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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.

Human CD300a is known to promote the infection by dengue and other enveloped viruses and is overexpressed on CD4+ T cells from HIV-1-infected patients. We found that infected CD4+RA− T cells from untreated HIV-1-infected patients were mostly CD300a+. Furthermore, CD300a expressing CD4+RA− T cells from healthy donors were significantly more infected by HIV-1 in vitro than CD300a− cells. CD300a might represent a biomarker of susceptibility to HIV-1 infection on memory CD4+ T lymphocytes.

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.

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.

Natural Killer (NK) cells are characterized by their potential to kill tumor cells by different means without previous sensitization and have, therefore, become a valuable tool in cancer immunotherapy. However, their efficacy against solid tumors is still poor and further studies are required to improve it. One of the major restrictions for NK cell activity is the immunosuppressive tumor microenvironment (TME). There, tumor and other immune cells create the appropriate conditions for tumor proliferation while, among others, preventing NK cell activation. Furthermore, NK cell metabolism is impaired in the TME, presumably due to nutrient and oxygen deprivation, and the higher concentration of tumor-derived metabolic end products, such as lactate. This metabolic restriction of NK cells limits their effector functions, and it could represent a potential target to focus on to improve the efficacy of NK cell-based therapies against solid tumors. In this review, we discuss the potential effect of TME into NK cell metabolism and its influence in NK cell effector functions.

The human IL22RA2 gene co-produces three protein isoforms in dendritic cells [IL-22 binding protein isoform-1 (IL-22BPi1), IL-22BPi2, and IL-22BPi3]. Two of these, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame 32-amino acid insertion provided by an alternatively spliced exon, remains unknown. Using transfected human cell lines, we demonstrate that IL-22BPi1 is secreted detectably, but at much lower levels than IL-22BPi2, and unlike IL-22BPi2 and IL-22BPi3, is largely retained in the endoplasmic reticulum (ER). As opposed to IL-22BPi2 and IL-22BPi3, IL-22BPi1 is incapable of neutralizing or binding to IL-22 measured in bioassay or assembly-induced IL-22 co-folding assay. We performed interactome analysis to disclose the mechanism underlying the poor secretion of IL-22BPi1 and identified GRP78, GRP94, GRP170, and calnexin as main interactors. Structure-function analysis revealed that, like IL-22BPi2, IL-22BPi1 binds to the substrate-binding domain of GRP78 as well as to the middle domain of GRP94. Ectopic expression of wild-type GRP78 enhanced, and ATPase-defective GRP94 mutant decreased, secretion of both IL-22BPi1 and IL-22BPi2, while neither of both affected IL-22BPi3 secretion. Thus, IL-22BPi1 and IL-22BPi2 are bona fide clients of the ER chaperones GRP78 and GRP94. However, only IL-22BPi1 activates an unfolded protein response (UPR) resulting in increased protein levels of GRP78 and GRP94. Cloning of the IL22RA2 alternatively spliced exon into an unrelated cytokine, IL-2, bestowed similar characteristics on the resulting protein. We also found that CD14(++)/CD16(+) intermediate monocytes produced a higher level of IL22RA2 mRNA than classical and non-classical monocytes, but this difference disappeared in immature dendritic cells (moDC) derived thereof. Upon silencing of IL22RA2 expression in moDC, GRP78 levels were significantly reduced, suggesting that native IL22RA2 expression naturally contributes to upregulating GRP78 levels in these cells. The IL22RA2 alternatively spliced exon was reported to be recruited through a single mutation in the proto-splice site of a Long Terminal Repeat retrotransposon sequence in the ape lineage. Our work suggests that positive selection of IL-22BPi1 was not driven by IL-22 antagonism as in the case of IL-22BPi2 and IL-22BPi3, but by capacity for induction of an UPR response. This work was supported by the following grants: Grupos de Investigacion (IT512-10, PPG17/44) and MINECO (SAF2012-32118, SAF2016-74891R).

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.

Background: Sepsis is a life-threatening illness with a challenging diagnosis. Current serum biomarkers are not sensitive enough for diagnosis. With the aim of finding proteins associated with sepsis, serum protein profile was compared between patients and healthy donors and serum classical inflammatory proteins were analyzed in both groups. Methods: Serum protein profiles were characterized by two-dimensional electrophoresis (2DE). Identification of the proteins was carried out by mass spectrophotometry and their validation was performed by Enzyme-Linked-ImmunoSorbent Assay (ELISA) in a cohort of 85 patients and 67 healthy donors. Seven classical inflammatory proteins were analyzed in the same cohort by ELISA: interleukin-2 receptor α-chain (sCD25), scavenger receptor cysteine-rich-type-1 (sCD163), tumor-necrosis factor receptor superfamily-member-6 (sFas), hemeoxigenase-1 decycling (HO-1), interleukin-6 (IL-6), interleukin-18 (IL-18) and intercellular adhesion-molecule-1 (sICAM-1). Results: After 2DE, 20 significantly differently expressed spots were identified by mass spectrometry analysis, revealing deregulation of six different proteins upon sepsis and 50% were validated by ELISA: Antithrombin-III (AT-III), Clusterin (CLUS) and Serum amyloid A-1 (SAA-1). Serum concentration of AT-III and CLUS was significantly lower in patients′ serum, whereas SAA-1 showed higher values in septic patients. Serum concentration of the seven inflammatory proteins was significantly increased in septic patients. Functional analysis of the ten deregulated proteins revealed an enrichment of proteins related mainly to the activation of the immune response. Conclusion: We have identified a panel of ten potential sepsis marker proteins biologically connected and validated in a large number of patients, whose analysis could be considered as a complementary tool for the diagnosis of sepsis. Keywords: ELISA, Mass spectrometry analysis, Proteome, Sepsis, Septic shock, Serum markers

