help to identify a memory B cell subset that contributes to T cell-independent responses to pneumococcal infections and may explain the increase in severe pneumococcal infections and the impaired responses to pneumococcal vaccination in PLWH.

E2f2 Attenuates Apoptosis of Activated T Lymphocytes and Protects from Immune-Mediated Injury through Repression of Fas and FasL.

Targeted disruption of E2f2 in mice causes T-cell hyperactivation and a disproportionate cell cycle entry upon stimulation. However, E2f2-/- mice do not develop a lymphoproliferative condition. We report that E2f2 plays a Fas-dependent anti-apoptotic function in vitro and in vivo. TCR-stimulated murine E2f2-/- T cells overexpress the proapoptotic genes Fas and FasL and exhibit enhanced apoptosis, which is prevented by treatment with neutralizing anti-FasL antibodies. p53 pathway is activated in TCR-stimulated E2f2-/- lymphocytes, but targeted disruption of p53 in E2f2-/- mice does not abrogate Fas/FasL expression or apoptosis, implying a p53-independent apoptotic mechanism. We show that E2f2 is recruited to Fas and FasL gene promoters to repress their expression. in vivo, E2f2-/- mice are prone to develop immune-mediated liver injury owing to an aberrant lymphoid Fas/FasL activation. Taken together, our results suggest that E2f2-dependent inhibition of Fas/FasL pathway may play a direct role in limiting the development of immune-mediated pathologies.

CD300 receptor family in viral infections.

The CD300 molecules constitute an evolutionarily significant family of receptors that are expressed on myeloid and lymphoid cells, but also on other cell types, such as tuft cells. Many of the CD300 receptors have been shown to recognize lipids, e.g. phosphatidylserine and phosphatidylethanolamine. Over the past couple of years, accumulating evidence has shown that this family of receptors is involved in the pathogenesis of many diseases. Specifically, CD300 molecules participate in the mechanisms that viruses employ to develop immune evasion strategies and to infect host cells. The participation of CD300 molecules in viral infection includes both lipid dependent and independent mechanisms, as for example in infections with dengue virus (DENV) and murine norovirus (MNV), respectively. CD300 receptors are also involved in viral escape mechanisms, for instance inhibiting NK cell-mediated cytotoxicity against infected cells. Moreover, it is becoming increasingly recognized that the expression of CD300 receptors is altered during viral diseases. Here, we review the involvement of human and murine CD300 molecules in viral binding and entry and in cellular responses to viruses, which highlights the potential of CD300 molecules in the search of new biomarkers for various stages of infection and therapeutic targets for the treatment of viral infections.

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.

The Expression and Function of CD300 Molecules in the Main Players of Allergic Responses: Mast Cells, Basophils and Eosinophils.

Allergy is the host immune response against non-infectious substances called allergens. The prevalence of allergic diseases is increasing worldwide. However, while some drugs counteract the symptomatology caused by allergic reactions, no completely effective treatments for allergic diseases have been developed yet. In this sense, the ability of surface activating and inhibitory receptors to modulate the function of the main effector cells of allergic responses makes these molecules potential pharmacological targets. The CD300 receptor family consists of members with activating and inhibitory capabilities mainly expressed on the surface of immune cells. Multiple studies in the last few years have highlighted the importance of CD300 molecules in several pathological conditions. This review summarizes the literature on CD300 receptor expression, regulation and function in mast cells, basophils and eosinophils, the main players of allergic responses. Moreover, we review the involvement of CD300 receptors in the pathogenesis of certain allergic diseases, as well as their prospective use as therapeutic targets for the treatment of IgE-dependent allergic responses.

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.

IL-12/15/18-induced cell death and mitochondrial dynamics of human NK cells.

Natural killer (NK) cells are lymphocytes with potent antitumor functions and, consequently, several NK cell-based strategies have been developed for cancer immunotherapy. A remarkable therapeutic approach is the adoptive transfer of NK cells stimulated with IL-12, IL-15 and IL-18. This cytokine stimulation endows NK cells with properties that resemble immunological memory and, for this reason, they are known as cytokine-induced memory-like (CIML) NK cells. Very promising results have been reported in clinical trials and yet, there are still unknown aspects of CIML NK cells. Here, we have conducted a preliminary study of their mitochondrial dynamics. Our results show that upon IL-12/15/18 stimulation the viability of NK cells decreased and an increment in mitochondrial superoxide levels was observed. In addition, we found that mitochondria appeared slightly elongated and their cristae density decreased following IL-12/15/18 stimulation, possibly in a process mediated by the low levels of optic atrophy type 1 (OPA1) protein. Interestingly, although mitophagy was slightly impaired, an increase in autophagic flux was observed, which might explain the reduced viability and the accumulation of unfit mitochondria. Our findings could be of relevance in order to design new strategies intended to improve the mitochondrial fitness of IL-12/15/18-stimulated NK cells with the aim of improving their therapeutic efficacy.

Turning universal O into rare Bombay type blood.

