Natural Killer Cell Subsets Research Articles

ARC-12: Phase 1/1b Study to Evaluate Safety and Tolerability of AB308 + Zimberelimab (AB122) in Advanced Malignancies

Background: PD-(L)1 pathway inhibitors have demonstrated durable clinical benefit in both solid tumors and hematologic malignancies; however, as less than 1/3 of patients respond to anti-PD-(L)1 monotherapy, combination therapies may be needed for improved response rates and survival. T cell Immunoglobulin and ITIM domain (TIGIT) is expressed on activated T cell and natural killer (NK) cell subsets; TIGIT-CD155 binding suppresses T and NK cell function and inhibits the antitumor immune response. AB308, an Fc-enabled anti-TIGIT humanized IgG1 monoclonal antibody (mAb), has reversed TIGIT-CD155-mediated T cell inhibition in preclinical models. ARC-12 will assess whether TIGIT pathway blockade by AB308 can augment zimberelimab (zim; anti-PD-1 mAb) activity in patients with solid and hematologic malignancies.Study Design and Methods: ARC-12 is a phase 1/1b, first-in-human, open-label, dose-escalation study of AB308 + zim in patients with advanced solid tumors or hematologic malignancies. Dose expansion cohorts include relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM). For the dose-escalation, eligible adults have solid tumors for which no standard-of-care therapy exists or have pathologically-confirmed R/R non-Hodgkin lymphoma (NHL) and have not received or are ineligible for allogeneic stem cell transplant or adoptive cell transfer; ≤5 lines of prior systemic therapy; ≥1 measurable lesion per RECIST (solid tumors) or Lugano Classification (NHL) criteria; and ECOG performance score of 0-1. As shown in the figure, patients will receive AB308 + zim intravenously (IV) once every 3 weeks (Q3W; Part A) or once every 4 weeks (Q4W; Part B). In Part A, the first 6 patients of Cohort 1 will follow 3+3+6 rules, allowing for AB308 dose adjustment if dose-limiting toxicities (DLTs) warrant; all other dose-escalation cohorts will use 3+3 rules. After the Cohort 2 DLT period has been cleared, Part B enrollment will begin in parallel with Part A to determine the recommended dose for expansion (RDE) for AB308 + zim for both the Q3W (Part A) and Q4W (Part B) schedules. Each part can independently proceed to dose-expansion once the RDE is determined for that dose schedule. In the dose-expansion, patients will be enrolled into disease-specific cohorts for non-small cell lung cancer (NSCLC), melanoma, gastrointestinal cancer, cervical cancer, and hematologic malignancies (DLBCL or MM). AB308 + zim may be dosed on either a Q3W or Q4W schedule; all patients in each expansion cohort will receive the same dose schedule. For both the dose-escalation and dose-expansion, the primary endpoints assess safety and tolerability of AB308 + zim; secondary endpoints include pharmacokinetics (PK), immunogenicity, and objective response rate for AB308 + zim. Additional secondary endpoints in the dose-expansion are disease control rate and duration of response. Safety and efficacy data will be analyzed using summary statistics and Kaplan Meier estimates as appropriate; PK parameters will be estimated using noncompartmental methods. ARC-12 is actively recruiting at sites in the United States (NCT04772989). [Display omitted] DisclosuresAilawadhi: Pharmacyclics: Consultancy, Research Funding; Takeda: Consultancy; Sanofi: Consultancy; Amgen: Consultancy, Research Funding; Ascentage: Research Funding; Genentech: Consultancy; AbbVie: Consultancy; BMS: Consultancy, Research Funding; Xencor: Research Funding; Janssen: Consultancy, Research Funding; Medimmune: Research Funding; Cellectar: Research Funding; Karyopharm: Consultancy; Beigene: Consultancy; GSK: Consultancy, Research Funding. Foster: Arcus Biosciences, Inc.: Current Employment. Ryba: Arcus Biosciences, Inc.: Current Employment. Scharville: Arcus Biosciences, Inc.: Current Employment. El-Baghdady: Arcus Biosciences, Inc.: Current Employment. OffLabel Disclosure:AB308 and zimberelimab (AB122) are in clinical development and are not approved in the United States for the use under discussion.