The ability of the CD300a inhibitory receptor to modulate immune cell functions and its involvement in the pathogenesis of many diseases has aroused a great interest in this molecule. Within human CD4+ T lymphocytes from healthy donors, the inhibitory receptor CD300a is differentially expressed among different T helper subsets. However, there are no data about the expression and regulation of CD300a receptor on CD4+ T cells from human immunodeficiency virus (HIV)-infected patients. The objective of this study was to investigate the expression of CD300a on CD4+ T cells from HIV-infected patients on suppressive combined antiretroviral therapy (cART) and cART naïve patients. Our results have demonstrated that the expression levels of this inhibitory receptor were higher on CD4+ T cells from HIV infected subjects compared with healthy donors, and that cART did not reverse the altered expression of CD300a receptor in these patients. We have observed an increase of CD300a expression on both PD1+CD4+ and CD38+CD4+ T cells from HIV infected people. Interestingly, a triple positive (CD300a+PD1+CD38+) subset was expanded in naïve HIV infected patients, while it was very rare in healthy donors and patients on cART. Finally, we found a negative correlation of CD300a expression on CD4+ T lymphocytes and some markers associated with HIV disease progression. Thus, our results show that HIV infection has an impact in the regulation of CD300a inhibitory receptor expression levels, and further studies will shed light into the role of this cell surface receptor in the pathogenesis of HIV infection., This study was supported by a grant from “Plan Estatal de I + D + I 2013–2016, ISCIII-Subdirección de Evaluación y Fomento de la Investigación-Fondo Europeo de Desarrollo Regional (FEDER) (Grant PI13/00889)” and Marie Curie Actions, Career Integration Grant, European Commission (Grant CIG 631674). JV is recipient of a predoctoral contract funded by the Department of Education, Language Policy and Culture, Basque Government (PRE_2017_2_0242). JV and IT are recipients of a fellowship from the Jesús de Gangoiti Barrera Foundation (FJGB15/008 and FJGB17/003). LT-D was supported by Instituto de Salud Carlos III, PFIS (FI00/00431). OZ is recipient of a postdoctoral contract funded by “Instituto de Salud Carlos III-Contratos Sara Borrell 2017 (CD17/0128)” and the European Social Fund (ESF)-The ESF invests in your future. ER-M is supported by Programa Nicolás Monardes, C0032-2017, Consejería de Salud, Junta de Andalucía. FB is an Ikerbasque Research Professor, Ikerbasque, Basque Foundation for Science.

A brief in vitro stimulation of natural killer (NK) cells with interleukin (IL)-12, IL-15 and IL-18 endow them a memory-like behavior, characterized by higher effector responses when they are restimulated after a resting period of time. These preactivated NK cells, also known as cytokine-induced memory-like (CIML) NK cells, have several properties that make them a promising tool in cancer immunotherapy. In the present study, we have described the effect that different combinations of IL-12, IL-15 and IL-18 have on the generation of human CIML NK cells. Our data points to a major contribution of IL-15 to CIML NK cell mediated cytotoxicity against target cells. However, the synergistic effect of the three cytokines grant them the best polyfunctional profile, that is, cells that simultaneously degranulate (CD107a) and produce multiple cytokines and chemokines such as interferon (IFN)γ, tumor necrosis factor (TNF)α and C-C motif chemokine ligand (CCL)3. We have also analyzed the involvement of each cytokine and their combinations in the expression of homing receptors CXCR4 and CD62L, as well as the expression of CD25 and IL-2-induced proliferation. Furthermore, we have tested the effects of the Jak1/2 inhibitor ruxolitinib in the generation of CIML NK cells. We found that ruxolitinib-treated CIML NK cells expressed lower levels of CD25 than non-treated CIML NK cells, but exhibited similar proliferation in response to IL-2. In addition, we have also found that ruxolitinib-treated NK cells displayed reduced effector functions after the preactivation, which can be recovered after a 4 days expansion phase in the presence of low doses of IL-2. Altogether, our results describe the impact that each cytokine and the Jak1/2 pathway have in the phenotype, IL-2 induced proliferation and effector functions of human CIML NK cells.

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.

Background Basophils express high-affinity IgE receptors (FceRI), 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/FceRI axis alone. Coligation of FceRI and CD300c increased intracellular calcium mobilization and phosphorylation of signaling intermediates evoked only by FceRI 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.

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.

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of mature and functional NK cells. An option for future immunotherapy treatments is to use large amounts of NK cells derived and differentiated from umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), mainly because UCB is one of the most accessible HSC sources. In our study, we compared the potential of two stromal cell lines, OP9 and M2-10B4, for in vitro generation of mature and functional CD56+ NK cells from UCB CD34+ HSC. We generated higher number of CD56+ NK cells in the presence of the OP9 cell line than when they were generated in the presence of M2-10B4 cells. Furthermore, higher frequency of CD56+ NK cells was achieved earlier when cultures were performed with the OP9 cells than with the M2-10B4 cells. Additionally, we studied in detail the maturation stages of CD56+ NK cells during the in vitro differentiation process. Our data show that by using both stromal cell lines, CD34+ HSC in vitro differentiated into the terminal stages 4-5 of maturation resembled the in vivo differentiation pattern of human NK cells. Higher frequencies of more mature NK cells were reached earlier by using OP9 cell line than M2-10B4 cells. Alternatively, we observed that our in vitro NK cells expressed similar levels of granzyme B and perforin, and there were no significant differences between cultures performed in the presence of OP9 cell line or M2-10B4 cell line. Likewise, degranulation and cytotoxic activity against K562 target cells were very similar in both culture conditions. The results presented here provide an optimal strategy to generate high numbers of mature and functional NK cells in vitro, and point toward the use of the OP9 stromal cell line to accelerate the culture procedure to obtain them. Furthermore, this method could establish the basis for the generation of mature NK cells ready for cancer immunotherapy.

CD16 (FcγRIIIa), the low-affinity receptor for IgG, expressed by the majority of human NK cells, is a potent activating receptor that facilitates Ab-dependent cell-mediated cytotoxicity (ADCC). ADCC dysfunction has been linked to cancer progression and poor prognosis for chronic infections, such as HIV; thus, understanding how CD16 expression is regulated by NK cells has clinical relevance. Importantly, CD16 cell-surface expression is downmodulated following NK cell activation and, in particular, exposure to stimulatory cytokines (IL-2 or IL-15), likely owing to the action of matrix metalloproteinases (MMPs). In this article, we identify membrane-type 6 (MT6) MMP (also known as MMP25) as a proteinase responsible for CD16 downmodulation. IL-2–induced upregulation of MT6/MMP25 cell-surface expression correlates with CD16 downmodulation. MT6/MMP25, sequestered in intracellular compartments in unstimulated NK cells, translocates to the cell surface after stimulation; moreover, it polarizes to the effector–target cell interface of the CD16-mediated immunological synapse. siRNA-mediated disruption of MT6/MMP25 expression enhances the ADCC capacity of NK cells, emphasizing the important functional role of MT6/MMP25 in the regulation of ADCC activity. Thus, this study uncovers a previously unknown role of MT6/MMP25 in human NK cells, and suggests that inhibition of MT6/MMP25 activity could improve ADCC efficacy of therapeutically administered NK cells that require IL-2 for culture and expansion.

