Human Natural Killer Cell Subsets Research Articles

Expansion of the Innate Lymphocyte Family: Discovery of IL-22-Producing ILC3s.

This Pillars of Immunology article is a commentary on “A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity,” a pivotal article written by M. Cella, A. Fuchs, W. Vermi, F. Facchetti, K. Otero, J. K. M. Lennerz, J. M. Doherty, J. C. Mills, and M. Colonna, and published in Nature, in 2009. https://www.nature.com/articles/nature07537.

SOT101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity.

SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.

CXCR6+ and NKG2C+ Natural Killer Cells Are Distinct With Unique Phenotypic and Functional Attributes Following Bone Marrow Transplantation.

Reactivation of human cytomegalovirus (HCMV) is a life-threatening complication in transplant patients. Natural Killer (NK) cells are the first lymphocyte lineage to reconstitute following an allogeneic hematopoietic stem cell transplant (HSCT). Amongst them, NK cell Group 2 isoform C/Killer cell lectin-like receptor subfamily C, member 2 (NKG2C)-expressing NK cells contribute significantly to patient protection upon HCMV reactivation. NKG2C+ NK cells are capable of immunological memory, albeit NK cell memory is not restricted to them. Hepatic C-X-C Motif Chemokine Receptor 6 (CXCR6)-expressing NK cells also mediate memory responses in mice and humans. Small numbers of them circulate and can thus be studied in peripheral blood samples. We hypothesize that NKG2C+ and CXCR6+ NK cell subsets are distinct. To test our hypothesis, we used multi-parametric flow cytometry to determine the phenotypes and effector functions of CD56bright vs. CD56dim and NKG2C+ vs. CXCR6+ human NK cell subsets in the peripheral blood (PB) of pediatric transplant recipients monthly while monitoring patients for HCMV reactivation. Interestingly, we did not find any NKG2C+CXCR6+ NK cells in the transplant recipients’ peripheral blood, suggesting that NKG2C+ and CXCR6+ NK cells are distinct. Also, NKG2C–CXCR6– NK cells, rather than NKG2C+ NK cells, made up most NK cells post-transplant, even in transplant recipients with HCMV viremia. In contrast to NKG2C+ NK cells, CXCR6+ NK cells appeared phenotypically less differentiated but were highly proliferative and produced IFN-γ and TNF α . Our findings contribute to our understanding of post-transplant NK cell development and its implications for human health.

Assessing the Response of Human NK Cell Subsets to Infection by Clinically Isolated Virus Strains.

Natural killer (NK) cells play a critical role in defending against virus infections.Investigating human NK cell antiviral functions is of prime importance; however, there are challenges such as the human-specific nature of many viruses and differences in NK cell surface markers between humans and rodents. Research on the antivirus response of human NK cells must therefore be carefully planned around species tropism of the viruses of interest and the specific biological questions to be answered. The initial site of many virus infections is a mucosal/epithelial surface. In this context, a clinical virus infection at the ocular surface enables direct analyses on the mechanisms and consequences of infection and immune reactions in situ over the course of disease. For example, the site of infection of a clinical infection in the conjunctiva and cornea can be directly observed in real-time, utilizing split-lamp microscopy, and specimens are readily accessed with minimally invasive techniques.In this chapter, we describe protocols for investigating NK cell responses using clinically isolated viruses in co-culture assays. We also describe procedures for ex vivo analysis of conjunctiva-derived NK cells in adenovirus infection.

CD200R1 Distinguishes Uncommitted Precursors from Functionally Mature NK Cells within the Human Tonsil Stage 4A NK Cell Population

