Adaptive Natural Killer Cells Research Articles

Abstract IA007: Tumor HLA-E expression and cytomegalovirus infection modulate NK cell activity in human bladder cancer

Abstract The intersection of viral infections and cancer therapy presents a complex landscape for immunotherapy strategies, particularly in solid tumors, where treatments such as systemic chemotherapy and targeted radiation are standard. Largely unexplored in solid tumors are the effects of human herpes virus-5 (HHV-5) also known as HCMV that imprints both lifelong effects on natural killer (NK) cells as well progressive effects on T cells with aging. HCMV is an extremely successful virus as it rarely kills the host outside of settings of compromised immunity, e.g. transplantation and advanced HIV-1 infection. HCMV is not considered a comorbidity in patients with solid tumors. Our group has identified reactivated cytomegalovirus (HCMV), found in bladder tissues post treatment, including infection of monocytes, fibroblasts, and even tumor cells, suggesting a viral influence that extends beyond traditional perspectives on cancer treatment resistance and response. We have observed spread of infection in both settings of BCG-treated non-muscle-invasive bladder cancer (NMIBC) as well as MIBC treated with gemcitabine and cisplatin chemotherapy. HCMV infection leads to the development of “adaptive” NK cells that express the activating isoform of NKG2A: NKG2C, which are likely expanded in response to HCMV reactivation and have established functions for controlling virus reactivation. Our central hypothesis is that certain treatments may reactivate HCMV, potentially greatly altering the biology within the tumor microenvironment (TME) and providing a framework to exploit NK cells for immunotherapy strategies. In a cohort of NMIBC patients (N=90) receiving BCG therapy at Mount Sinai, we profiled patients’ serum for HCMV-IgG (N=47 HCMV seropositive; N=43 HCMV seronegative) and observed a significantly improved recurrence-free survival in HCMV seropositive patients (HR=0.58, p=0.03). Further, HCMV-seropositive patients with circulating NKG2C+ adaptive NK cells experienced a near perfect response to BCG therapy (p=0.0002). Strikingly, in situ imaging and RNAscope analyses of patient tumors before and after BCG therapy revealed strong presence of active viremia in tumors. Further, analysis of on-treatment bladder biopsies after tumor resection demonstrated the presence of adaptive NK cells in HCMV+ bladder tissues, but only after exposure to BCG therapy and tissue-derived adaptive NK cells expressed high levels of chemokine receptors, CXCR3 and CXCR4 as well as tissue-residency markers, suggesting a mechanisms for trafficking to the bladder and taking up residence for subsequent antitumor control against recurrence. Impact and Translation: Our findings suggest that adaptive NK cells mediate response to immunotherapies for solid tumors as well as provide preclinical models for adoptive transfer therapies targeting NK cells. Additionally, if HCMV reactivation is widespread across solid tissues in response to systemic therapies, it may prove a critical comorbidity to consider in design of treatment strategies and in clinical trials. Citation Format: Amir Horowitz. Tumor HLA-E expression and cytomegalovirus infection modulate NK cell activity in human bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA007.

ITAM-based receptors in natural killer cells.

The ability of cells of the immune system to acquire features such as increased longevity and enhanced secondary responses was long thought to be restricted to cells of the adaptive immune system. Natural killer (NK) cells have challenged this notion by demonstrating that they can also gain adaptive features. This has been observed in both humans and mice during infection with cytomegalovirus (CMV). The generation of adaptive NK cells requires antigen-specific recognition of virally infected cells through stimulatory NK receptors. These receptors lack the ability to signal on their own and rather rely on adaptor molecules that contain ITAMs for driving signals. Here, we highlight our understanding of how these receptors influence the production of adaptive NK cells and propose areas in the field that merit further investigation.

