Natural Killer Cell Receptors Research Articles

Single-Cell Transcriptomics Reveals Adaptive NK Cell and Antigen-Specific T Cell Responses Shaped by CMV Viremia Post-Hematopoietic Stem Cell Transplant

Background Impaired immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) poses risks for both disease recurrence and treatment-related complications, such as cytomegalovirus (CMV) reactivation. CMV strongly shapes the immune system, but the development of anti-CMV T cells and CMV-associated adaptive NK cells in vivo is still largely uncharacterized. Understanding these dynamics post-allo-HSCT may have clinical implications, since NK cells mediate a graft-versus-leukemia effect, particularly in myeloid malignancies, and adaptive NK cells have recently been associated with favorable immunotherapy responses in both hematological and solid cancers. Here, with longitudinal sample collection pre and post-allo-HSCT, we aimed to determine how patient and donor-derived NK and T cells synergistically contribute to immune system regeneration, how clonotypes develop and persist, and whether donor/recipient CMV serostatus and CMV reactivation post-allo-HSCT influence NK and T cell phenotypes. Methods We sequenced 32 longitudinal CD45+ sorted peripheral blood samples with single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-seq) from 5 allo-HSCT donor-recipient pairs (pre-transplant sample from donor and recipient and 3-4 follow-up samples per patient over 3 months) and 6 healthy controls (Figure). All recipients were CMV seropositive, while 2 and 3 donors were seropositive and negative, respectively. CMV reactivation occurred in 4 patients during follow-up, on average 32 days post-allo-HSCT. We screened 32 common viral epitopes from post-transplant samples (n=3, 5 samples) with scRNA+TCRαβ+pMHC-seq (Immudex dCODE Dextramers). We then profiled T cells specific to CMV pp65 NLV-multimer with TCRβ-seq (allo-HSCT donor-recipient pairs n=3, 13 samples, healthy n=4), and traced CMV-recognizing TCRs in the scRNA+TCRαβ-seq data. Results We analyzed a total of 197,887 immune cells, including 37,123 NK cells and 59,852 CD8+ T cells. After allo-HSCT, an early increase in classical CD14+ monocytes was observed, followed by expansion of NK and T cell populations. At the time of CMV viremia, NK cell and CD8+ T effector memory/terminally differentiated effector memory (CD8+ T EM/EMRA) populations increased regardless of donor CMV serology. CD56 bright, CD56 dim and adaptive NK cells were present in all patients post-transplant, with patient-specific heterogeneity in phenotype and temporal dynamics. The proportion of adaptive NK cells out of CD45+ cells was higher post-viremia in 2 patients with CMV+ donors (18.9% and 7.4%), compared to 2 patients with CMV- donors (3.1% and 1.7%). Surprisingly, the expansion of adaptive NK cells (3.8%) was also noted in a patient with a CMV- donor without CMV reactivation post-allo-HSCT. After viremia, adaptive NK cells upregulated IFNG production, KLRC2/NKG2C and KLRC3/NKG2E and downregulated KLRC1/NKG2A. CD8+ T cell clonality markedly increased after CMV viremia in all patients with CMV reactivation, as measured by proportion of large and hyperexpanded clones in the scTCRαβ-seq data. Hyperexpanded clones consisted mostly of CD8+ T EM/EMRA phenotypes and were present only in samples collected during or after viremia. With scRNA+TCRαβ+pMHC-seq, pp65 NLV was discovered to be the immunodominant CMV epitope. Characterizing pp65 NLV-specific TCRs in the scRNA+TCRαβ data, CMV-specific CD8+ T cells belonged to highly clonal T EM/EMRA clusters. Further, a subset enriched to a T EMRA phenotype expressing NK cell receptors, including KIR3DL2, KIR2DL3, KLRC2/NKG2C KLRC3/NKG2E, and FCGR3A/CD16, and the exhaustion markers TIGIT, TOX, and CD160 at a higher level than other CD8+ T cells. The donor-derived CMV-specific TCR repertoire evolved in recipients after viremia. For example, in patient 2 we noted an 18.5-fold expansion of a donor-derived CMV-specific clone during viremia that was not originally the most dominant anti-CMV T cell clone in the donor. Conclusions We delineated NK cell and antigen-specific T cell dynamics in the unique setting of allo-HSCT and CMV reactivation, highlighting the manyfold expansion of donor-derived T cell clones and the expansion and evolution in the transcriptional profile of adaptive NK cells during CMV viremia. Our study sheds light on the immunobiology of these two cell types with implications in transplant outcome and translational relevance as cancer immunotherapy.