Selinexor Enhances NK Cell Activation Against Lymphoma Cells Via Downregulation of HLA-E

Introduction: Natural killer (NK) cells are powerful immune effectors which induce direct cytotoxicity, promote adaptive immune responses and mediate antibody dependent cellular cytotoxicity (ADCC). Enhancement of NK cell activity against cancer is currently the focus of intense research efforts and strategies include CAR-NK, stimulatory antibodies, cytokines and checkpoint inhibitors.Upregulation of exportin-1 (XPO1) is common in human cancers and high expression is negatively associated with survival in various cancers including diffuse large B cell lymphoma (DLBCL). Targeted inhibition of XPO1 by the selective inhibitor selinexor leads to cancer cell death via accumulation of tumour suppressor proteins in the nucleus, dysregulation of growth regulatory proteins and blockade of oncogene protein translation. The therapeutic efficacy of XPO1 inhibition has led to FDA approval of the oral XPO1 inhibitor selinexor for the treatment of multiple myeloma and DLBCL. The effect of selinexor on NK cell activity has not previously been investigated and was therefore addressed in this study.Methods: The B lymphoma cell lines JeKo-1, SU-DHL-4 and Ramos were incubated with selinexor (50-2000nM) for 18 hours before analysis. Flow cytometry was used to assess cell surface expression of activating and inhibitory ligands for NK cells. For NK based assays, peripheral blood derived NK cells were isolated from healthy donors and incubated with IL-15 (1ng/ml) overnight prior to co-culture with target lymphoma cells for a further 4 hours. Cytotoxicity was assessed using propidium iodide staining of target cells and degranulation of NK cells was assessed by measurement of CD107a. Whole blood samples from colorectal cancer patients (n=11) at pre-treatment and 3 weeks post selinexor monotherapy were assessed by flow cytometry for CD45+CD3-CD19-CD56+ NK cells.Results: Selinexor pre-treatment of target lymphoma cells significantly increased NK cell mediated cytotoxicity against SU-DHL-4 (2.2 Fold increase, p<0.01), JeKo-1 (2 Fold increase, p<0.01) and Ramos (1.7 Fold increase, p<0.01) cells. In accordance with this, selinexor pre-treatment of target cells also increased the activation of NK cells against SU-DHL-4, JeKo-1 and Ramos cells as measured by CD107a expression in both CD56 bright and CD56 dim NK cell sub-groups.To identify the mechanism behind this, we measured expression of activating and inhibitory ligands for NK cells on SU-DHL-4 cells after incubation with selinexor. No significant changes in expression of activating ligands (MICA/B, ULBP-2/5/6, ULBP-1, Vimentin, B7H6, CD54) were evident. In contrast, selinexor significantly (p<0.001) reduced the surface expression of HLA-E on SU-DHL-4 cells by 50%. Selinexor mediated downregulation of HLA-E was also evident in Ramos (60% reduction, p<0.001) and JeKo-1 cells (20% reduction, p<0.01). HLA-E binds the ITIM containing receptor NKG2A, a key inhibitory receptor for NK cells and subsets of T cells. In accordance with this, selinexor pre-treatment of SU-DHL-4 cells selectively increased NKG2A+ NK cell activation (p<0.01) following co-culture.To examine the effect of selinexor on NK cells in patients, we assessed the proportion of NK cells in the peripheral blood of 11 colorectal cancer patients at pre-treatment and three weeks post selinexor monotherapy. % NK cells of CD45+ peripheral blood lymphocytes following treatment with selinexor was increased 2-fold (Median 5% pre-treatment vs 10% post selinexor). In addition, increased abundance of the less mature and less cytotoxic CD56 bright subset of NK cells was associated with poor response to therapy (Median 4% responders (n=3) vs 20% non-responders (n=8)). Larger patient datasets are required to confirm these effects and this analysis is currently ongoing. The effect of selinexor on NK cells in patients with lymphoma is also currently under investigation.Conclusions:The NKG2A:HLA-E axis is a novel immune checkpoint target and our data identifies that selinexor sensitises lymphoma cells to NK cell mediated killing via disruption of this interaction. In addition, we provide initial evidence that NK cells may be associated with clinical response to selinexor. This data indicates that NK cells may contribute to the therapeutic efficacy of selinexor and that selinexor may synergise with NK cell targeted therapies for the treatment of lymphoma. DisclosuresWalker: Karyopharm Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Johnson: Morphosys: Honoraria; Kymera: Honoraria; Kite Pharma: Honoraria; Incyte: Honoraria; Genmab: Honoraria; Celgene: Honoraria; Bristol-Myers: Honoraria; Epizyme: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy; Novartis: Honoraria; Takeda: Honoraria; Oncimmune: Consultancy; Janssen: Consultancy. Cragg: BioInvent International: Consultancy, Research Funding; GSK: Research Funding; UCB: Research Funding; iTeos: Research Funding; Roche: Research Funding. Forconi: Novartis: Honoraria; Roche: Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Gilead: Research Funding. Landesman: Karyopharm Therapeutics: Current Employment, Current equity holder in publicly-traded company. Blunt: Karyopharm Therapeutics: Research Funding.