Pgam1 Regulate Adaptive NK Cells in Anti-Viral and Anti-Tumor Response

Natural killer (NK) cells are the innate immune cells that play a role in the anti-viral infections and tumor cells. The adaptive NK cells are characterized with expression of the HLA-E-specific CD94/NKG2C activating receptor. The reconstitution of NKG2C+ adaptive NK cells post HSCT is much faster in NKG2C homozygous(NKG2C wt/wt) donors than NKG2C heterozygous donors (NKG2C wt/del), which resulted in lower incidence of CMV infection. In this study, we aimed to find out the mainly regulators of adaptive NK cells between NKG2C wt/wt and NKG2C wt/del donors. RNA-sequencing analysis were performed. Results shown that NK cells from NKG2C wt/wt donors constant upregulated glycolysis and biosynthesis-related genes, while downregulated in TGF-beta signaling and FOXO signaling pathway(Figure A and B). In order to determine the role of glycolysis in adaptive NK cells, upregulated glycolysis related genes were assessed by q-PCR, and PGAM1 is considered as the most important gene in the expansion of adaptive NK cells(Figure C). PGAM1 is upregulated upon NK cells stimulation via cytokine or target cells, which is related to the CD107a expression. HKB99 is an allosteric inhibitor for PGAM1, which is considered as a potential therapeutic target for many cancers nowadays. NK cells were added with HKB99 in vitro for 12h hours to evaluate the anti-K562 function. After that, Glycolysis is inhibited with the decreasing of ECAR while the OCR is not affected (FigureD). The adding of PGAM1 inhibitor resulted in decreasing of CD107a expression in NK cells, especially in the adaptive NK cells. NK cells activation signaling pathway is also inhibited, including the phosphorylation of mTORC1 protein S6, Syk, zap70, and the PLC-gamma. The PI3K/AKT is inhibited after the PGAM1 inhibition. After that,supplementation of pyruvate is efficient to rescue the inhibition caused by PGAM1 inhibitor. 5mM pyruvate adding can restore the cytokine release of NK cells and the phosphorylation of S6 and syk/zap70(Figure E). PGAM1-NCR-cre mice was constructed in which PGAM1 is knock out in NK cells specifically. In KO group, the ratio of NK cells and the maturation of NK cells are decreased. Upon cytokine stimulation, the activator receptors expression is less in KO mice. Besides, PGAM1 deficiency NK cells exhibited glucose uptake deficiency and disadvantages in proliferation both in vivo and in vitro (Figure F). The differentiation (CD11b+CD27-) and activation marker (DNAM1, Ly49H) are decreased in the competing transplantation mouse model, while the exhaustion markers such as PD-1 and NKG2A is upregulated. Further, MCMV infection models shown that PGAM1 deficiency mice with lower ratio of Ly49H+ adaptive NK cells at the early stage of infection, while Ly49G2 and Ly49C+ cells are not affected(Figure G). Further, Ly49H+ adaptive NK cells shown less expression of Ki67 in PB-NK cells, with the activating markers CD25 is decreased. Higher MCMV load induced inflammation is occurred in PGAM1 deficiency NK cells. Anti-tumor effect was also evaluated. The apoptosis of YAC-1 cells is decrease in coincidence with CD107a expression decreasing. MLL-AF9 leukemia mice model shown in day 18, the Ly49H+ adaptive NK cells in PGAM1 deficiency mice is less than WT mice, and the tumor burden is higher and accompanied with shorter survival under tumor circumstances(Figure H). RNA seq analysis shown compared to WT mice, the downregulated genes in PGAM1 deficiency NK cells are enriched in leukocyte activation and innate immune response also interfered the cytokine-mediated signaling pathway. At last, we monitored expression of PGAM1 and the function of NK cells in patients whose CMV is reactive post HSCT. PGAM1 is elevated post CMV infection, which is correlated with CD107a secretion. ROC curve suggests that PGAM1 may act as a predictor for refractory recurrence CMV infection in patients post HSCT(Figure I). In summary, our studies shown the glycolysis is effectively support the faster adaptive NK cells expansion and CMV response. PGAM1-mediated glycolysis is significant for NK cells anti-tumor and anti-viral function.

NK cell-induced damage to P.falciparum-infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG.

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMβ2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free vesicles similar in size to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.