Summary Activation of human natural killer (NK) cells is associated with the cleavage of CD16 from the cell surface, a process mediated by matrix metalloproteinases (MMPs). In this report, we examined whether inhibition of MMPs would lead to improved NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) function. Using an in-vitro ADCC assay, we tested the anti-tumour function of NK cells with three different therapeutic monoclonal antibodies (mAbs) in the presence of MMPs inhibitor GM6001 or its control. Loss of CD16 was observed when NK cells were co-cultured with tumour targets in the presence of specific anti-tumour antibodies, and was found particularly on the majority of degranulating NK responding cells. Treatment with MMPs inhibitors not only prevented CD16 down-regulation, but improved the quality of the responding cells significantly, as shown by an increase in the percentage of polyfunctional NK cells that are capable of both producing cytokines and degranulation. Furthermore, MMPs inhibition resulted in augmented and sustained CD16-mediated signalling, as shown by increased tyrosine phosphorylation of CD3ζ and other downstream signalling intermediates, which may account for the improved NK cell function. Collectively, our results provide a foundation for combining MMPs inhibitors and therapeutic mAbs in new clinical trials for cancer treatment.

FcR specific for pentameric IgM (FCMR) 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 that lack FCMR. FCMR transcripts are expressed at different levels by various B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic follicular and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. After 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.

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.

• BIOEF-EiTB Maratoia Pediatric Cancer. Beca BIO13/CI/011, para Francisco Borrego Rabasco • BIOEF-EiTB Maratoia Transplantation. Beca BIO14/TP/001, para Susana Larrucea Bilbao • Ministerio de Economia y Competitividad. Beca SAF2013-46161-R, para Raquel Tarazona Rabasco • Ministerio de Salud. Beca PI13/02691, para Rafael Solana Lara

We find that the cell surface receptor Toso is dramatically downregulated 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 those of naive T cells, indicating that activation in vivo correlates with the downregulation of Toso. Moreover, in vitro activation of memory T cells through TCR dramatically downregulates Toso expression compared with that of naive CD4 T cells. However, overexpression 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 antiapoptotic molecule.

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.

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 article, we investigate the effect of a conservative R65K mutation on LAIR-1 ligand binding and function. Compared with 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 analyses showed that LAIR-1 R65K protein has markedly reduced avidity for collagen type I compared with LAIR-1 wt. Likewise, LAIR-1 R65K protein has decreased avidity for cells expressing transmembrane collagen XVII. Thus, a single residue, Arg65, is critical for the interaction of LAIR-1 with collagens.

Fc receptor–like (FCRL) 5 is a novel IgG binding protein expressed on B cells, with the capacity to regulate Ag receptor signaling. We assessed FCRL5 expression on circulating B cells from healthy donors and found that FCRL5+ cells are most enriched among atypical CD21−/lo/CD27− tissue-like memory (TLM) B cells, which are abnormally expanded in several autoimmune and infectious diseases. Using multicolor flow cytometry, FCRL5+ TLM cells were found to express more CD11c and several inhibitory receptors than did the FCRL5− TLM subset. The homing receptor profiles of the two TLM subsets shared features consistent with migration away from lymphoid tissues, but they also displayed distinct differences. Analysis of IgH V regions in single cells indicated that although both subsets are diverse, the FCRL5+ subset accumulated significantly more somatic mutations. Furthermore, the FCRL5+ subset had more switched isotype expression and more extensive proliferative history. Microarray analysis and quantitative RT-PCR demonstrated that the two TLM subsets possess distinct gene expression profiles, characterized by markedly different CD11c, SOX5, T-bet, and RTN4R expression, as well as differences in expression of inhibitory receptors. Functional analysis revealed that the FCRL5+ TLM subset responds poorly to multiple stimuli compared with the FCRL5− subset, as reflected by reduced calcium mobilization and blunted cell proliferation. We propose that the FCRL5+ TLM subset, but not the FCRL5− TLM subset, underwent Ag-driven development and is severely dysfunctional. The present study elucidates the heterogeneity of TLM B cells and provides the basis to dissect their roles in the pathogenesis of inflammatory and infectious diseases.

An adequate immune response is the result of the fine balance between activation and inhibitory signals. The exact means by which inhibitory signals obviate activation signals in immune cells are not totally elucidated. Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and some CD8+ T cells that recognize HLA-E. We show that the engagement of this receptor prevents NK cell activation by disruption of the actin network and exclusion of lipid rafts at the point of contact with its ligand (inhibitory NK cell immunological synapse, iNKIS). CD94/NKG2A engagement leads to recruitment and activation of src homology 2 domain-bearing tyrosine phosphatase 1. This likely explains the observed dephosphorylation of guanine nucleotide exchange factor and regulator of actin, Vav1, as well as ezrin-radixin-moesin proteins that connect actin filaments to membrane structures. In contrast, NK cell activation by NKG2D induced Vav1 and ezrin-radixin-moesin phosphorylation. Thus, CD94/NKG2A prevents actin-dependent recruitment of raft-associated activation receptors complexes to the activating synapse. This was further substantiated by showing that inhibition of actin polymerization abolished lipid rafts exclusion at the iNKIS, whereas cholesterol depletion had no effect on actin disruption at the iNKIS. These data indicate that the lipid rafts exclusion at the iNKIS is an active process which requires an intact cytoskeleton to maintain lipid rafts outside the inhibitory synapse. The net effect is to maintain an inhibitory state in the proximity of the iNKIS, while allowing the formation of activation synapse at distal points within the same NK cell.