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.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

Deciphering the Role of Mucosal Immune Responses and the Cervicovaginal Microbiome in Resistance to HIV Infection in HIV-Exposed Seronegative (HESN) Women.

ABSTRACTThe female genital tract (FGT) is an important site of human immunodeficiency virus (HIV) infection. Discerning the nature of HIV-specific local immune responses is crucial for identifying correlates of protection in HIV-exposed seronegative (HESN) individuals. The present study involved a comprehensive analysis of soluble immune mediators, secretory immunoglobulins (sIg), natural killer (NK) cells, CXCR5+ CD8+ T cells, T follicular helper (Tfh) cells, and T regulatory cells (Tregs) in the vaginal mucosa as well as the nature and composition of the cervicovaginal microbiome in HESN women. We found significantly elevated antiviral cytokines, soluble immunoglobulins, and increased frequencies of activated NK cells, CXCR5+ CD8+ T cells, and Tfh cells in HESN females compared to HIV-unexposed healthy (UH) women. Analysis of the genital microbiome of HESN women revealed a greater bacterial diversity and increased abundance of Gardnerella spp. in the mucosa. The findings suggest that the female genital tract of HESN females represents a microenvironment equipped with innate immune factors, antiviral mediators, and critical T cell subsets that protect against HIV infection.IMPORTANCE The vast majority of human immunodeficiency virus (HIV) infections across the world occur via the sexual route. The genital tract mucosa is thus the primary site of HIV replication, and discerning the nature of HIV-specific immune responses in this compartment is crucial. The role of the innate immune system at the mucosal level in exposed seronegative individuals and other HIV controllers remains largely unexplored. This understanding can provide valuable insights to improve vaccine design. We investigated mucosal T follicular helper (Tfh) cells, CXCR5+ CD8+ T cells, natural killer (NK) cells subsets, soluble immune markers, and microbiome diversity in HIV-exposed seronegative (HESN) women. We found a significantly higher level of mucosal CXCR5+ CD8+ T cells, CD4+ Tfh cells, activated NK cell subsets, and antiviral immune cell mediators in HESN women. We also found a higher abundance of Gardnerella spp., microbiome dysbiosis, and decreased levels of inflammatory markers to be associated with reduced susceptibility to HIV infection. Our findings indicate that increased distribution of mucosal NK cells, CXCR5+ CD8+ T cells, Tfh cells, and soluble markers in HIV controllers with a highly diverse cervicovaginal microbiome could contribute effectively to protection against HIV infection. Overall, our findings imply that future vaccine design should emphasize inducing these highly functional cell types at the mucosal sites.

Making a Killer: Selecting the Optimal Natural Killer Cells for Improved Immunotherapies.

Over the past 20 years natural killer (NK) cell-based immunotherapies have emerged as a safe and effective treatment option for patients with relapsed or refractory leukemia. Unlike T cell-based therapies, NK cells harbor an innate capacity to eliminate malignant cells without prior sensitization and can be adoptively transferred between individuals without the need for extensive HLA matching. A wide variety of therapeutic NK cell sources are currently being investigated clinically, including allogeneic donor-derived NK cells, stem cell-derived NK cells and NK cell lines. However, it is becoming increasingly clear that not all NK cells are endowed with the same antitumor potential. Despite advances in techniques to enhance NK cell cytotoxicity and persistence, the initial identification and utilization of highly functional NK cells remains essential to ensure the future success of adoptive NK cell therapies. Indeed, little consideration has been given to the identification and selection of donors who harbor NK cells with potent antitumor activity. In this regard, there is currently no standard donor selection criteria for adoptive NK cell therapy. Here, we review our current understanding of the factors which govern NK cell functional fate, and propose a paradigm shift away from traditional phenotypic characterization of NK cell subsets towards a functional profile based on molecular and metabolic characteristics. We also discuss previous selection models for NK cell-based immunotherapies and highlight important considerations for the selection of optimal NK cell donors for future adoptive cell therapies.

Dynamics of Adaptive Immune Cell and NK Cell Subsets in Patients With Ankylosing Spondylitis After IL-17A Inhibition by Secukinumab.