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells

ObjectivePrimary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class...

Upregulation of the NKG2D Ligand ULBP2 by JC Polyomavirus Infection Promotes Immune Recognition by Natural Killer Cells.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a potentially fatal complication of severe immune suppression with no effective treatment. Natural killer (NK) cells play critical roles in defense against viral infections; however, NK-cell response to JCPyV infection remains unexplored. NK- and T-cell responses against the JCPyV VP1 were compared using intracellular cytokine staining upon stimulation with peptide pools. A novel flow cytometry-based assay was developed to determine NK-cell killing efficiency of JCPyV-infected astrocyte-derived SVG-A cells. Blocking antibodies were used to evaluate the contribution of NK-cell receptors in immune recognition of JCPyV-infected cells. In about 40% of healthy donors, we detected robust CD107a upregulation and IFN-γ production by NK cells, extending beyond T-cell responses. Next, using the NK-cell-mediated killing assay, we showed that coculture of NK cells and JCPyV-infected SVG-A cells leads to a 60% reduction in infection, on average. JCPyV-infected cells had enhanced expression of ULBP2-a ligand for the activating NK-cell receptor NKG2D, and addition of NKG2D blocking antibodies decreased NK-cell degranulation. NKG2D-mediated activation of NK cells plays a key role in controlling JCPyV replication and may be a promising immunotherapeutic target to boost NK-cell anti-JCPyV activity.

In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy.

Treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%. In this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months. At the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (p<0.0001). This NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response.

Genomic view of the origins of cell-mediated immunity.

NKp30 is an activating natural killer cell receptor (NKR) with a single-exon variable (VJ)-type immunoglobulin superfamily (IgSF) domain. Such VJ-IgSF domains predate the emergence of the antigen receptors (immunoglobulin and T cell receptor), which possess the same domain but undergo gene rearrangement. NCR3, the gene encoding NKp30, is present in jawed vertebrates from sharks to mammals; thus, unlike most NKR that are highly divergent among vertebrate taxa, NKp30 is uniquely conserved. We previously hypothesized that an ancestral NCR3gene was encoded in the proto-major histocompatibility complex (MHC), the region where many immune-related genes have accumulated. Herein, we searched in silico databases to identify NCR3 paralogues and examined their genomic locations. We found a paralogue, NCR3H, in many vertebrates but was lost in mammals. Additionally, we identified a set of voltage-gated sodium channel beta (SCNB) genes as NCR3-distantly-related genes. Like NCR3, both NCR3H and SCNB proteins contain a single VJ-IgSF domain followed by a transmembrane region. These genes map to MHC paralogous regions, originally described in an invertebrate, along with genes encoding cell adhesion molecules involved in NK cell recognition networks. Other genes having no obvious relationship to immunity also map to these paralogous regions. These gene complexes were traced to several invertebrates, suggesting that the foundation of these cellular networks emerged before the genome-wide duplications in early gnathostome history. Here, we propose that this ancestral region was involved in cell-mediated immunity prior to the emergence of adaptive immunity and that NCR3 piggybacked onto this primordial complex, heralding the emergence of vertebrate NK cell/T cells.

Therapeutic Anti-KIR Antibody of 1-7F9 Attenuates the Antitumor Effects of Expanded and Activated Human Primary Natural Killer Cells on In Vitro Glioblastoma-like Cells and Orthotopic Tumors Derived Therefrom.