Natural killer cell responses during SARS-CoV-2 infection and vaccination in people living with HIV-1

Natural killer (NK) cell subsets with adaptive properties are emerging as regulators of vaccine-induced T and B cell responses and are specialized towards antibody-dependent functions contributing to SARS-CoV-2 control. Although HIV-1 infection is known to affect the NK cell pool, the additional impact of SARS-CoV-2 infection and/or vaccination on NK cell responses in people living with HIV (PLWH) has remained unexplored. Our data show that SARS-CoV-2 infection skews NK cells towards a more differentiated/adaptive CD57+FcεRIγ− phenotype in PLWH. A similar subset was induced following vaccination in SARS-CoV-2 naïve PLWH in addition to a CD56bright population with cytotoxic potential. Antibody-dependent NK cell function showed robust and durable responses to Spike up to 148 days post-infection, with responses enriched in adaptive NK cells. NK cell responses were further boosted by the first vaccine dose in SARS-CoV-2 exposed individuals and peaked after the second dose in SARS-CoV-2 naïve PLWH. The presence of adaptive NK cells associated with the magnitude of cellular and humoral responses. These data suggest that features of adaptive NK cells can be effectively engaged to complement and boost vaccine-induced adaptive immunity in potentially more vulnerable groups such as PLWH.

CHARACTERIZATION OF THE ADAPTIVE NKG2C+ NK CELL IN HUMAN CYTOMEGALOVIRUS INFECTED INDIVIDUALS: A SYSTEMATIC REVIEW

The significance of adaptive natural killer (NK) cells in the subject pertaining to human cytomegalovirus (HCMV) infection is emphasized by the preferential expansion of a specific subset, termed NKG2C+ which can recognize an array of HCMV-encoded proteins. Until today, the subject of adaptive or ‘memory-like’ NK cells remains controversial, but more emerging evidence shows the cells' ability to ‘remember’. The purpose of this systematic review was to appraise recent literature that centered on the expansion of adaptive NKG2C+ NK cells driven by HCMV infection, specific to ‘human’. Databases including PubMed, Scopus, and JURN were systematically searched to source relevant journal articles published between 2011 and 2021, in a period of 10 years. Full-text terms such as ‘NKG2C+ adaptive NK cells’ AND ‘human cytomegalovirus’ were applied to retrieve related articles. This systematic review is comprised of continuous data on the expansion of NKG2C+ NK cells driven by HCMV and other HCMV-affiliated receptors, immune cells, and diseases with appropriate implementation of distinct inclusion and exclusion criteria. The authors evaluated the risk of bias and quality evidence of the data independently. The study is beneficial by gathering the evidence to conclude the occurrence of adaptive or ‘memory-like’ properties in human NK cells.

Natural killer cells- from innate cells to the discovery of adaptability.

Natural Killer (NK) cells have come a long way since their first description in the 1970's. The most recent reports of their adaptive-like behavior changed the way the immune system dichotomy is described. Adaptive NK cells present characteristics of both the innate and adaptive immune system. This NK cell subpopulation undergoes a clonal-like expansion in response to an antigen and secondary encounters with the same antigen result in an increased cytotoxic response. These characteristics can be of extreme importance in the clinical setting, especially as adoptive immunotherapies, since NK cells present several advantages compared other cell types. This review will focus on the discovery and the path to the current knowledge of the adaptive NK cell population.

Single cell transcriptomes from HCMV +donors reveal a unique NKG2C Highsubset that represents true memory NK cells