Background: Anti-IL-17A therapy is generally effectively applied in patients with Ankylosing Spondylitis (AS) to achieve and maintain remission. However, the influence of anti-IL-17A on the composition of the immune system is not apparent. Our prospective study was to explore the changes in immune imbalance regarding T cell, B cell and natural killer (NK) cell subsets after secukinumab treatment in AS patients. Methods: Immune cell distribution of 43 AS patients treated with secukinumab for 12 weeks and 47 healthy controls (HC) were evaluated. Flow cytometry using monoclonal antibodies against 25 surface markers was accomplished to explore the frequencies of lineage subsets. The differences between HC, AS pre-treatment, and post-treatment were compared using the paired Wilcoxon test, Mann-Whitney U test, and ANOVA. Results: AS patients had altered immune cell distribution regarding T cell and B cell subsets. Apart from activated differentiation of CD4+ T cell, CD8+ T cell and B cell, higher levels of cytotoxic T (Tc) two cells and Tc17 cells were noted in AS patients. We confirmed that helper T (Th) one cell became decreased; however, Th17 cells and T follicular helper (Tfh) 17 cells went increased in AS. After 12 weeks of secukinumab therapy, CRP and ASDAS became significantly decreased, and meanwhile, the proportions of Th1 cells, Tfh17 cells and classic switched B cells were changed towards those of HC. A decreased CRP was positively correlated with a decrease in the frequency of naïve CD8+ T cells (p = 0.039) and B cells (p = 0.007) after secukinumab treatment. An elevated level of T cells at baseline was detected in patients who had a good response to secukinumab (p = 0.005). Conclusion: Our study confirmed that AS patients had significant multiple immune cell dysregulation. Anti-IL-17A therapy (Secukinumab) could reverse partial immune cell imbalance.

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

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

P-080: Single-cell transcriptomic analysis of bone marrow NK cells reveals loss of activated cytotoxic NK cells in Multiple Myeloma

<h3>Background</h3> Multiple Myeloma (MM) disease progression and therapy response are influenced by cues of the microenvironment including tumor control by a cytotoxic immune response. Natural killer (NK) cells are notable mediators of the cytotoxic immune response to MM, and important effector cells in recent immune-mediated therapies. NK cells are drivers of antibody-dependent-cellular cytotoxicity in therapies based on anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided based upon CD56 expression into a cytokine-producing CD56bright subset, releasing cytokines such as IFN-γ, TNF-a and GM-CSF, and a cytotoxic CD56dim subset. However, accumulating evidence suggests much larger heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact response to NK cell-driven immunotherapies. Here, we used single-cell RNA sequencing to investigate the heterogeneity and MM-driven alterations of the NK cell compartment in the bone marrow of newly diagnosed MM patients undergoing first-line Daratumumab-containing therapy. <h3>Methods</h3> We performed single-cell RNA sequencing of the CD38+ and CD38- fractions of viably frozen bone marrow aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. <h3>Results</h3> After integration of the datasets, bone marrow NK cells formed eight distinct transcriptomic clusters. Conventional CD56bright and CD56dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK+CD56bright NK cells contained both a cluster of naive and a cluster of activated NK cells. The GZMB+CD56dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets we compared GZMK+CD56bright vs GZMB+CD56dim cluster composition and distribution between control and MM patients. Control bone marrow was dominated by GZMB-transcribing cytotoxic CD56dim NK cells, represented by a low cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim ratio. In contrast, MM bone marrow was characterized by heterogeneity in the ratio of cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim NK cells. A subset of patients presented with a complete reversal of this ratio. This altered composition was due to a loss of cytotoxic GZMB+CD56dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation. <h3>Conclusion</h3> Here we present a transcriptomic overview of NK cells in MM bone marrow at the single-cell level. A subset of MM patients has a loss of activated cytotoxic GZMB+CD56dim NK cells, suggestive of reduced cytotoxic anti-tumor responses. Current analyses are focused on clinical implications of NK cell alterations in response to Daratumumab-based therapies.

Functions of NK and iNKT cells in pediatric and adult CVID, ataxia telangiectasia and agammaglobulinemia patients

Primary immune deficiencies (PID) are known to be more than 400 genetic defects caused by the impairment in development and/or functions of the immune system. Common Variable Immunodeficiency (CVID), Ataxia Telangiectasia (AT) and Agammaglobulinemia (AG) are examples of the most common immunodeficiency syndrome. Natural killer (NK) cells are a component of innate immune system and play a major role in the host-rejection of both tumors and virally infected cells. iNKT cells have a role in autoimmune and infectious diseases and controlling of tumor rejection. In this study, NK and iNKT cells and their functions, and intracellular cytokine amount are aimed to determine in patients that suffer CVID, AT and AG. NKp30, NKp46, NKG2D, perforin and granzyme mRNA expression levels were analyzed using RT-PCR. Receptors, cytokine amount of NK cell subset and iNKT were analyzed by flow cytometry. Decreased CD3+ T and elevated NK cell subset in pediatric AT were found. Expression of NKp44 was decreased in adult AG, but not in pediatric patients. Low NKp44 expression in CD3−CD16+CD56dim NK cell subset was found in pediatric AT patients. High HLA-DR, perforin and granzyme expression were found in CD3−CD16+CD56dim NK cell subset of pediatric CVID and AT patients. Alteration of the number of NK subsets, NK receptor expression and cytokine production were observed in pediatric patients compared to healthy subjects.