IL-21 is a recently described cytokine, produced by activated Th cells, that shares significant homology with members of the IL-2 family of cytokines. IL-21 mediates its biological effects via the IL-21R in conjunction with the common receptor γ-chain that is also shared by members of the IL-2 family. We report that culture of human primary NK and CD8+ T cells with IL-21 in combination with IL-2 results in significant reduction of the cell surface expression of NKG2D, compared with that in cells treated with IL-2 alone. The reduced expression of NKG2D after IL-21 culture had functional consequences for NK cell function, as assessed by NKG2D-mediated redirected lysis assays and degranulation assays, compared with NK cells treated with IL-2 alone. IL-21-mediated NKG2D down-regulation in human NK cells correlated with a marked reduction of DNAX-activating protein of 10 kDa (DAP10) transcription in cells treated with IL-2 in combination with IL-21 compared with cells stimulated with only IL-2. This was attributed to a dramatic reduction in DAP10 promoter activity, as assessed by a DAP10 luciferase reporter construct. In contrast to NKG2D expression, IL-21 was able to induce the expression of the NK activation receptors NKp30 and 2B4 as well as the costimulatory receptor CD28 on CD8+ T cells. These data indicate that IL-21 is able to channel NK and CD8+ T cell function by altering the expression pattern of activation/costimulatory receptors.

CD94/NKG2A is an inhibitory receptor expressed by most human NK cells and a subset of T cells that recognizes HLA-E on potential target cells. To study the transcriptional regulation of the human NKG2A gene, we cloned a 3.9-kb genomic fragment that contains a 1.65-kb region upstream of the exon 1, as well as exon 1 (untranslated), intron 1 and exon 2. Using deletion mutants, we identified a region immediately upstream from the most upstream transcriptional initiation site that led to increased transcriptional activity from a luciferase reporter construct in YT-Indy (NKG2A positive) cells relative to Jurkat and K562 (both NKG2A negative) cells. We also localized a DNase I hypersensitivity site to this region. Within this 80-bp segment, we identified two GATA binding sites. Mutation of GATA binding site II (−2302 bp) but not GATA binding site I (−2332 bp) led to decreased transcriptional activity. Pull-down assays revealed that GATA-3 could bind oligonucleotide probes containing the wild type but not a mutated GATA site II. Using chromatin immunoprecipitation assays, we showed that GATA-3 specifically binds to the NKG2A promoter in situ in NKL and primary NK cells, but not in Jurkat T cells. Moreover, coexpression of human GATA-3 with an NKG2A promoter construct in K562 cells led to enhanced promoter activity, and transfection of NKL cells with small interfering RNA specific for GATA-3 reduced NKG2A cell surface expression. Taken together, our data indicate that GATA-3 is an important transcription factor for regulating NKG2A gene expression.

Natural killer (NK) cell lines are useful for studying facets of NK cell biology. Such cell lines are notoriously difficult to transfect by traditional methods, a fact that has hampered NK cell biology studies for a long time. To overcome this, we investigated the use of the Amaxa nucleofection system that directly transfers DNA into the nucleus of the cell. This technology has revolutionized transfection studies with heretofore relatively transfection resistant cell types such as T cells, B cells and dendritic cells. Despite these advances, NK cells and NK cell lines have remained relatively resistant to transfection, including nucleofection. In this study we employed cDNA for SHP1 and various Rab proteins cloned in enhanced green/yellow fluorescent protein (EGFP/EYFP) expression plasmids for transient transfections into NKL cells. The expression of EGFP/EYFP fusion proteins was analyzed by flow cytometry, immunoblot and confocal microscopic analyses. We achieved 40-70% transfection efficiency with high levels of expression in this cell line with 85-90% viability. The method used in this report proves to be far superior to existing methods for delivering DNA into this well studied NK cell line and, consequently, provides new experimental opportunities.

CD94 is a C-type lectin required for the dimerization of the CD94/NKG2 family of receptors, which are expressed on NK cells and T cell subsets. Little is known about CD94 gene expression and the elements that regulate CD94 transcription. In this study, we report that CD94 gene expression is regulated by distal and proximal promoters that transcribe unique initial exons specific to each promoter. This results in two species of transcripts; the previously described CD94 mRNA and a novel CD94C mRNA. All NK cells and CD94+, CD8+ αβ T cells transcribe CD94 mRNA. Stimulation of NK and CD8+ αβ T cells with IL-2 or IL-15 induced the transcription of CD94C mRNA. The distal and proximal promoters both contain elements with IFN-γ-activated and Ets binding sites, known as GAS/EBS. Additionally, an unknown element, termed site A, was identified in the proximal promoter. EMSA analyses showed that constitutive factors could bind to oligonucleotide probes containing each element. After treatment of primary NK cells with IL-2 or IL-15, separate inducible complexes could be detected with oligonucleotide probes containing either the proximal or distal GAS/EBS elements. These elements are highly conserved between mice and humans, which suggests that both species regulate CD94 gene expression via mechanisms that predate their evolutionary divergence.

Natural killer (NK) cells play an essential role in the fight against tumor development. Over the last years, the progress made in the NK cell biology field and in deciphering how NK cell function is regulated, is driving efforts to utilize NK cell-based immunotherapy as a promising approach for the treatment of malignant diseases. Therapies involving NK cells may be accomplished by activating and expanding endogenous NK cells by means of cytokine treatment or by transferring exogenous cells by adoptive cell therapy and/or by hematopoietic stem cell transplantation (HSCT). NK cells that are suitable for adoptive cell therapy can be derived from different sources, including ex vivo expansion of autologous NK cells, unstimulated or expanded allogeneic NK cells from peripheral blood, derived from CD34+ hematopoietic progenitors from peripheral blood and umbilical cord blood, and NK cell lines. Besides, genetically modified NK cells expressing chimeric antigen receptors (CARs) or cytokines genes may also have a relevant future as therapeutic tools. Recently, it has been described the derivation of large numbers of functional and mature NK cells from pluripotent stem cells (PSCs), both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which adds another tool to the expanding NK cell-based cancer immunotherapy arsenal.