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) whose development and anti-tumor functions can be critical for the successful treatment and long-term disease-free survival of patients with hematologic malignancies. In humans, NK cells derive from bone marrow resident hematopoietic progenitor cells that traffic to secondary lymphoid tissues (SLTs) where they complete their terminal differentiation and maturation through a series of developmental stages before returning to the blood as mature NK cells. Although major stages of human NK cell development in SLTs have been clearly defined according to the differential surface expression of CD34, CD117, CD94, NKp80, CD16, and CD57 among lineage antigen (Lin) negative lymphocytes, continued investigation has revealed additional phenotypic and functional heterogeneity at each developmental stage. Through extensive ex vivo single-cell RNA sequencing and flow cytometry analyses we have identified two subsets of tonsil-resident Lin -CD34 -CD117 +/-CD94 +NKp80 -CD16 -CD57 - stage 4A NK cells. These two subsets differ in their expression of the inhibitory receptor, CD200R1, which is not expressed by mature NK cells in the peripheral blood from healthy individuals. The majority of stage 4A cells expressed high amounts of surface CD200R1, which correlated with low gene and undetectable protein expression of intracellular cytolytic granules (perforin and granzymes A, B, K, and M), killer immunoglobulin-like receptors (KIRs), and transcription factors required for terminal NK cell maturation (EOMES, T-BET). In addition, upon ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, CD200R1 + stage 4A NKDIs did not produce interferon-gamma (IFN-g), a hallmark feature of mature NK cells. In contrast, many CD200R1 - stage 4A cells constitutively expressed perforin, granzymes, EOMES, and/or T-BET; many expressed KIRs; and many produced IFN-g upon ex vivo stimulation. Furthermore, the frequency of KIR + cells among CD200R1 - stage 4A cells was significantly higher than that among autologous tonsil stage 4B NK cells (Lin -CD34 -CD117 +/-CD94 +NKp80 +CD16 -CD57 -) (20.8 ± 1.65 vs. 8.12 ± 1.66; p < 0.01; n = 14), suggesting that as a population CD200R1 - stage 4A cells are potentially out of sequence in terms of the linear NK cell developmental pathway. Based on these ex vivo findings, we hypothesized that CD200R1 + stage 4A cells represent NK cell precursors, whereas the CD200R1 - stage 4A population contains more mature NK cells that lack NKp80, CD16, and CD57. To further address this hypothesis and to determine their ex vivo potentials for NK cell and non-NK ILC differentiation, we cultured CD200R1 + and CD200R1 - stage 4A cells in vitro in the presence of OP9-DL1 stroma and recombinant human IL-7 and IL-15, conditions previously shown to support all human ILC and NK cell subset differentiation. Under these conditions, both stage 4A populations generated NKp80 + NK cells in bulk and single-cell clonal assays, whereas neither population gave rise to ILC2s (CD294 +) which precede stage 4A NK cells in the developmental scheme. However, while the majority of cultures derived from CD200R1 + stage 4A clones contained ILC3s (CD94 -NKp44 +), significantly fewer clones from CD200R1 - stage 4A cells produced ILC3s (7 of 26 CD200R1 - clones vs. 20 of 23 CD200R1 + clones; p = 0.000587). Moreover, none of the CD200R1 - stage 4A-derived clonal cultures that contained KIR + NK cells contained ILC3s, suggesting that the majority of CD200R1 - stage 4A cells are lineage committed NK cells. Collectively, these data further characterize the heterogeneity of the human tonsil stage 4A NK cell population and identify CD200R1 as a marker distinguishing uncommitted precursor cells from a minor population of cells with otherwise mature NK-associated phenotype and function. In light of the role of CD200R1 in regulating lymphocyte functions in the setting of cancer, further research is warranted to determine its potential role(s) in regulating human NK cell development. DisclosuresBlachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

Characterization of Surface Receptor Expression and Cytotoxicity of Human NK Cells and NK Cell Subsets in Overweight and Obese Humans.

Obesity is associated with an increased risk for several cancer types and an altered phenotype and functionality of natural killer (NK) cells. This study aimed to investigate the association of overweight and obesity with NK cell functions and receptor expression profiles in humans. Therefore, peripheral blood mononuclear cells were isolated from normal weight, overweight, and obese healthy blood donors. In depth analysis of immune cell populations and 23 different surface markers, including NK cell receptors, NK-cell-related markers as well as functional intracellular markers on total NK cells and NK subgroups were performed by multicolor flow cytometry. The data revealed a decreased expression of the activating NK cell receptors KIR2DS4 and NKp46 as well as an increased expression of the inhibitory NK cell receptors NKG2A and Siglec-7 in overweight and obese compared to normal weight individuals. Additionally, the expression of the adhesion molecule CD62L and the maturation and differentiation marker CD27 was downregulated in NK cells of overweight and obese subjects. Furthermore, the cytotoxicity of NK cells against colorectal cancer cells was decreased in overweight and obese subjects. Investigations on underlying killing mechanisms demonstrated a reduced TRAIL expression on NK cells of obese subjects suggesting an impaired death receptor pathway in obesity. The present study gives new insights into an impaired functionality and phenotype of NK cells and NK cell subsets in overweight and obesity. These phenotypic alterations and dysfunction of NK cells might be an explanation for the increased cancer risk in obesity.