Abstract Infection with human cytomegalovirus (HCMV) can result in severe morbidity and mortality to the developing fetus and immunocompromised individuals. Natural killer (NK) cells are cytotoxic lymphocytes that are required to manage and control HCMV infections. The NKG2C receptor expressed by NK cells recognizes and responds to infected cells expressing HLA-E loaded with HCMV UL40-derived peptide. These responding NKG2C +NK cells have been referred to as ‘adaptive NK’ because they possess features of adaptive immunity and are found at greater percentages in HCMV seropositive individuals. The molecular mechanisms by which adaptive NK cells are generated and their transcriptomic signatures remain unknown. To investigate this, we obtained healthy adult spleens from four HCMV +and four HCMV −donors and their NK cells were sorted and captured for single-cell RNA-sequencing (scRNA-seq). Donor median age was 59 [IQR 48.5–56.5], 50% (n=4) were female, 50% (n=2) of females were HCMV seropositive and 50% (n=2) of females were HCMV seronegative. Using scRNA-seq, we observed elevated expression and cell numbers of NKG2C in HCMV +donors when compared to HCMV-donors. We identified a distinct population of NKG2C +adaptive NK cells displaying elevated levels of CD52, B3GAT1, FCGR3A, CD3E, and IL7R. In addition, our developmental trajectory analysis of adaptive NKG2C +NK cells revealed a unique branchpoint of true memory NK cells expressing elevated levels of NKG2C and IL7R. Here, we demonstrate that HCMV infection can induce the formation of adaptive NKG2C +NK cells that display a unique transcriptional and developmental profile. These findings have the potential to influence the future application of adaptive NK cells in cellular immunotherapies. This work is supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant number TL1TR001437

Brief IL-12, IL-15, IL-18 activation drives the acquisition of two distinct memory-like NK cell states

Abstract IL-12/15/18 activation induces natural killer (NK) cell differentiation into cytokine-induced memory-like (ML) NK cells with enhanced IFNγ and killing of tumor targets that is antigen independent, rendering them distinct from conventional (cNK) and CMV-induced adaptive NK cells. In leukemia patients, ML NK cells have an excellent safety profile and 47% CR rate (PMID: 32826231). Despite their clinical promise, little is known about the biology underlying ML NK cells. Based on individual variability of human ML NK cell function, we hypothesized that ML NK cells manifest molecular heterogeneity. To this end, we applied Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) to profile RNA and protein expression on the single-cell level, and evaluated NK cell function. Overnight IL-12/15/18 stimulation induced broad activation with distinct transcriptional programs evident in CD56 brightcompared to CD56 dimNK cells. In contrast, following in vitrodifferentiation for 7 days, IL-12/15/18 activated NK cells acquired a unique transcriptional and protein signature, and manifest one of two ML NK cell states (ML-1, ML-2), or remained similar to cNK cells. ML-1 was marked by increased activating receptor expression (NKp46, NKp30, NKp44) while ML-2 had increased expression of inhibitory receptors, CD25, HLA-DR, MKI67, and IFNG. In functional assays, ML-2 cells produced more IFNγ following cytokine stimulation than ML-1 cells. Further, ML-1 and ML-2 had divergent transcription factor profiles suggesting distinct mechanisms of regulation. Collectively, our findings reveal molecular and functional heterogeneity of ML NK cells, and investigations into ML subsets in patients receiving ML NK cell therapy are ongoing. AAI Intersect Fellowship for Computational Scientists & Immunologists (JAF, TAF, AAP), T32GM139799 (JAF), P50CA171963 (TAF, MMB-E), R01CA205239 (TAF), P30CA91842 (TAF)

267 Defining the single-cell transcriptomes of splenic adaptive Natural Killer cells in donors with latent human cytomegalovirus infection

OBJECTIVES/GOALS: The primary objective of this study was to define the transcriptomes and transcriptional regulatory network required for the development and function of adaptive Natural Killer (NK) cells in donors with latent human cytomegalovirus (HCMV) infection. METHODS/STUDY POPULATION: Eight healthy adult human spleens were obtained from four HCMV seropositive and four HCMV seronegative donors. Spleens were provided by the Versiti Organ Donor Center of Wisconsin and were processed to a single cell suspension. CD7+ CD3E- CD14- CD19- CD20- NK cells were isolated, using the BD FACSAria sorter. Following cell sorting, single-cell RNA sequencing (scRNA-seq) was performed, and cDNA libraries were constructed and sequenced via NextSeq 550. Cell Ranger was then used to algin the cDNA reads and the Seurat R package was used to analyze the transcriptional data. Cells were filtered and clustered based on the number of uniquely expressed genes. The monocle software was used for single cell trajectory analysis and the SCENIC software was used to decipher gene regulatory networks. RESULTS/ANTICIPATED RESULTS: Eight healthy spleens from four HCMV seropositive and four HCMV seronegative donors were obtained and their NK cells were sorted and captured for scRNA-seq. Donor median age was 59 [IQR 48.5-56.5], 50% (n=4) were female and all donors were not experiencing any acute or chronic symptoms. Using scRNA-seq, we observed elevated numbers of NKG2C+ adaptive NK cells in HCMV seropositive individuals when compared to HCMV seronegative individuals. In addition, we identify a set of transcription markers and regulators that are responsible for the development and function of adaptive NKG2C+ NK cells. Finally, our trajectory analysis of adaptive NKG2C+ NK cells revealed a unique developmental pathway. DISCUSSION/SIGNIFICANCE: Here, we demonstrate that HCMV infection can induce the formation of adaptive NKG2C+ NK cells that display a unique transcriptional and developmental profile. These findings have the potential to influence the future application of adaptive NK cells in cellular immunotherapies.