EZH2 identifies the precursors of human natural killer cells with trained immunity.

Objective:Trained immunity of natural killer (NK) cells has shown great potential in the treatment of cancers by eliciting enhanced effector responses to restimulation by cytokines or cancer cells for long time periods after preactivation. However, the human NK cells responsible for the generation and maintenance of trained immunity are largely unknown. We hypothesized that heterogeneous human NK cells would respond differentially to stimulation with a combination of IL-12, IL-15, and IL-18, and that an NK cell subset might exist that is mainly responsible for the induction of trained immunity. On the basis of our hypothesis, we aimed to identify the subset from which cytokine-trained human NK cells originate and to explore possible regulatory targets for drug intervention.Methods:Flow cytometry assays were performed to analyze the functions of cytokine-trained NK cells and examine cell division and protein expression in NK cell subsets. Single-cell RNA sequencing (scRNA-seq) plus TotalSeq™ technology was used to track the heterogeneity of NK cells during the induction of trained immunity.Results:Traditional developmental markers for peripheral NK cells were unable to identify the precursors of human NK cells with trained immunity. Therefore, we used scRNA-seq plus TotalSeq™ technology to track the heterogeneity of NK cells during the induction of trained immunity and identified a unique cluster of CD57−NKG2A+EZH2+IFNG+MKI67+IL12R+IL15R+IL18R+ NK cells. Enrichment and pseudotime trajectory analyses suggested that this cluster of NK cells contained the precursor of trained NK cells. We then used flow cytometry to further investigate the role of EZH2 in trained NK precursors and found that CD57−NKG2A+EZH2+ NK cells had faster cell cycles and an enhanced trained phenotype, and EZH2 inhibition significantly impaired the induction of trained immunity in NK cells. These results suggested that EZH2 is a unique epigenetic marker of precursors of human NK cells with trained immunity.Conclusions:Our work revealed human NK heterogeneity in the induction of trained immunity, identified the precursor subset for trained NK cells, and demonstrated the critical role of EZH2 in the induction of trained immunity in human NK cells.

Activation status dictates the function of unlicensed natural killer cells in mice and humans

Natural killer (NK) cells are involved in innate defense against viral infection and cancer. NK cells can be divided into subsets based on the ability of different receptors to bind to major histocompatibility (MHC) class 1 molecules, resulting in differential responses upon activation in a process called “licensing” or “arming.” NK cells expressing receptors that bind self-MHC are considered licensed due to an augmented effector lytic function capability compared with unlicensed subsets. However, we demonstrated that unlicensed NK subsets instead positively regulate the adaptive T-cell response during viral infections that are related to localization and cytokine production. In this study, the differential effects of the two types of NK subsets were contingent on the environment in viral infection and hematopoietic stem cell transplantation (HSCT) models. Infection of mice with high-dose (HD) murine cytomegalovirus (MCMC) led to a loss of licensing-associated differences, as compared with mice with low-dose (LD) infection: the unlicensed NK subset no longer localized in lymph nodes (LNs), but instead remained at the site of infection. Similarly, the patterns observed during HD infection paralleled the phenotypes of both human and mouse NK cells in an HSCT setting where NK cells exhibit an activated phenotype. However, in contrast to the effects of subset depletion in T-cell replete models, the licensed NK cell subsets still dominated antiviral responses after HSCT. Overall, our results highlight the intricate tuning of NK cells and how it affects overall immune responses with regard to licensing patterns and their dependency on the level of stimulation and activation status.

Circulating NKG2C + NK cell expressing CD107a/LAMP-1 subsets at the onset of CMV reactivation in seropositive kidney transplant recipients

Cytomegalovirus (CMV) infection contributes to morbidity and mortality among kidney transplant recipients. Natural killer (NK) cells can battle against CMV in kidney transplant recipients (KTRs). This study aimed to analyze the association between CMV reactivation and the proportion of NK cell subsets and their activity. In a cross-sectional study, ten CMV reactivated KTRs, and ten non- CMV reactivated ones were recruited. Ten matched healthy controls were also included in this cohort. The presence of anti-CMV-IgG Ab in both KTR subgroups from seronegative donors and healthy controls was determined. The frequency of distinct subsets of memory-like NK cells was analyzed through NKG2C, NKG2A, and CD57 using flow cytometry. The activity of NK cells was evaluated after stimulation via coculture with K562 cell line and then assessment of the frequency of CD107a and granzyme B. The mRNA levels of transcription factors, including T-bet, EAT, and inflammatory proteins, including IFN-γ and perforin contributing to NK cell activation, were also evaluated.Results showed a significantly lower frequency of NKG2C + NKG2A-CD57+ NK cell population in CMV-reactivated KTRs compared to non-reactivated ones (P-value:0.003). NKG2C+ NK cells expressing CD107a/LAMP-1 significantly was increased in CMV-reactivated KTRs compared to non-reactivated ones (P-value: 0.0002). The mRNA level of IFN-γ had a significant increase in the CMV-reactivated KTRs vs. nonreactive ones (P-value: 0.004). Finally, evaluation of the NK cells' cytotoxicity and activity through assessment of CD107a/LAMP-1 expression and IFN-γ secretion may be helpful for the identification of the risk of CMV reactivation in KTRs.