Specialized Roles of Human Natural Killer Cell Subsets in Kidney Transplant Rejection.

Background: Human natural killer (NK) cells are key functional players in kidney transplant rejection. However, the respective contributions of the two functionally distinct human NK cell subsets (CD56bright cytokine-producing vs. CD56dim cytotoxic effector) in episodes of allograft rejection remain uncertain, with current immunohistochemical methods unable to differentiate these discrete populations. We report the outcomes of an innovative multi-color flow cytometric-based approach to unequivocally define and evaluate NK cell subsets in human kidney allograft rejection.Methods: We extracted renal lymphocytes from human kidney transplant biopsies. NK cell subsets were identified, enumerated, and phenotyped by multi-color flow cytometry. Dissociation supernatants were harvested and levels of soluble proteins were determined using a multiplex bead-based assay. Results were correlated with the histopathological patterns in biopsies—no rejection, borderline cellular rejection, T cell-mediated rejection (TCMR), and antibody-mediated rejection (AMR).Results: Absolute numbers of only CD56bright NK cells were significantly elevated in TCMR biopsies. In contrast, both CD56bright and CD56dim NK cell numbers were significantly increased in biopsies with histopathological evidence of AMR. Notably, expression of the activation marker CD69 was only significantly elevated on CD56dim NK cells in AMR biopsies compared with no rejection biopsies, indicative of a pathogenic phenotype for this cytotoxic NK cell subset. In line with this, we detected significantly elevated levels of cytotoxic effector molecules (perforin, granzyme A, and granulysin) in the dissociation supernatants of biopsies with a histopathological pattern of AMR.Conclusions: Our results indicate that human NK cell subsets are differentially recruited and activated during distinct types of rejection, suggestive of specialized functional roles.

Soy isoflavones and their metabolites modulate cytokine-induced natural killer cell function

Soybeans are a rich source of isoflavones that have been linked with anti-inflammatory processes and various health benefits. However, specific mechanisms whereby soy bioactives impact immune cell subsets are unclear. Isoflavones, such as genistein and daidzein, are metabolized by microbes to bioactive metabolites as O-desmethylangolensin (O-DMA) and equol, whose presence has been linked to health benefits. We examined how soy isoflavones and metabolites impact natural killer (NK) cell signaling and function. We observe no impact of isoflavones on viability of healthy donor peripheral blood mononuclear cells (PBMCs) or NK cells, even at high (25 µM) concentrations. However, pre-treatment of PBMCs with physiologically-relevant concentrations of genistein (p = 0.0023) and equol (p = 0.006) decreases interleukin (IL)-12/IL-18-induced interferon-gamma (IFN-γ) production versus controls. Detailed cellular analyses indicate genistein and equol decrease IL-12/IL-18-induced IFN-γ production by human NK cell subsets, but do not consistently alter cytotoxicity. At the level of signal transduction, genistein decreases IL-12/IL-18-induced total phosphorylated tyrosine, and phosphorylation MAPK pathway components. Further, genistein limits IL-12/IL-18-mediated upregulation of IL-18Rα expression on NK cells (p = 0.0109). Finally, in vivo studies revealed that C57BL/6 mice fed a soy-enriched diet produce less plasma IFN-γ following administration of IL-12/IL-18 versus control-fed animals (p < 0.0001). This study provides insight into how dietary soy modulates NK cell functions.

Porcine NK Cells Stimulate Proliferation of Pseudorabies Virus-Experienced CD8+ and CD4+CD8+ T Cells

Natural killer (NK) cells belong to the innate immune system and play a central role in the defense against viral infections and cancer development, but also contribute to shaping adaptive immune responses. NK cells are particularly important in the first line defense against herpesviruses, including alphaherpesviruses. In addition to their ability to kill target cells and produce interferon-γ, porcine and human NK cell subsets have been reported to display features associated with professional antigen presenting cells (APC), although it is currently unclear whether NK cells may internalize debris of virus-infected cells and whether this APC-like activity of NK cells may stimulate proliferation of antiviral T cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we show that vaccination of pigs with a live attenuated PRV vaccine strain triggers expression of MHC class II on porcine NK cells, that porcine NK cells can internalize debris from PRV-infected target cells, and that NK cells can stimulate proliferation of CD8+ and CD4+CD8+ PRV-experienced T cells. These results highlight the potential of targeting these NK cell features in future vaccination strategies.