The Frequency and Function of NKG2C+CD57+ Adaptive NK Cells in Cytomagalovirus Co-Infected People Living with HIV Decline with Duration of Antiretroviral Therapy.

Human cytomegalovirus (CMV) infection drives the expansion and differentiation of natural killer (NK) cells with adaptive-like features. We investigated whether age and time on antiretroviral therapy (ART) influenced adaptive NK cell frequency and functionality. Flow cytometry was used to evaluate the frequency of adaptive and conventional NK cells in 229 CMV+ individuals of whom 170 were people living with HIV (PLWH). The frequency of these NK cell populations producing CD107a, CCL4, IFN-γ or TNF-α was determined following a 6-h antibody dependent (AD) stimulation. Though ART duration and age were correlated, longer time on ART was associated with a reduced frequency of adaptive NK cells. In general, the frequency and functionality of NK cells following AD stimulation did not differ significantly between treated CMV+PLWH and CMV+HIV- persons, suggesting that HIV infection, per se, did not compromise AD NK cell function. AD activation of adaptive NK cells from CMV+PLWH induced lower frequencies of IFN-γ or TNF-α secreting cells in older persons, when compared with younger persons.

Alzheimer's disease alters the transcriptomic profile of natural killer cells at single-cell resolution.

Alzheimer's disease (AD) is the most common dementia without an effective cure at least partially due to incomplete understanding of the disease. Inflammation has emerged as a central player in the onset and progression of AD. As innate lymphoid cells, natural killer (NK) cells orchestrate the initiation and evolution of inflammatory responses. Yet, the transcriptomic features of NK cells in AD remain poorly understood. We assessed the diversity of NK cells using web-based single-cell RNA sequencing data of blood NK cells from patients with AD and control subjects and flow cytometry. We identified a contraction of NK cell compartment in AD, accompanied by a reduction of cytotoxicity. Unbiased clustering revealed four subsets of NK cells in AD, i.e., CD56bright NK cells, CD56dim effector NK cells, adaptive NK cells, and a unique NK cell subset that is expanded and characterized by upregulation of CX3CR1, TBX21, MYOM2, DUSP1, and ZFP36L2, and negatively correlated with cognitive function in AD patients. Pseudo-temporal analysis revealed that this unique NK cell subset was at a late stage of NK cell development and enriched with transcription factors TBX21, NFATC2, and SMAD3. Together, our study identified a distinct NK cell subset and its potential involvement in AD.

Clonal expansion and epigenetic inheritance of long-lasting NK cell memory

Clonal expansion of cells with somatically diversified receptors and their long-term maintenance as memory cells is a hallmark of adaptive immunity. Here, we studied pathogen-specific adaptation within the innate immune system, tracking natural killer (NK) cell memory to human cytomegalovirus (HCMV) infection. Leveraging single-cell multiomic maps of ex vivo NK cells and somatic mitochondrial DNA mutations as endogenous barcodes, we reveal substantial clonal expansion of adaptive NK cells in HCMV+ individuals. NK cell clonotypes were characterized by a convergent inflammatory memory signature enriched for AP1 motifs superimposed on a private set of clone-specific accessible chromatin regions. NK cell clones were stably maintained in specific epigenetic states over time, revealing that clonal inheritance of chromatin accessibility shapes the epigenetic memory repertoire. Together, we identify clonal expansion and persistence within the human innate immune system, suggesting that these mechanisms have evolved independent of antigen-receptor diversification.

Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E.

Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Adaptive NK cell expansion and activity in ovarian cancer patients with cytomegalovirus infection and inflammation (258)

Objectives: Cytomegalovirus (CMV) infection has been shown to expand a subset of natural killer (NK) cells called adaptive NK cells (CD57+NKG2C+) that are long-lived, have enhanced cytotoxicity, and are thought to contribute to cancer control. CMV reactivation is associated with increased inflammation, which can impact patient outcomes. We sought to evaluate the impact of CMV exposure and inflammation, as measured by C-reactive protein (CRP), on adaptive NK cells in patients with high-grade serous ovarian cancer (HGSOC) using serum and ascites samples collected at the time of surgery. Methods: A total of 30 serum samples, collected at the time of cytoreductive surgery in women with HGSOC, were measured for CMV serostatus using an immunoassay and measured for CRP with an immunoturbidimetric method. The samples were categorized as reactive or non-reactive for CMV and low or high for CRP (≤10, >10 mg/L). Thirty serum and 23 ascites samples were evaluated by flow cytometry to quantify adaptive NK cells. A CyTOF panel of 42 antibodies was used to identify adaptive NK cells, differences in NK cell maturation and functional mediators, activation markers, a number of other immune subsets (CD8 T cells, T regulatory cells, B cells, monocytes), and proliferation signatures. Functional assays were performed to assess natural cytotoxicity and antibody-dependent cytokine cytotoxicity (ADCC) by CD1O7a degranulation and IFNy production. Data were analyzed using GraphPad Prism 8.2.0, and p-values ≤ 0.05 were considered statistically significant. Results: Patients with HGSOC with evidence of CMV exposure had increased percentages of CD57+NKG2C+ adaptive NK cells in the blood and ascites compared to those without CMV exposure (Figure 1, p=0.03). CRP blood levels were not significantly different (p=0.30). The increased percentages of adaptive NK cells, observed in the blood and ascites samples that were CMV IgG+, were not significantly altered by the presence (p=0.90) or absence (p=0.90) of CRP. Conclusions: CMV exposure influences the presence of an adaptive NK cell population in HGSOC patients. CMV expanded adaptive NK cells represent a unique NK cell population that, with their enhanced cytotoxicity and memory-like attributes, could be manipulated for future NK cell-based therapies in ovarian cancer. Objectives: Cytomegalovirus (CMV) infection has been shown to expand a subset of natural killer (NK) cells called adaptive NK cells (CD57+NKG2C+) that are long-lived, have enhanced cytotoxicity, and are thought to contribute to cancer control. CMV reactivation is associated with increased inflammation, which can impact patient outcomes. We sought to evaluate the impact of CMV exposure and inflammation, as measured by C-reactive protein (CRP), on adaptive NK cells in patients with high-grade serous ovarian cancer (HGSOC) using serum and ascites samples collected at the time of surgery. Methods: A total of 30 serum samples, collected at the time of cytoreductive surgery in women with HGSOC, were measured for CMV serostatus using an immunoassay and measured for CRP with an immunoturbidimetric method. The samples were categorized as reactive or non-reactive for CMV and low or high for CRP (≤10, >10 mg/L). Thirty serum and 23 ascites samples were evaluated by flow cytometry to quantify adaptive NK cells. A CyTOF panel of 42 antibodies was used to identify adaptive NK cells, differences in NK cell maturation and functional mediators, activation markers, a number of other immune subsets (CD8 T cells, T regulatory cells, B cells, monocytes), and proliferation signatures. Functional assays were performed to assess natural cytotoxicity and antibody-dependent cytokine cytotoxicity (ADCC) by CD1O7a degranulation and IFNy production. Data were analyzed using GraphPad Prism 8.2.0, and p-values ≤ 0.05 were considered statistically significant. Results: Patients with HGSOC with evidence of CMV exposure had increased percentages of CD57+NKG2C+ adaptive NK cells in the blood and ascites compared to those without CMV exposure (Figure 1, p=0.03). CRP blood levels were not significantly different (p=0.30). The increased percentages of adaptive NK cells, observed in the blood and ascites samples that were CMV IgG+, were not significantly altered by the presence (p=0.90) or absence (p=0.90) of CRP. Conclusions: CMV exposure influences the presence of an adaptive NK cell population in HGSOC patients. CMV expanded adaptive NK cells represent a unique NK cell population that, with their enhanced cytotoxicity and memory-like attributes, could be manipulated for future NK cell-based therapies in ovarian cancer.

Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19.

The kinetics of NKG2C+ adaptive natural killer (ANK) cells and NKG2A+inhibitory NK (iNK) cells with respect to the incidence of SARS-CoV-2 infection were studied for 6 months in a cohort of healthcare workers following the administration of the heat-killed Mycobacterium w (Mw group) in comparison to a control group. In both groups, corona virus disease 2019 (COVID-19) correlated with lower NKG2C+ANK cells at baseline. There was a significant upregulation of NKG2C expression and IFN-γ release in the Mw group (p=0.0009), particularly in those with a lower baseline NKG2C expression, along with the downregulation of iNK cells (p<0.0001). This translated to a significant reduction in the incidence and severity of COVID-19 in the Mw group (incidence risk ratio-0.15, p=0.0004). RNA-seq analysis at 6 months showed an upregulation of the ANK pathway genes and an enhanced ANK-mediated antibody-dependent cellular cytotoxicity (ADCC) signature. Thus, Mw was observed to have a salutary impact on the ANK cell profile and a long-term upregulation of ANK-ADCC pathways, which could have provided protection against COVID-19 in a non-immune high-risk population.

Biology and Clinical Relevance of HCMV-Associated Adaptive NK Cells.

Natural killer (NK) cells are an important component of the innate immune system due to their strong ability to kill virally infected or transformed cells without prior exposure to the antigen (Ag). However, the biology of human NK (hNK) cells has largely remained elusive. Recent advances have characterized several novel hNK subsets. Among them, adaptive NK cells demonstrate an intriguing specialized antibody (Ab)-dependent response and several adaptive immune features. Most adaptive NK cells express a higher level of NKG2C but lack an intracellular signaling adaptor, FcϵRIγ (hereafter abbreviated as FcRγ). The specific expression pattern of these genes, with other signature genes, is the result of a specific epigenetic modification. The expansion of adaptive NK cells in vivo has been documented in various viral infections, while the frequency of adaptive NK cells among peripheral blood mononuclear cells correlates with improved prognosis of monoclonal Ab treatment against leukemia. This review summarizes the discovery and signature phenotype of adaptive NK cells. We also discuss the reported association between adaptive NK cells and pathological conditions. Finally, we briefly highlight the application of adaptive NK cells in adoptive cell therapy against cancer.

Delayed B cell repopulation after rituximab treatment in multiple sclerosis patients with expanded adaptive natural killer cells.

Background and purposeThe aim was to evaluate whether adaptive NKG2C+ natural killer (NK) cells, characterized by enhanced antibody‐dependent cell cytotoxicity (ADCC), may influence time to B cell repopulation after rituximab treatment in multiple sclerosis (MS) patients.MethodsThis was a prospective observational study of MS patients treated with rituximab monitoring peripheral B cells for repeated doses. B cell repopulation was defined as CD19+ cells above 2% of total lymphocytes, classifying cases according to the median time of B cell repopulation as early or late (≤9 months, >9 months, respectively). Basal NK cell immunophenotype and in vitro ADCC responses induced by rituximab were assessed by flow cytometry.ResultsB cell repopulation in 38 patients (24 relapsing–remitting MS [RRMS]; 14 progressive MS) was classified as early (≤9 months, n = 19) or late (>9 months, n = 19). RRMS patients with late B cell repopulation had higher proportions of NKG2C+ NK cells compared to those with early repopulation (24.7% ± 16.2% vs. 11.3% ± 10.4%, p < 0.05), and a direct correlation between time to B cell repopulation and percentage of NKG2C+ NK cells (R 0.45, p < 0.05) was observed. RRMS cases with late repopulation compared with early repopulation had a higher secretion of tumor necrosis factor α and interferon γ by NK cells after rituximab‐dependent NK cell activation. The NK cell immunophenotype appeared unrelated to B cell repopulation in progressive MS patients.ConclusionsAdaptive NKG2C+ NK cells in RRMS may be associated with delayed B cell repopulation after rituximab, a finding probably related to enhanced depletion of B cells exerted by NK‐cell‐mediated ADCC, pointing to the use of personalized regimens with anti‐CD20 monoclonal antibody therapy in some patients.