CD69 is a differentiation antigen expressed shortly after activation on T lymphocytes and other cells of haematopoietic origin, including natural killer (NK) cells. The function of CD69 on T lymphocytes acting as a costimulatory molecule in proliferation and lymphokine secretion is well established. NK cells express CD69 after activation by different stimuli such as phorbol 12-myristate 13-acetate (PMA), interleukin (IL)-2, IL-12, interferon-alpha (IFN-alpha) or anti-CD16 monoclonal antibodies (mAbs). However, although it has been shown that CD69 triggers NK-cell-mediated cytolytic activity, its effect on other NK-cell functions has not been studied. Furthermore, the possible interaction of CD69 triggering with other C-lectin type inhibitory receptors is not known. Thus, the objective of this work is to determine whether CD69-mediated NK cytotoxicity can be regulated by CD94 inhibitory receptor and the role of CD69 on other NK-cell functions different of cytotoxicity. The results show that CD69-mediated NK cytotoxicity can be abrogated by CD94 stimulation in NK cells expressing the CD94 inhibitory form of the receptor, indicating that CD94 regulates the cytotoxic events initiated by a wide variety of NK activatory receptors. We also show that anti-CD69 mAbs, not only triggered NK cytotoxicity, but also induce NK-cell proliferation, CD25 and intracellular adhesion molecule-1 (ICAM-1) expression, TNF-alpha production and Ca2+ mobilization in preactivated NK cells. These results suggest that CD69 plays a crucial role in NK-cell function contributing to sustain NK-cell activation, as it has been previously demonstrated in T cells.

The CD94/NKG2 receptors expressed by subpopulations of NK cells and T cells have been implicated as receptors for a broad range of both classical and nonclassical HLA class I molecules. To examine the ligand specificity of CD94/NKG2 proteins, a soluble heterodimeric form of the receptor was produced and used in direct binding studies with cells expressing defined HLA class I/peptide complexes. We confirm that CD94/NKG2A specifically interacts with HLA-E and demonstrate that this interaction is dependent on the association of HLA-E with peptide. Moreover, no interaction between CD94/NKG2A and classical HLA class I molecules was observed, as assayed by direct binding of the soluble receptor or by functional assays using CD94/NKG2A+ NK cells. The role of the peptide associated with HLA-E in the interaction between HLA-E and CD94/NKG2A was also assessed. All class I leader sequence peptides tested bound to HLA-E and were recognized by CD94/NKG2A. However, amino acid variations in class I leader sequences affected the stability of HLA-E. Additionally, not all HLA-E/peptide complexes examined were recognized by CD94/NKG2A. Thus CD94/NKG2A recognition of HLA-E is controlled by peptide at two levels; first, peptide must stabilize HLA-E and promote cell surface expression, and second, the HLA-E/peptide complex must form the ligand for CD94/NKG2A.

Cross-linking of the collagen binding receptor leukocyte-associated Ig-like receptor-1 (LAIR-1) in vitro delivers an inhibitory signal that is able to downregulate 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, regulatory T, and dendritic cells. As LAIR-1−/− mice age, the splenic T cell population shows a higher frequency of activated and memory T cells. Because 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 trinitrophenyl-OVA, switch less efficiently to Ag specific IgG2a and IgG2b, whereas switching to IgG1 is not affected. Several mouse disease models, including experimental autoimmune encephalitis and colitis, were used 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.

Human NKG2D/DAP10 is an activation receptor expressed by NK and subsets of T cells, whose ligands include MHC class I chain-related (MIC) protein A and protein B and UL16-binding proteins that are often up-regulated by stress or pathological conditions. DAP10 is required for NKG2D/DAP10 cell surface expression and signaling capacity. Little is known about the mechanisms that regulate DAP10 gene expression. We describe the existence of multiple transcriptional start sites upstream of DAP10 exon 1 and identify the location of the basic promoter upstream of these starting sites. The promoter is active in NK and CD8+ T cells, but not in CD4+ T cells. We demonstrate TCR-mediated up-regulation of DAP10 transcription and found that a 40 bp region within the DAP10 promoter, containing an Ap-1 binding site, is largely responsible for this increased transcription. Using pull-down and chromatin immunoprecipitation assays, we show that the DAP10 promoter interacts with Ap-1 transcription factors in primary CD8+ T and NK cells in vitro and in vivo. Overexpression of c-Jun or c-Fos in NK and T cells led to enhanced DAP10 promoter activity and DAP10 protein expression. Taken together, our data indicate that Ap-1 is an important transcription factor for regulating DAP10 gene expression in human NK and T cells, and that Ap-1 plays a key role in the transactivation of DAP10 promoter following TCR stimulation.

SummaryThe CD94-NKG2 receptor family that regulates NK and T cells is unique among the lectin-like receptors encoded within the natural killer cell complex. The function of the CD94-NKG2 receptors is dictated by the pairing of the invariant CD94 polypeptide with specific NKG2 isoforms to form a family of functionally distinct heterodimeric receptors. However, the structural basis for this selective pairing and how they interact with their ligand, HLA-E, is unknown. We describe the 2.5 Å resolution crystal structure of CD94-NKG2A in which the mode of dimerization contrasts with that of other homodimeric NK receptors. Despite structural homology between the CD94 and NKG2A subunits, the dimer interface is asymmetric, thereby providing a structural basis for the preferred heterodimeric assembly. Structure-based sequence comparisons of other CD94-NKG2 family members, combined with extensive mutagenesis studies on HLA-E and CD94-NKG2A, allows a model of the interaction between CD94-NKG2A and HLA-E to be established, in which the invariant CD94 chain plays a more dominant role in interacting with HLA-E in comparison to the variable NKG2 chain.

Human CD94/NKG2A is an inhibitory receptor that recognizes HLA-E and is expressed by NK cells and a subset of T cells. We have analyzed the cellular trafficking of the CD94/NKG2A receptor using the NKL cell line and peripheral blood NK cells. Flow cytometric, confocal microscopic, and biochemical analyses show that CD94/NKG2A continuously recycles in an active process that requires the cytoskeleton between the cell surface and intracellular compartments that are distinguishable from recycling compartments used by well-characterized receptors, such as transferrin receptor (CD71). CD94/NKG2A, an inhibitory receptor, traffics differently from the closely related CD94/NKG2C molecule, an activating receptor. Using transfection/expression analyses of wild-type and mutant CD94/NKG2A molecules in the HLA-E negative rat basophilic cell line RBL-2H3, we demonstrate that CD94/NKG2A internalization is independent of ligand cross-linking or the presence of functional immunoreceptor tyrosine-based inhibition motifs. Thus, the mechanisms that control cell surface homeostasis of CD94/NKG2A are independent of functional signaling.