Inhibition of histone H3K27 demethylases selectively modulates inflammatory phenotypes of natural killer cells

Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic JMJD3/UTX (Jumonji domain–containing protein 3) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte–macrophage colony-stimulating factor (GM-CSF), and interleukin-10 levels in cytokine-stimulated NK cells while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function.

Gradual Increase of FcγRIIIa/CD16a Expression and Shift toward IFN-γ Secretion during Differentiation of CD56dim Natural Killer Cells.

Natural killer (NK) cell effector functions include cytotoxicity and secretion of cytokines such as interferon-γ (IFN-γ). The immature CD56bright subset of human NK cells lacks expression of FcγRIIIa/CD16a, one of the low-affinity immunoglobulin G receptors, or exhibits low-density expression (CD56brightCD16−/dim) and produces IFN-γ in response to cytokine stimulation, whereas the mature CD56dimCD16+ subset is the most cytotoxic one. A further differentiation/maturation of the latter subset according to the gradual loss of NKG2A and/or gain of KIR2DL (CD158a and CD158b) has been demonstrated and the ability to produce IFN-γ in response to activating receptor (AR) co-engagement is gradually acquired during terminal differentiation. In the course of flow cytometry analysis of CD56dim NK cells, we noted a substantial intraindividual heterogeneity of expression of FcγRIIIa. FcγRIIIa is unique among ARs: it does not require the co-engagement of other ARs to induce substantial cytotoxicity or cytokine synthesis in CD56dim cells. We, therefore, investigated whether individual differentiation/maturation of polyclonal CD56dim NK cells defined by expression of NKG2A/KIR2DL is related to FcγRIIIa expression and to the heterogeneity of NK cell responses upon FcγRIIIa engagement. When we analyzed unstimulated CD56dim cells by increasing level of FcγRIIIa expression, we found that the proportion of the more differentiated CD158a,h+ and/or CD158b,j+ cells and that of the less differentiated NKG2A+ cells gradually increased and decreased, respectively. FcγRIIIa engagement by using plate-bound murine anti-CD16 monoclonal antibody (mAb) or rituximab or trastuzumab (two therapeutic mAbs), resulted in donor-dependent partial segregation of IFN-γ-producing and/or degranulating CD56dim cells. Importantly, the proportion of CD158a,h/b,j+ cells and that of NKG2A+ cells was increased and decreased, respectively, IFN-γ-producing cells, whereas these proportions were poorly modified in degranulating cells. Similar results were observed after engagement of ARs by a combination of mAbs targeting NKG2D, NKp30, NKp46, and 2B4. Thus, the gradual increase of FcγRIIIa expression is an important feature of the differentiation/maturation of CD56dim cells and this differentiation/maturation is associated with a shift in functionality toward IFN-γ secretion observed upon both FcγRIIIa-dependent and FcγRIIIa-independent stimulation. The functional heterogeneity related to the differentiation/maturation of CD56dim NK cells could be involved in the variability of the clinical responses observed in patients treated with therapeutic mAbs.

Humanized mouse model supports development, function, and tissue residency of human natural killer cells

Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2-/- Il2rg-/- background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg-/- (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt's lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.

Licensing delineates helper and effector NK cell subsets during viral infection.

Natural killer (NK) cells can be divided into phenotypic subsets based on expression of receptors that bind self-MHC-I molecules, a concept termed licensing or education. Here we show NK cell subsets with different migratory, effector, and immunoregulatory functions in dendritic cell and antigen (ag)-specific CD8+ T cell responses during influenza and murine cytomegalovirus infections. Shortly after infection, unlicensed NK cells localized in draining lymph nodes and produced GM-CSF, which correlated with the expansion and activation of dendritic cells, and resulted in greater and sustained ag-specific T cell responses. In contrast, licensed NK cells preferentially migrated to infected tissues and produced IFN-γ. Importantly, human NK cell subsets exhibited similar phenotypic characteristics. Collectively, our studies demonstrate a critical demarcation between the functions of licensed and unlicensed NK cell subsets, with the former functioning as the classical effector subset and the latter as the stimulator of adaptive immunity helping to prime immune responses.