Glioblastoma (GBM) is the leading malignant intracranial tumor, where prognosis for which has remained extremely poor for two decades. Immunotherapy has recently drawn attention as a cancer treatment, including for GBM. Natural killer (NK) cells are immune cells that attack cancer cells directly and produce antitumor immunity-related cytokines. The adoptive transfer of expanded and activated NK cells is expected to be a promising GBM immunotherapy. We previously established an efficient expansion method that produced highly purified, activated primary human NK cells, which we designated genuine induced NK cells (GiNKs). The GiNKs demonstrated antitumor effects in vitro and in vivo, which were less affected by blockade of the inhibitory checkpoint receptor programmed death 1 (PD-1). In the present study, we assessed the antitumor effects of GiNKs, both alone and combined with an antibody targeting killer Ig-like receptor 2DLs (KIR2DL1 and DL2/3, both inhibitory checkpoint receptors of NK cells) in vitro and in vivo with U87MG GBM-like cells and the T98G GBM cell line. Impedance-based real-time cell growth assays and apoptosis detection assays revealed that the GiNKs exhibited growth inhibitory effects on U87MG and T98G cells by inducing apoptosis. KIR2DL1 blockade attenuated the growth inhibition of the cell lines in vitro. The intracranial administration of GiNKs prolonged the overall survival of the U87MG-derived orthotopic xenograft brain tumor model. The KIR2DL1 blockade did not enhance the antitumor effects; rather, it attenuated it in the same manner as in the in vitro experiment. GiNK immunotherapy directly administered to the brain could be a promising immunotherapeutic alternative for patients with GBM. Furthermore, KIR2DL1 blockade appeared to require caution when used concomitantly with GiNKs.

Human NCR3 gene variants rs2736191 and rs11575837 alter longitudinal risk for development of pediatric malaria episodes and severe malarial anemia

BackgroundPlasmodium falciparum malaria is a leading cause of pediatric morbidity and mortality in holoendemic transmission areas. Severe malarial anemia [SMA, hemoglobin (Hb) < 5.0 g/dL in children] is the most common clinical manifestation of severe malaria in such regions. Although innate immune response genes are known to influence the development of SMA, the role of natural killer (NK) cells in malaria pathogenesis remains largely undefined. As such, we examined the impact of genetic variation in the gene encoding a primary NK cell receptor, natural cytotoxicity-triggering receptor 3 (NCR3), on the occurrence of malaria and SMA episodes over time.MethodsSusceptibility to malaria, SMA, and all-cause mortality was determined in carriers of NCR3 genetic variants (i.e., rs2736191:C > G and rs11575837:C > T) and their haplotypes. The prospective observational study was conducted over a 36 mos. follow-up period in a cohort of children (n = 1,515, aged 1.9–40 mos.) residing in a holoendemic P. falciparum transmission region, Siaya, Kenya.ResultsPoisson regression modeling, controlling for anemia-promoting covariates, revealed a significantly increased risk of malaria in carriers of the homozygous mutant allele genotype (TT) for rs11575837 after multiple test correction [Incidence rate ratio (IRR) = 1.540, 95% CI = 1.114–2.129, P = 0.009]. Increased risk of SMA was observed for rs2736191 in children who inherited the CG genotype (IRR = 1.269, 95% CI = 1.009–1.597, P = 0.041) and in the additive model (presence of 1 or 2 copies) (IRR = 1.198, 95% CI = 1.030–1.393, P = 0.019), but was not significant after multiple test correction. Modeling of the haplotypes revealed that the CC haplotype had a significant additive effect for protection against SMA (i.e., reduced risk for development of SMA) after multiple test correction (IRR = 0.823, 95% CI = 0.711–0.952, P = 0.009). Although increased susceptibility to SMA was present in carriers of the GC haplotype (IRR = 1.276, 95% CI = 1.030–1.581, P = 0.026) with an additive effect (IRR = 1.182, 95% CI = 1.018–1.372, P = 0.029), the results did not remain significant after multiple test correction. None of the NCR3 genotypes or haplotypes were associated with all-cause mortality.ConclusionsVariation in NCR3 alters susceptibility to malaria and SMA during the acquisition of naturally-acquired malarial immunity. These results highlight the importance of NK cells in the innate immune response to malaria.

Association of inhibitory NKG2A and activating NKG2D natural killer cell receptor genes with resistance to SARS-CoV-2 infection in a western Indian population.