FcεRIγ-negative NK cells persist in vivo and enhance efficacy of therapeutic monoclonal antibodies in multiple myeloma

AbstractMonoclonal antibodies (mAbs) are a central component of therapy for hematologic malignancies. Widely used mAb agents in multiple myeloma (MM) include daratumumab and elotuzumab. However, not all patients respond to these agents, and resistance is a significant clinical issue. A recently discovered subset of human natural killer (NK) cells lacking expression of FcεRIγ (g-NK cells) was found to have a multifold increase in antibody-dependent effector functions after CD16 crosslinking. In this study, we tested the capacity of g-NK cells to enhance the efficacy of therapeutic mAbs against MM. In vitro, we found that g-NK cells have strikingly superior anti-myeloma cytotoxicity compared with conventional NK (cNK) cells when combined with daratumumab or elotuzumab (∼sixfold; P < .001). In addition, g-NK cells naturally expressed minimal surface CD38 and SLAMF7, which reduced the incidence of therapeutic fratricide. In tumor-naïve murine models, the persistence of g-NK cells in blood and spleen was >10 times higher than that of cNK cells over 31 days (P < .001). In vivo efficacy studies showed that the combination of daratumumab and g-NK cells led to a >99.9% tumor reduction (by flow cytometry analysis) compared with the combination of daratumumab and cNK cells (P < .001). Moreover, treatment with daratumumab and g-NK cells led to complete elimination of myeloma burden in 5 of 7 mice. Collectively, these results underscore the unique ability of g-NK cells to potentiate the activity of therapeutic mAbs and overcome limitations of current off-the-shelf NK cell therapies without the need for cellular irradiation or genetic engineering.

The Impact of High-Fat Diet and Restrictive Feeding on Natural Killer Cells in Obese-Resistant BALB/c Mice.

Background: The association of obesity and an increased risk for severe infections and various cancer types is well-described. Natural killer (NK) cells are circulating lymphoid cells and promoters of the immune response toward viruses and malignant cells. As demonstrated in previous studies the phenotype and functionality of NK cells is impaired in obesity. So far, the majority of animal studies were exclusively performed using ad libitum feeding regimes and it remained unclear whether NK cell alterations are mediated by obesity-associated immunological changes or by direct effects of the dietary composition. Therefore, the aim of the present study was to characterize NK cells in the peripheral blood of obese-resistant BALB/c mice supplied a normal-fat diet (NFD) or high-fat diet (HFD), ad libitum or in a restrictive manner.Methods: Twenty-eight BALB/c-mice were fed a NFD or HFD either ad libitum or in a restrictive feeding regime with 90% of the mean daily diet supply of the corresponding ad libitum group (each group n = 7). Blood and visceral adipose tissue were collected for flow cytometric analysis, analysis of plasma cytokine concentrations by multiplex immunoassay and real-time RT-PCR analyses. For statistical analyses two-way ANOVA with the factors “feeding regime” and “diet” was performed followed by a post-hoc Tukey's multiple comparison test and to compare means of the four mouse groups.Results: Ad libitum-feeding of a HFD in BALB/c mice has no influence on body weight gain, visceral fat mass, plasma cytokine concentrations, immune cell populations as well as the number, frequency and phenotype of NK cells. In contrast, restrictive feeding of a HFD compared to NFD led to significantly higher body weights, visceral fat mass and plasma interferon-γ concentrations which was associated with changes in the frequencies of granulocytes and NK cell subsets as well as in the surface expression of NK cell maturation markers.Conclusion: Results demonstrate for the first time that HFD-induced alterations in NK cells are consequences of the obese associated immunological profile rather than a direct effect of the dietary composition. These data can help to clarify the increased risk for cancer and severe infections in obesity.

Innate Immune Response to Viral Infections at the Maternal-Fetal Interface in Human Pregnancy.