The pursuit of better vaccines: uncovering the functional capacity of human cytomegalovirus-induced subsets of NK cells

Abstract Cytomegalovirus infection (CMV) poses a considerable burden to public health due to significant morbidity and mortality in immunocompromised patients and babies born with the infection, thus highlighting the priority of developing a successful CMV vaccine. Whereas previous CMV vaccination strategies have aimed to induce T- and B-cell responses, we speculate that natural killer (NK) cells hold the key to protective immunity based on the evidence that patients lacking NK cells suffer recurrent, severe infections with multiple herpesviruses, including CMV. Notably, CMV infections in both mice and humans triggered accumulation of unique adaptive NK-cell subsets that putatively possess enhanced effector functions against CMV. This suggests that a pioneering approach to creating an efficacious CMV vaccine would include stimulation of the accumulation and long-term maintenance of these CMV-reactive NK cells. It remains unknown, however, whether vaccines can be designed to stimulate the expansion of these NK-cell subsets. Here, using a unique set of samples collected during a CMV vaccine trial, we aim to determine whether vaccination promotes accumulation of these NK cells. Moreover, we examined the relationship between the activity of NK cells and stimulation of virus-specific T and B cell responses during immunization and in cell culture. Preliminary analysis revealed a supportive role for NK cells in stimulation of CMV-specific T cells during in vitro culture with peptide which correlates to the presence of adaptive NK cells. Our studies represent a key step in understanding the potential role of subsets of human NK cells in primary CMV infection and vaccine-induced immune responses against CMV.

Natural Killer Cell Assessment in Peripheral Circulation and Bronchoalveolar Lavage Fluid of Patients with Severe Sepsis: A Case Control Study.

Sepsis is a complex systemic inflammatory syndrome, the most common cause of which is attributed to systemic underlying bacterial infection. The complete mechanisms of the dynamic pro- and anti-inflammatory processes underlying the pathophysiology of sepsis remain poorly understood. Natural killer (NK) cells play a crucial role in the pathophysiology of sepsis, leading to exaggerated inflammation due their rapid response and production of pro-inflammatory cytokines such as interferon gamma (IFN-γ). Several studies have already shown that NK cells undergo lymphopenia in the peripheral blood of patients with sepsis. However, our understanding of the mechanisms behind its cellular trafficking and its role in disease development is restricted to studies in animal models. In this study, we aimed to compare the human NK cell subset (CD56bright or dim) levels in the peripheral blood and bronchoalveolar lavage (BAL) fluid of sepsis patients. We conducted a case-control study with a sample size consisting of 10 control patients and 23 sepsis patients enrolled at the Hospital Cajuru (Curitiba/PR, Brazil) from 2013 to 2015. Although we were able to confirm previous observations of peripheral blood lymphopenia, no significant differences were detected in NK cell levels in the BAL fluid of these patients. Overall, these findings strengthened the evidence that peripheral blood lymphopenia is likely to be associated with cell death as a consequence of sepsis.

Requirements for human natural killer cell development informed by primary immunodeficiency.

Human natural killer (NK) cell development is poorly understood; however, we gain important insight from cases of human primary immunodeficiency that affect the generation of mature human NK cell subsets. In this review, monogenic primary immunodeficiencies that highlight critical requirements for human NK cell development are discussed. Increased resolution in NK cell phenotyping has revealed NK cell deficiencies that affect the distribution of NK cell subsets found in peripheral blood. These, in addition to those that prevent the generation of mature NK cells, identify important requirements for the maturation and homeostasis of human NK cells. The serious and often life-threatening viral susceptibility that accompanies these highlights the nonredundant role that NK cells play in human health and disease. Human NK cell deficiency may occur in cases where NK cells are present in normal numbers but subsets are affected.

Cell surface cathepsin G activity differs between human natural killer cell subsets

Natural killer (NK) cells are critical in diverse defense mechanisms, including elimination of viral infected cells and destruction of tumor cells. NK cells are characterized by the ability to initiate apoptosis in target cells when their cell surface major histocompatibility complex class I (MHC I) repertoire is missing. On the other hand, NK cells are not activated when MHC I or non-classical MHC molecules are found on the respective cells. It was demonstrated that cathepsin G (CatG) binds to the cell surface of NK cells; however, the distribution of this protease on the cell surface of NK cell subsets has not been identified. Here, we show that CatG cell surface level differs between NK cell subsets. CatG was determined on the protein- and activity level (activity-based probe MARS116) by using flow cytometry. Thus, MARS116 is a novel reporter of cell surface CatG activity and can be used to differentiate between distinct NK cell subsets.

Leukocyte Immunoglobulin-Like Receptor 1-Expressing Human Natural Killer Cell Subsets Differentially Recognize Isolates of Human Cytomegalovirus through the Viral Major Histocompatibility Complex Class I Homolog UL18.