The human NKG2A chain of the CD94/NKG2A receptor contains two immunoreceptor Tyr-based inhibitory motifs (ITIMs) in its cytoplasmic tail. To determine the relative importance of membrane-distal (residues 6–11) and membrane-proximal (residues 38–43) ITIMs in mediating the inhibitory signal, we made site-directed mutants of NKG2A at the Y (Y8F, Y40F, Y8F/Y40F) and the residues two positions N-terminal (Y-2) of Y (V6A, I38A, V6A/I38A) in each motif. Wild-type (wt) and mutated NKG2A were then cotransfected with CD94 into rat basophilic leukemia 2H3 cells. Immunochemical analyses after pervanadate treatment showed that each of the mutant molecules could be phosphorylated to expected levels relative to wt NKG2A and that all the mutations significantly reduced the avidity of SH2 domain-bearing tyrosine phosphatase-1 for NKG2A. Confocal microscopy was used to determine whether SH2 domain-bearing tyrosine phosphatase-1 and CD94/NKG2A colocalized intracellularly after receptor ligation. Only the Y8F/Y40F and Y8F mutant NKG2A molecules failed to show a dramatic colocalization. In agreement with this result, the Y8F/Y40F mutant was unable to inhibit FcεRI-mediated serotonin release and the Y8F mutant was relatively ineffective compared with wt NKG2A. In contrast, the Y40F mutant was 70% as effective as wt in mediating inhibition, and the Y-2 mutations did not remarkably affect inhibitory function. These results show that, like KIR, both NKG2A ITIMs are required for mediating the maximal inhibitory signal, but opposite to KIR, the membrane-distal ITIM is of primary importance rather than the membrane-proximal ITIM. This probably reflects the opposite orientation of the ITIMs in type II vs type I proteins.

To the Editor: Natural killer (NK) cells are lymphocytes that are important for host defense against viral and bacterial infections as well as malignant transformation. They have the capacity to kill target cells and to produce cytokines upon activation without previous sensitization. Although NK cells have been traditionally categorized as members of the innate immune system, recent studies have shown that NK cells have the ability to modify their effector function based on previous cytokine and/or activating receptor-mediated stimulation (1–3). The first evidence that NK cells can remember came from studies showing that they mediated the recall responses to haptens in mice lacking T and B cells using a contact hypersensitivity model (4). NK cell memory has also been demonstrated in viral infections using murine models (3). In these models, NK cells acquired long-lived memory to influenza A, vesicular stomatitis virus and human immunodeficiency virus antigens (5). Additionally, development of long-lived NK cell memory to mouse cytomegalovirus (MCMV) infection was shown to be dependent on IL-12 mediated signals (3, 6). Even without exposure to specific antigens, ex vivo short exposure of mouse and human NK cells to activating cytokines, such as IL-12, IL-15 and IL-18, elicits “memory-like” properties that are defined as enhanced effector functions after restimulation (1, 7, 8). In this study we tested the role of IL-12 mediated signals in the in vitro generation of human NK cells with enhanced effector function after restimulation. In our opinion, the best method to address this issue would involve the use of PBMCs from patients with a deficiency in IL-12 or IL-12 receptor (IL-12R), since we could then exclude that the NK cells used in the experiments had been activated at any time in vivo by IL-12. We have previously described a 19-month-old patient with an IL-12Rβ1 deficiency due to a complex mutation at exon 14 in the IL12RB1 gene (c.1623_1624delinsTT; p.Q541X) (9). We showed that IFN-γ production was markedly decreased after stimulation of PBMCs with phytohemagglutinin (PHA) or PHA plus IL-12 and that there was diminished STAT4 phosphorylation after IL-12 stimulation (9). Here, we specifically tested if the NK cells from this patient responded to IL-12 stimulation. Flow cytometric analyses showed this patient’s NK cells did not produce IFN-γ when they were stimulated with IL- 12 + IL-15 or IL-12 + IL-18 (Fig 1). On the other hand, approximately 20% of the NK cells from the age-matched healthy control produced IFN-γ when they were stimulated with IL-12 + IL-15, and 85% of the NK cells produced IFN-γ when they were stimulated with IL-12 + IL-18 (Fig 1). When NK cells were stimulated with a combination of the three cytokines, almost all NK cells from the healthy control produced IFN-γ, while only 12% of the NK cells from the patient expressed IFN-γ. Furthermore, on a per cell basis the NK cells from the patient produced less IFN-γ than the NK cells from the control, as shown by median fluorescence intensity (MFI) of 921 for the IFN-γ producing NK cells from the patient versus a MFI of 35205 for the IFN-γ producing NK cells from the healthy control. We excluded that the patient had a defect in the IFNG gene or its regulation because her NK cells and those from the control produced similar amounts of IFN-γ in response to phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Fig 1). As expected, these results corroborated that NK cells from an IL-12Rβ1 deficient patient do not respond to IL-12. FIG. 1 Decreased IFN-γ production by IL-12Rβ1 deficient NK cells in response to IL-12 in combination with IL-15 and/or IL-18. PBMCs were stimulated with different combinations of IL-12 (10 ng/mL), IL-15 (10 ng/mL) and IL-18 (50 ng/mL) for 16 ... Next, we evaluated the role of IL-12-mediated signals in the generation of NK cells with enhanced effector functions after restimulation, or “memory-like” NK cells as reported (1, 8), following preactivation with cytokines (Fig 2). We stimulated PBMCs with IL-12 + IL-15 + IL-18 for 16-hours, followed by washes and a 7-day rest period in vitro with survival supported by low concentrations of IL-15. After the resting period, cells were harvested and co-incubated with 721.221 target cells (EBV-transformed B cells). The expression of CD107 (CD107a and CD107b), as surrogates for degranulation, triggered by 721.221 tumor cells was measured by flow cytometry. Preactivated NK cells from the healthy control degranulated more than non-preactivated NK cells after co-incubation with 721.221 cells (15.4% versus 9.4%) (Fig 2A, left panel). However, preactivated and non-preactivated NK cells from the patient showed similar degranulation after co-incubation with 721.221 cells (4.3% versus 4%) (Fig 2A, left panel). These results suggest there is no significant increase in the degranulation capabilities after pre-activation with cytokines in NK cells lacking IL-12 mediated signaling. FIG. 2 Absence of enhanced effector functions after re-stimulation by IL-12Rβ1 deficient NK cells preactivated with IL-12 + IL-15 + IL-18. A) PBMCs were cultured for 16 hours with either low concentrations of IL-15 (1 ng/mL) (non-preactivated) or with ... We also measured TNF-α production by NK cells after co-incubation with 721.221 cells (Fig 2A, right panel). Similar to the degranulation results, preactivated NK cells from the healthy control produced more TNF-α than non-preactivated NK cells (0.51% versus 0.1%) (Fig 2A, right panel), while there were no differences in the TNF-α production when preactivated and non-preactivated NK cells from the patient were compared (0.02% versus 0.04%) (Fig 2A, right panel). In order to confirm that the observed results were related with the impaired IL-12 signaling rather than with a particular mutation, a patient with a different homozygous null IL12RB1 mutation (c.1495C>T; p. Q499X) and impaired STAT4 phosphorylation upon IL-12 stimulation (see Fig E1 in this article´s Online Repository at www.jacionline.org), was also tested. Similar to the results obtained with cells from the first patient, preactivated NK cells from the healthy control produced more IFN-γ than non-preactivated NK cells after restimulation with 721.221 cells (20.4% versus 14.7%) and IL-15 + IL-18 (54.1% versus 29.7%) (Fig 2B). However, preactivated and non-preactivated NK cells from the second patient showed similar IFN-γ production after restimulation with 721.221 cells (1.29% versus 3.06%) and IL-15 + IL-18 (3.60% versus 1.94%) (Fig. 2B). These results indicate that IL-12Rβ1-deficient NK cells do not have the ability to acquire enhanced effector functions after restimulation. In addition to the defect in restimulation, the IL-12Rβ1- deficient NK cells also have a defect in primary activation. The deficiency in IFN-γ production after target and IL-15 + IL-18 stimulation, and in the absence of preactivation, suggests that human NK cells require in vivo priming with IL-12 to acquire full functional reactivity, and is consistent with a study by Guia et al (10). Our observations are partly in contradiction to those published by Romee et al. (8), who showed that after preactivation of human NK cells with IL-12 + IL-15, re- stimulation with K562 target cells led to an increase in IFN-γ production without an increase in the cell surface expression of CD107a on NK cells. Although we do not fully understand the reasons for this discrepancy, the use of different target cells and the staining with both anti-CD107a and anti-CD107b mAbs in our experiments may explain the differences. In fact, our results are in accordance with those obtained with the MCMV infection model which demonstrated that degranulation by mouse memory NK cells was enhanced after stimulation with anti-NK1.1 mAb, (3) and the generation of memory NK cells was dependent on IL-12-mediated signals (6). Here, using human IL-12Rβ1-deficient NK cells, we have demonstrated the essential role of IL-12-mediated signals in the generation of human NK cells with enhanced effector function after restimulation. These findings provide the rationale for including brief stimulations or preactivation with IL-12, in combination with other cytokines such as IL-15 and IL-18, to generate NK cells with enhanced effector functions, i.e. “memory-like”, that may be of great relevance in the field of NK-cell based immunotherapy and may also unveil a new aspect of pathophysiology to be considered in IL-12Rβ1 deficient patients.