The placenta, the first and largest organ to develop after conception, not only nurtures and promotes the development of the conceptus, but, it also functions as a barrier against invading pathogens. Early phases of pregnancy are associated with expansion of specific subsets of Natural Killer cells (dNK) and macrophages (dMφ) at the maternal uterine mucosa, the basal decidua. In concert with cells of fetal origin, dNK cells, and dMφ orchestrate all steps of placenta and fetus development, and provide the first line of defense to limit vertical transmission. However, some pathogens that infect the mother can overcome this protective barrier and jeopardize the fetus health. In this review, we will discuss how members of the classical TORCH family (Toxoplasma, Other, Rubella, Cytomegalovirus, and Herpes simplex virus) and some emerging viruses (Hepatitis E virus, Zika virus, and SARS-CoV2) can afford access to the placental fortress. We will also discuss how changes in the intrauterine environment as a consequence of maternal immune cell activation contribute to placental diseases and devastating pregnancy outcomes.

Effect of gMDSCs on natural killer cell functionality in chronic hepatitis C patients treated with direct-acting antiviral agents

Objective: To investigate the changes of natural killer(NK) cell function, and clarify the effect of granulocytic myeloid derived suppressor cells (G-MDSCs) on NK cell functionality in patients with treatment-naive chronic hepatitis C (CHC) who were cured by direct-acting antiviral agents (DAAs). Methods: Thirteen treatment-naive CHC patients and 13 healthy controls were prospectively included in this study from March 2016 to January 2017. They were divided into case group and control group, respectively. The patients of case group,6 males and 7 females aged 21-65 years old with an average of (37±14),were treated with daclatasvir and asunaprevir combination (DCV/ASV) at the Department of Infectious Diseases, Fifth Medical Center of Chinese PLA General Hospital. While 13 healthy individuals, 6 males and 7 females aged 21-57 (36±11) years old, were enrolled as healthy controls(control group). Flow cytometry was used to determine the immunological characteristics of peripheral blood NK cells subset, and detect the frequencies of gMDSCs in peripheral blood of people in two groups. It was specifically notes that CHC patients of case group would be detected before, during and after treatment. The correlations between gMDSCs and each NK cell subset function were also examined. The impact of gMDSCs on NK cell functionalities and the relevant regulatory mechanisms were explored using co-culture experiments of sorted NK cells and gMDSCs in vitro. Results: Compared with healthy controls, the decreased IFN-γ production[M(Q1,Q3)] [3.182 (2.757, 4.237) vs 6.675 (4.476, 8.280),1.434 (1.127, 2.434) vs 3.045 (1.680, 4.856), 2.611 (1.749, 3.498) vs 5.160 (4.232, 7.683)] and increased CD107a degranulation [9.314 (7.838, 13.543) vs 3.480 (2.938, 6.824), 2.544 (1.366, 4.768) vs 0.552 (0.408, 1.560), 10.339 (9.145, 12.534) vs 3.488 (3.117, 5.651)] (all P<0.05) were found on NK cell and its subsets. The frequencies of gMDSCs and plasma concentration of arginase-1 in CHC patients was significantly higher than that in healthy controls [7.050 (4.180, 12.538) vs 1.440 (0.444, 2.261), 114.278 (68.492, 163.724) vs 64.753 (50.809, 93.278)](all P<0.05). The production of IFN-γ was increased and the secretion of CD107a was decreased in NK cell and its subsets after DAAs treatment (P<0.05). The frequencies of gMDSCs and plasma arignase I levels were also decreased in CHC patients treated with DAAs (P<0.05).The results of the study indicated that the frequencies of G-MDSCs were inversely associated with the levels of IFN-γproduction of NK cells and CD56dim NK cells in CHC patients (r=0.668, -0.750, respectively, both P<0.05). In addition, the frequencies of gMDSCs were positively associated with the expression of CD107a in the CD56bright NK cell subset (r=0.711, P=0.021). In vitro, the inhibition of gMDSCs on the IFN-γ production of NK cells was demonstrated in the co-culture experiments of sorted NK cells and gMDSCs, and blocking arginase I can significantly increase the ability of NK cells to produce IFN-γ, restore NK cell IFN-γ production. Conclusions: gMDSCs in peripheral blood of CHC patients has been shown to suppress NK cell IFN-γ production in an arginase I-dependent manner. Direct-acting antiviral-mediated clearance of HCV is associated with the normalization of NK cell function and gMDSCs frequency.