ABSTRACTImmune responses of natural killer (NK) cell are controlled by the balance between activating and inhibitory receptors, but the expression of these receptors varies between cells within an individual. Although NK cells are a component of the innate immune system, particular NK cell subsets expressing Ly49H are positively selected and increase in frequency in response to cytomegalovirus infection in mice. Recent evidence suggests that in humans certain NK subsets also have an increased frequency in the blood of human cytomegalovirus (HCMV)-infected individuals. However, whether these subsets differ in their capacity of direct control of HCMV-infected cells remains unclear. In this study, we developed a novel in vitro assay to assess whether human NK cell subsets have differential abilities to inhibit HCMV growth and dissemination. NK cells expressing or lacking NKG2C did not display any differences in controlling viral dissemination. However, when in vitro-expanded NK cells were used, cells expressing or lacking the inhibitory receptor leukocyte immunoglobulin-like receptor 1 (LIR1) were differentially able to control dissemination. Surprisingly, the ability of LIR1+ NK cells to control virus spread differed between HCMV viral strains, and this phenomenon was dependent on amino acid sequences within the viral ligand UL18. Together, the results here outline an in vitro technique to compare the long-term immune responses of different human NK cell subsets and suggest, for the first time, that phenotypically defined human NK cell subsets may differentially recognize HCMV infections.IMPORTANCE HCMV infection is ubiquitous in most populations; it is not cleared by the host after primary infection but persists for life. The innate and adaptive immune systems control the spread of virus, for which natural killer (NK) cells play a pivotal role. NK cells can respond to HCMV infection by rapid, short-term, nonspecific innate responses, but evidence from murine studies suggested that NK cells may display long-term, memory-like responses to murine cytomegalovirus infection. In this study, we developed a new assay that examines human NK cell subsets that have been suggested to play a long-term memory-like response to HCMV infection. We show that changes in an HCMV viral protein that interacts with an NK cell receptor can change the ability of NK cell subsets to control HCMV while the acquisition of another receptor has no effect on virus control.

Enhancement of Natural Killer Cell Interferon-Gamma Production By the Derivatives of the Natural Product Phyllanthusmins Via TLR-Mediated NF-Kb and STAT3 Signaling Pathways

Natural killer (NK) cells are known to play a pivotal role in regulating immune defense against tumors and viruses. However, very few studies describe the effects of natural products or their derivatives on NK cell function. We previously showed that the natural product lignan phyllanthusmin C enhances human NK cell IFN-gamma production. In this study, we successfully synthesized dozens of phyllanthusmin derivatives and screened their activities to enhance IFN-gamma production by NK cells. We found that three of these derivatives possessed capacities to induce IFN-gamma production by NK cells. Among them, derivative No. 17 had the highest efficacy and was significantly superior (P < 0.05) to that of the original phyllanthusmin C. These three phyllanthusmin derivatives had no adverse effect on human NK cell survival or proliferation. All derivatives alone or in combination with IL-12 or IL-15 induced human NK cell interferon (IFN)-gamma in comparison with the corresponding vehicle control or cytokine alone (P < 0.05). These derivatives stimulated IFN-gamma production in both CD56bright and CD56dim human NK cell subsets (P < 0.01). Mechanistically, immunoblotting assays and chromatin immunoprecipitation (ChIP) analyses combined with promoter-reporter luciferase assays revealed that these phyllanthusmin derivatives induced the phosphorylation of NF-κB and STAT3, resulting in their increased binding on theIFN G promoter, which was dependent on the Toll-like receptor (TLR) 1 and the TLR3 signaling pathways, respectively. STAT3 knockdown with lentivirus shRNA and inhibition of NF-kB signaling with a specific inhibitor (TPCK) significantly attenuated IFN-gamma production induced by these phyllanthusmin derivatives in human NK cells (P < 0.05). Blockade of TLR1 or TLR3 with their corresponding neutralizing antibodies nearly abolished activation of NF-κB or STAT3 as well as IFN-γ induction by the phyllanthusmin derivatives (P < 0.05). In conclusion, we have successfully synthesized and screened phyllanthusmin derivatives and identified three compounds that induce IFN-gamma production in human NK cells, one of which had optimal potential. The induction of human NK cell IFN-gamma production by phyllanthusmin derivatives is mediated by TLR1-NF-κB and TLR3-STAT3 signaling pathways, rendering their potential for use as agents for cancer prevention or treatment. DisclosuresNo relevant conflicts of interest to declare.

OMIP-028: activation panel for Rhesus macaque NK cell subsets.

OMIP-028: activation panel for Rhesus macaque NK cell subsets.