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.

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.

CD16 (FcgammaRIIIa), the low affinity receptor for IgG is a potent activating receptor expressed by the majority of human natural killer (NK) cells that functions to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Since the bound IgG can be exchanged, the target cell recognition through CD16 gives NK cells flexibility in receptor specificity that can be manipulated for therapeutic purposes. ADCC dysfunction has been linked to cancer progression, poor prognosis and chronic infections; thus, understanding how CD16 expression is regulated by NK cells has clinical relevance. It is well established that upon activation NK cells enter into a refractory period in which CD16 expression is dramatically down-modulated, mainly due to the action of matrix metalloproteinases (MMPs). The aim of this project is to identify the MMP(s) responsible for CD16 cell surface down-modulation on activated NK cells and to determine if this down-modulation can be inhibited. Our data indicate that CD16 is down-modulated upon cell activation in primary NK cells, the released CD16 can be detected in the supernatant of the activated cells and a membrane-type MMP is involved in the process of CD16 shedding.

FCMR is a Fc receptor specific for pentameric IgM 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 highest levels by follicular (FO) B cells and at much lower levels by developing B cells and other mature 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 resistant to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR functions are critical for B cell homeostasis, the prevention of autoreactive B cells and responsiveness to antigenic challenge.

CD300a is an immuno-receptor tyrosine based inhibitory motif (ITIM) containing molecule that belongs to the CD300 family of paired activating/inhibitory receptors clustered in human chromosome 17. It has been shown that its ligation inhibits activation signals on cells of both myeloid and lymphoid lineages. To identify the ligand of CD300a, we generated an Fc fusion protein containing the extracellular domain of CD300a (CD300a-Ig). We show that CD300a-Ig specifically binds to dead cells that are evolutionary apart, such as human and insect cells, suggesting that the ligand has to be conserved. Using ELISA, ultracentrifugation, surface plasmon resonance, and reporter cell assays we identified phosphatidylethanolamine (PE) and phosphatidylserine (PS), two phospholipids that translocate to the outer leaflet of the plasma membrane of dead cells, as the ligands for CD300a. Our 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. Ectopic expression of CD300a, in the otherwise CD300a negative L929 cells, down-regulated the phagocytosis of dead cells. Furthermore, we also show that CD300a inhibits the uptake of apoptotic cells by human macrophages. Collectively, our results indicate that PE and PS are ligands for CD300a and that these interactions may play an important role in the removal of dead cells by phagocytes.