Restoring NK cells functionality via cytokine activation enhances cetuximab-mediated NK-cell ADCC: A promising therapeutic tool for HCC patients

Natural Killer (NK) cells are considered the first line of defense against viral infections and tumors. Several factors affect NK cytotoxic activity rendering it dysfunctional and thereby impeding the ability to scavenge abnormal cells as a part of immune escaping mechanisms induced by different types of cancers. NK cells play a crucial role augmenting the activity of various types of anticancer mAb since dysfunctional NK cells are the main reason for the low response to these therapies. To this light, we examined the phenotypic characters of the circulating NK cells isolated from HCC patients compared to healthy controls. Then, dysfunctional NK cells, from HCC patients, were reactivated with cytokines cocktail and their cytotoxic activity with the anti-EGFR mAb “cetuximab” was investigated. This showed a downregulation of patients NK cells activating receptors (NKP30, NKP46, NKG2D and CD16) as well as CD56 and up-regulation of NKG2A inhibitory receptor. We also reported an increase in aberrant CD56− NK cells subset in peripheral blood of HCC patients compared to healthy controls. Thus, confirming the dysfunctionality of peripheral NK cells isolated from HCC patients. Cytokines re-activation of those NK cells lead to upregulation of NK activating receptors and downregulation of inhibitory receptor. Moreover, the percentage of aberrant CD56− NK cells subset was reduced. Here, we proved that advanced HCC patients have an increased percentage of more immature and noncytotoxic NK cell subsets in their peripheral blood, which might account for the low cytotoxicity noticed in those patients. A significant improvement in the cytotoxicity against HCC was noticed upon using reactivated NK cells combined with cetuximab. Therefore, this study highlights the potential recruitment of NK immune cells along with cetuximab to enhance cytotoxicity against HCC.

Study of surface activation markers on CD3- CD16+ NK cells and their correlation with clinical manifestations in children with infectious mononucleosis.

This study aimed to investigate the proportion of surface activation markers on natural killer (NK) cells in children with infectious mononucleosis (IM) and to explore its clinical relevance. A total of 17 children hospitalized with IM were included in this study as the experimental group. Meanwhile, healthy children matched for age and gender served as controls. First, we isolated peripheral blood mononuclear cells from children with IM and healthy children. Then, NK cell surface markers were stained with monoclonal antibodies and analyzed by flow cytometry. The results showed that the percentage of CD3- CD16+ NK cells was higher in peripheral blood lymphocytes from children with IM than that from healthy children (t = -4.52, P < 0.05). And the expression of the surface activation markers CD69 and CD25 on CD3- CD16+ NK cells was also higher in children with IM (t = -7.729, P < 0.05; t = -5.068, P < 0.05). There was a positive correlation between the percentage of CD3- CD16+ NK cells in peripheral blood and the duration of fever in children with IM (r = 0.530, P < 0.05). Therefore, the proportion of NK cell subsets in children's peripheral blood changes in the acute phase of IM, suggesting that NK cells enhance their cytotoxicity and play a role in the control of infection in children with IM. Higher levels of CD3- CD16+ NK cells and the association with disease progression suggest that these cells might be a useful index to help evaluate the disease course.

Impact of high-dose rate radiotherapy on B and natural killer (NK) cell polarization in peripheral blood mononuclear cells (PBMCs) via inducing non-small cell lung cancer (NSCLC)-derived exosomes.

BackgroundNon-small cell lung cancer (NSCLC) is the most commonly diagnosed solid tumor. While it has been established that stereotactic body radiotherapy for NSCLC plays an important role in antitumor immune response, the possible effects of the dose rate on this response has not been fully clarified.MethodsIn vitro, A549 cells were irradiated on a Varian TrueBeam® Linear Accelerator with dose and dose rate escalation using the flattening filter-free (FFF) technique, which was followed by coculturing with peripheral blood mononuclear cells (PBMCs). The exosomes from irradiated A549 cells were isolated and then cocultured with PBMCs. Flow cytometry was performed to analyze the proportion of lymph cell clusters in PBMCs.ResultsThe proportion of CD3− immune cell clusters in PBMCs was significantly higher in the 10 Gy treatment group than in the nonirradiated group and other lower-dose (2, 6 Gy) treatment groups at the dose rate of 1,000 MU/min. However, no influence was observed on the proportion of CD3+ T cell subsets. Further results showed that both natural killer (NK) and B cell proportions reached peaks in the 14 Gy treatment group when a dose rate of 1,200 MU/min was used. Notably, the peak values of these two cell proportions were reached at a lower radiation dose of 10 Gy when a greater dose rate, ranging from 1,600 to 2,400 MU/min, was used. We further found that a single, high dose of irradiation (10 Gy), as compared with a single, low dose of irradiation (2 Gy), could markedly stimulate the A549-related exosome secretion in a radiation dose rate-dependent manner. The ultrahigh dose rate radiation–derived exosomes contributed to the polarization of B and NK cell subsets in PBMCs.ConclusionsThe optimized radiation regime, which depends on the appropriate radiation dose and dose rate, results in the production of exosomes derived from NSCLC cells and eventually the redistribution of immune cells in PBMCs.