Red blood cell antigens play critical roles in blood transfusion since donor incompatibilities can be lethal. Recipients with the rare total deficiency in H antigen, the Oh Bombay phenotype, can only be transfused with group Oh blood to avoid serious transfusion reactions. We discover FucOB from the mucin-degrading bacteria Akkermansia muciniphila as an α-1,2-fucosidase able to hydrolyze Type I, Type II, Type III and Type V H antigens to obtain the afucosylated Bombay phenotype in vitro. X-ray crystal structures of FucOB show a three-domain architecture, including a GH95 glycoside hydrolase. The structural data together with site-directed mutagenesis, enzymatic activity and computational methods provide molecular insights into substrate specificity and catalysis. Furthermore, using agglutination tests and flow cytometry-based techniques, we demonstrate the ability of FucOB to convert universal O type into rare Bombay type blood, providing exciting possibilities to facilitate transfusion in recipients/patients with Bombay phenotype.

Cytokine-Induced Memory-Like NK Cells: From the Basics to Clinical Applications.

Natural killer (NK) cells are lymphocytes with a key role in the defense against viral infections and tumor cells. Although NK cells are classified as innate lymphoid cells (ILCs), under certain circumstances they exhibit adaptive and memory-like features. The latter may be achieved, among others, by a brief stimulation with interleukin (IL)-12, IL-15 and IL-18. These cytokine-induced memory-like (CIML) NK cells resemble the trained immunity observed in myeloid cells. CIML NK cells undergo transcriptional, epigenetic and metabolic reprogramming that, along with changes in the expression of cell surface receptors and components of cytotoxic granules, are responsible for their enhanced effector functions after a resting period. In addition, these memory-like NK cells persist for a long time, which make them a good candidate for cancer immunotherapy. Currently, several clinical trials are testing CIML NK cells infusions to treat tumors, mostly hematological malignancies. In relapse/refractory acute myeloid leukemia (AML), the adoptive transfer of CIML NK cells is safe and complete clinical remissions have been observed. In our review, we sought to summarize the current knowledge about the generation and molecular basis of NK cell memory-like responses and the up-to-date results from clinical trials with CIML NK cells.

In vivo expansion of a CD9+ decidual-like NK cell subset following autologous hematopoietic stem cell transplantation.

Autologous hematopoietic stem cell transplantation (autoHSCT) is a treatment option for hematological disorders and pediatric solid tumors. After an autoHSCT, natural killer (NK) cells are the first lymphocyte subset returning to normal levels. To uncover global changes during NK cell reconstitution after autoHSCT, we performed RNA-sequencing on NK cells before and after autoHSCT. Results showed profound changes in the gene expression profile of NK cells immediately after autoHSCT. Several biological processes including cell cycle, DNA replication and the mevalonate pathway were enriched. Significantly, we observed that following autoHSCT, NK cells acquired a decidual-like gene expression profile, including the expression of CD9. By using multiparametric flow cytometry, we confirmed the expansion of NK cells expressing CD9 immediately after autoHSCT, which exhibited higher granzyme B and perforin expression levels than CD9- NK cells. These results provide insights into the physiopathology of NK cells during their reconstitution after autoHSCT.

Human NK Cells in Autologous Hematopoietic Stem Cell Transplantation for Cancer Treatment.

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes with the ability to recognize and kill malignant cells without prior sensitization, and therefore, they have a relevant role in tumor immunosurveillance. NK cells constitute the main lymphocyte subset in peripheral blood in the first week after hematopoietic stem cell transplantation (HSCT). Although the role that NK cells play in allogenic HSCT settings has been documented for years, their significance and beneficial effects associated with the outcome after autologous HSCT are less recognized. In this review, we have summarized fundamental aspects of NK cell biology, such as, NK cell subset diversity, their effector functions, and differentiation. Moreover, we have reviewed the factors that affect autologous HSCT outcome, with particular attention to the role played by NK cells and their receptor repertoire in this regard.

Metabolic changes of Interleukin-12/15/18-stimulated human NK cells.

Natural Killer (NK) cells acquire memory-like properties following a brief stimulation with IL-12, IL-15 and IL-18. These IL-12/15/18-preactivated NK cells, also known as cytokine-induced memory-like (CIML) NK cells, have been revealed as a powerful tool in cancer immunotherapy due to their persistence in the host and their increased effector functions. Several studies have shown that NK cells modulate their metabolism in response to cytokine-stimulation and other stimuli, suggesting that there is a link between metabolism and cellular functions. In this paper, we have analyzed metabolic changes associated to IL-12/15/18-stimulation and the relevance of glycolytic pathway for NK cell effector functions. We have found CIML NK cells are able to retain a metabolic profile shifted towards glycolysis seven days after cytokine withdrawal. Furthermore, we found that treatment with 2-DG differently affects distinct NK cell effector functions and is stimuli-dependent. These findings may have implications in the design of NK cell-based cancer immunotherapies.

Increased Frequency of CTLA-4 and PD-1 Expressing Regulatory T Cells and Basophils With an Activating Profile in Infants With Moderate-to-Severe Atopic Dermatitis Hypersensitized to Food Allergens.