Human peripheral blood natural killer (NK) cells (CD56+, CD16+, CD3 epsilon- lymphocytes) express CD69 after their stimulation by interleukin-2 (IL-2) or interferon-alpha (IFN-alpha). This activation antigen represents a triggering surface molecule in NK cell clones as its stimulation triggers the cytolytic machinery of these cells. However, the mechanisms regulating the expression of CD69 in NK cells are unknown despite the functional relevance of CD69 in NK cell activation. Thus, we have analyzed the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the expression of CD69 on purified NK cells activated by IL-2, IFN-alpha, anti Fc gamma RIII (CD16) monoclonal antibodies or by K562 target cells. We found that CD69 is induced on NK cells not only by IL-2 and IFN-alpha but also by activation of the CD16 pathway, the interaction with NK target cells and the direct activation of PKC by phorbol 12-myristate 13-acetate (PMA), indicating that CD69 induction is associated to different NK activation pathways. The treatment with the PKC inhibitor staurosporine abolished the induction of CD69 induced by PMA or K562. However, it did not significantly affect CD69 induction by IL-2, IFN-alpha or CD16 cross-linking. This demonstrates that whereas PKC can play a central role in the regulation of CD69 expression in some instances (response to K562 cells or PMA), it does not participate in others (response to IL-2, IFN-alpha or anti CD16 monoclonal antibodies). On the other hand genistein, a competitive inhibitor of PTK enzymes, blocked the expression of CD69 induced by activation of NK cells via IL-2 or IFN-alpha receptors, CD16 and K562 receptor(s), indicating that stimulation of PTK is a common step in the signal transduction events leading to the induction of CD69 antigens after the activation of NK cells via these receptors.

Fc receptors (FcRs) are key regulators of the immune response. A receptor specific for IgM (FcμR) has been recently identified and shown to be predominately expressed on human B cells. FcμR, known previously as TOSO and as Fas apoptosis inhibitory molecule 3 (Faim3), has no role in the inhibition of Fas-mediated apoptosis. We have examined the expression of FcμR in mouse splenocytes by real-time PCR. We found significantly higher levels of FcμR mRNA expression on B cells compared with FcμR mRNA expression on T cells, NK cells, granulocytes, macrophages and monocytes. After stimulation with LPS, anti-CD40 or anti-IgM, FcμR mRNA expression was dramatically down-regulated on splenic B cells. We have also found that bone marrow B cells have lower levels of FcμR mRNA expression than splenic B cells. Furthermore, we analyzed FcμR mRNA expression during B cell development in the bone marrow and we found that immature and mature B cells express high levels of FcμR mRNA compared with pro- and pre- B cells. Currently, we are investigating the role that FcμR plays in B cell development and signaling.

Homeostasis of both NK and T cells in the periphery is regulated by activation induced cell death (AICD) mediated by signaling through Fas, and other death receptors. It has been shown that IL-2 is required for priming cells for this type of apoptosis. Cytokine primed NK cells, in addition to killing their target cells, also undergo AICD upon activation by activating ligands expressed by the target cells. TOSO is an anti-apoptotic molecule known to inhibit apoptosis mediated by the death receptor pathway and we investigated the role of TOSO in regulating AICD in NK and T cells. We observed by realtime PCR that stimulation of human NK cells and T cells in vitro with IL-2 resulted in down regulation of TOSO. We further analyzed TOSO expression levels on sorted human CD4 T cells and found that cells with an effector/memory phenotype have markedly lower levels of TOSO than naïve cells, which suggests that activation in vivo correlates with down-regulation of TOSO. In agreement, we found that activation of CD45RO+ cells in vitro dramatically down regulated TOSO compared to naïve CD4 T cells. Further, overexpression of Toso in Jurkat cells was able to inhibit Fas-mediated cell death. We hypothesize that IL-2 has a dual action: first, it activates and induces the proliferation of NK and T-cells and second, it down-regulates TOSO, thereby sensitizing or priming them for apoptosis upon re-stimulation. Considering that IL-2 is being used as a therapeutic agent for cancer treatment and the recent study showing that HIV envelope protein can down regulate TOSO levels on CD4 T cells, unraveling how TOSO functions and is regulated might have potential for treating cancer and other diseases.

Regulated apoptosis is essential for both the development and homeostasis of the immune system. Fas-mediated cell death has been implicated in apoptosis during the development and homeostasis of lymphocytes, and this apoptosis pathway is regulated by the interplay of BCL-2-family members, as well as other factors, such as cytokine withdrawal. However, Toso (also known as Fas Apoptosis Inhibitory Molecule-3) expression has been reported to inhibit Fas-mediated cell death. We isolated immune cells from the spleen and found that B cells expressed high levels of Toso mRNA compared to T cells, NK cells, granulocytes, macrophages and monocytes. After stimulation by LPS, anti-CD40 or anti-IgM, Toso expression is dramatically down-regulated in splenic B cells. Further analysis showed that bone marrow B cells express lower levels of Toso mRNA than splenic B cells. We sorted the bone marrow B cells into development stages and found that only immature B cells expressed high levels of Toso mRNA compared with pro-, pre- and mature B cells. We hypothesize that Toso has a role in normal B cell development and maintenance. To examine this, we are using Toso knockout and transgenic mice. This work was supported by the NIAID intramural research program.

The NKG2D receptor is an activation receptor expressed on all NK cells and the majority of CD8+ T cells that can bind stress-inducible ligands such as MICA/B. Human NKG2D signals through its association with DAP10, which can couple to the PI3-K signaling pathway upon NKG2D ligation leading to cellular activation. Inappropriate activation of NK and/or T cells by NKG2D has been implicated in a variety of human diseases and thus the determination of the factors that regulate NKG2D/DAP10 expression is important. We have embarked on a study in which to understand the molecular mechanisms governing transcription of the DAP10 gene. We have identified a highly conserved intron between exons III and IV within DAP10. By cloning gene fragments of DAP10 into pGLbasic for luciferase reporter assays, we have shown that intron III contains a negative regulatory element capable of completely abolishing the promoter activity of DAP10. Progressive deletions of the conserved intron III allowed us to localize the negative regulatory element to a region about 80 base pairs in size that contains a putative GATA-3 binding site. The putative silencer within intron III appears to be both orientation and location dependent. Using ChIP assays, we have also shown that intron III contains high levels of acetylated chromatin in resting NK cells. Further studies are underway to further characterize the negative regulatory element contained within the conserved intron of the DAP10 gene.