CMV Triplex Vaccine to Enhance Adaptive NK and T-cell Reconstitution After Autologous Hematopoietic Cell Transplantation

Cytomegalovirus (CMV) reactivation after hematopoietic cell transplantation (HCT) augments adaptive (CD56dimNKG2C+CD57+) natural killer (NK) and CMV-specific T cells, with potential antitumor effects. Our recent work found an association between higher abundance of adaptive NK cells after auto-HCT and lower risk of relapse in patients with multiple myeloma. Triplex vaccine is a recombinant modified vaccinia Ankara expressing immunodominant CMV antigens, which significantly enhanced CMV-specific T-cell immune responses in allo-HCT recipients. We evaluated whether 2 doses of the vaccine after auto-HCT in patients with lymphoma or myeloma improves reconstitution of adaptive NK and CMV-specific T cells. The primary endpoint was the number of adaptive NK cells at day 100 (∼1 month after dose 2) relative to day 28 (before dose 1). We conducted a single-arm phase 2 clinical trial of 20 patients with lymphoma or myeloma undergoing auto-HCT. Two doses of the vaccine were given on days 28 and 56. Adaptive NK cells increased in CMV-seronegative patients (P=.02), a rise that was more substantial than in unvaccinated historical CMV-seronegative cohorts (P=.03 comparing the rise between the 2 cohorts). There was also an increase in both CD4+ and CD8+ CMV-specific T cells in CMV-seronegative patients (P=.01) and CMV-specific CD8+ effector T cells in CMV-seropositive patients (P=.03). Triplex vaccine improved reconstitution of adaptive NK and CMV-specific T cells after auto-HCT in patients with lymphoma and myeloma. Further study is needed to determine the clinical impact of this modulation of immune response.

Adaptive NK cells undergo a dynamic modulation in response to human cytomegalovirus and recruit T cells in in vitro migration assays

Human cytomegalovirus (HCMV) reactivation remains a relevant complication after hematopoietic stem cell transplantation (HSCT) despite the great progress made in prophylaxis and treatment. Adaptive Natural Killer (NK) cells undergo a persistent reconfiguration in response to HCMV reactivation however, the exact role of adaptive NK cells in HCMV surveillance is currently unknown. We studied the relationship between HCMV reactivation and adaptive NK cells in 70 patients monitored weekly until day +100 after HSCT. Absolute cell counts of adaptive NK cells increased significantly after resolution of HCMV-reactivation compared to patients without reactivation. Patients with HCMV-reactivation had an early reconstitution of adaptive NK cells (“Responders”) and had mainly a single reactivation (75% Responders vs 48% Non-Responders). Adaptive NK cells eliminated HCMV-infected human foreskin fibroblasts (HFF) in vitro and recruited T cells in an in vitro transwell migration assay. An extensive cytokine/chemokine panel demonstrated strongly increased secretion of CXCL10/IP-10, IFN-α, IL-1α, IL-1β, IL-5, IL-7 and CCL4. Thus, adaptive NK cells may control viral spread and T cell expansion and survival during HCMV-reactivation. Taken together, we have demonstrated the potential of adaptive NK cells in the control of HCMV reactivation both by direct cytotoxicity and by recruitment of other immune cells.