Background: Infants with severe atopic dermatitis (AD) may be sensitized to foods that have not been introduced into their diet, posing a risk for developing an immediate hypersensitivity reaction on the first exposure to the food to which they are sensitized. The aim of this work was to perform an analysis of the sensitization profile in infants with moderate-to-severe AD and to identify cellular and molecular markers for food allergy (FA). Methods: Blood samples from healthy donors and children with moderate-to-severe AD were studied. Specific IgE to several allergens were determined using ImmunoCAP FEIA system and ISAC technology. Furthermore, using flow cytometry-based studies, basophils and regulatory T (Treg) cells were phenotypically characterized. Results: 90% of children with AD were sensitized to food antigens before introducing them into the diet, and 100% developed FA. Phenotypic analysis showed a significantly higher percentage of CTLA-4 and PD-1 expressing Treg cells in AD patients than in healthy controls. Basophils from patients exhibited a marked reduction in the expression of CD300a, higher expression of FcεRI and CXCR4, and to some extent higher expression of CD63 and CD300c. Conclusions: Infants with moderate-to-severe AD are at high risk of being sensitized to food allergens. Therefore, to avoid allergic reactions, broad-spectrum sensitization studies are necessary before introducing complementary diet. Increased expression of CTLA-4 and PD-1 suggests greater suppressive potential of Treg cells in infants with AD than healthy controls. Furthermore, our results suggest a role for CD300 molecules on circulating basophils as possible biomarkers for FA susceptibility.

NK Cell Reconstitution After Autologous Hematopoietic Stem Cell Transplantation: Association Between NK Cell Maturation Stage and Outcome in Multiple Myeloma.

Autologous hematopoietic stem cell transplantation (autoHSCT) is a standard of care for transplant-eligible patients with multiple myeloma (MM). Among factors that influence outcome after autoHSCT, it has been suggested that the number of natural killer (NK) cells plays an important role. However, the impact that different NK cell subsets and their phenotype could have in disease progression after autoHSCT are less clear. For this reason, we have phenotypically and functionally characterized NK cells during immune system reconstitution after autoHSCT in 54 MM patients. Shortly after leukocyte recovery, an extensive redistribution of NK cell subsets occurs in these patients. In addition, NK cells undergo a profound phenotypic change characterized, among others, by their increased proliferative capacity and immature phenotype. Importantly, MM patients who showed lower frequencies of the mature highly differentiated NKG2A-CD57+ NK cell subset at +30 and +100 days after autoHSCT experienced superior progression-free survival and had a longer time to the next treatment than those with higher frequencies. Our results provide significant insights into NK cell reconstitution after autoHSCT and suggest that the degree of NK cell maturation after autoHSCT affects the clinical outcome of MM patients treated with this therapeutic strategy.

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.

CFSE dilution to study human T and NK cell proliferation in vitro.

Lymphocytes proliferate in response to several stimuli. In many situations, a rapid lymphocyte expansion, or the identification of a slow dividing cell subpopulation may be of great interest. Thus, it is necessary to perform reliable assays to study and compare lymphocyte subsets proliferation. For this purpose, carboxifluorescein diacetate succinimidyl ester (CFSE) dilution assay has been stablished as a very useful tool that provides cumulative information about cell proliferation. Unlike other techniques that measure a static parameter of a specific time-point, CFSE staining allows to distinguish between subsequent cell divisions. Here, we show a simple protocol to study human T and NK cell proliferation with CFSE dilution assay by flow cytometry.

Identification and Functional Analysis of Human CD56neg NK Cells by Flow Cytometry.

Although scarce in the peripheral blood of healthy people, CD56neg NK cells are known to be expanded in some pathological conditions. However, studies on CD56neg NK cells had revealed contradictions, probably due to the lack of a specific NK cell surface marker that helps to identify this subset. This protocol details the step-by-step procedure for the identification and functional analysis of CD56neg NK cells, providing an improved gating strategy for the selection of this intriguing population. For complete details on the use and execution of this protocol, please refer to Orrantia et al. (2020).

Modulating NK cell metabolism for cancer immunotherapy.

Natural killer (NK) cells are lymphocytes with potent antitumor functions and, therefore, multiple NK cell-based cancer immunotherapies have been developed and are currently being tested. However, there is a necessity to find new means to improve these therapies, and immunometabolism represents an attractive target. NK cell effector functions are intricately linked to their metabolism, and modulating the latter could be the key to release their full potential. In this review, we have summarized how NK cell metabolism is regulated during some processes, such as maturation, viral infection, and cytokine stimulation. Additionally, we provide an overview of how NK cell metabolism is affected by current therapeutic approaches aimed to promote NK cell expansion and/or to increase their effector functions. We have also recapitulated several strategies that could help alleviating the metabolic impairment that characterizes tumor-infiltrating NK cells, and thus increase or restore their effector functions. Furthermore, we have reviewed several therapeutic approaches targeting cancer metabolism that could synergize with NK cell-based cancer immunotherapies, and thus enhance their efficacy.