We have evaluated the association of polymorphisms in the intronic variable-number tandem repeat (VNTR) regions of the human NKG2D, NKG2A, and IL-1RN genes with resistance and/or susceptibility to SARS-CoV-2 infection in a total of 209 patients with SARS-CoV-2 infection (125 asymptomatic patients and 84 symptomatic patients with mild symptoms) and 355 healthy controls, using the PCR-RFLP method. The genotypic and allelic frequency distributions for an IL-1RN (VNTR) single-nucleotide polymorphism (SNP) were found to be comparable among the patient groups. Overall, in SARS-CoV-2 patients, NKG2A (rs2734440) showed a protective association in the codominant [(A/A vs. A/G): (OR = 0.53, 95% CI = 0.34-0.83, p = 0.006)], recessive [(A/A vs. A/G+G/G): (OR = 0.6, 95% CI = 0.39-0.92, p = 0.02)] and over-dominant [(A/A+G/G vs. A/G): (OR = 0.57, 95% CI = 0.38-0.84, p = 0.005)] models. Similarly, NKG2D (rs7980470) showed a protective association in the codominant [(A/A vs. A/G): (OR = 0.46, 95% CI = 0.3-0.7, p = 0.0003), codominant (A/A vs. G/G): (OR = 0.54, 95% CI = 0.31-0.71, p = 0.027)], recessive [(A/A vs. A/G+G/G): (OR = 0.47, 95% CI = 0.32-0.7, p = 0.0001) and over-dominant [(A/A+G/G vs. A/G): (OR = 0.56, 95% CI = 0.38-0.82, p = 0.003)] models. At the allelic level, there was a higher frequency of the "G" allele of NKG2D (rs7980470) in healthy controls than in patients with SARS-CoV-2 infection, suggesting that individuals with the "G" allele in the intronic region of NKG2D are likely to be protected against SARS-CoV-2 infection. Overall, our data suggest that polymorphisms in the host NKG2D and NKG2A genes have a protective role in SARS-CoV-2 infection, although the functional impact of these polymorphisms on control of SARS-CoV-2 infection remains unknown.

Polymer Micropatches as Natural Killer Cell Engagers for Tumor Therapy.

Natural killer (NK) cell therapies have emerged as a potential therapeutic approach to various cancers. Their efficacy, however, is limited by their low persistence and anergy. Current approaches to sustain NK cell persistence in vivo include genetic modification, activation via pretreatment, or coadministration of supporting cytokines or antibodies. Such supporting therapies exhibit limited efficacy in vivo, in part due to the reversal of their effect within the immunosuppressive tumor microenvironment and off-target toxicity. Here, we report a material-based approach to address this challenge. Specifically, we describe the use of polymeric micropatches as a platform for sustained, targeted activation of NK cells, an approach referred to as microparticles as cell engagers (MACE). Poly(lactide-co-glycolic) acid (PLGA) micropatches, 4-8 μm in diameter and surface-modified with NK cell receptor targeting antibodies, exhibited strong adhesion to NK cells and induced their activation without the need of coadministered cytokines. The activation induced by MACE was greater than that induced by nanoparticles, attesting to the crucial role of MACE geometry in the activation of NK cells. MACE-bound NK cells remained viable and exhibited trans-endothelial migration and antitumor activity in vitro. MACE-bound NK cells activated T cells, macrophages, and dendritic cells in vitro. Adoptive transfer of NK-MACE also demonstrated superior antitumor efficacy in a mouse melanoma lung metastasis model compared to unmodified NK cells. Overall, MACE offers a simple, scalable, and effective way of activating NK cells and represents an attractive platform to improve the efficacy of NK cell therapy.

Gluten induces rapid reprogramming of natural memory αβ and γδ intraepithelial T cells to induce cytotoxicity in celiac disease.

Celiac disease (CD) is an autoimmune disease in which intestinal inflammation is induced by dietary gluten. The means through which gluten-specific CD4+ T cell activation culminates in intraepithelial T cell (T-IEL)-mediated intestinal damage remain unclear. Here, we performed multiplexed single-cell analysis of intestinal and gluten-induced peripheral blood T cells from patients in different CD states and healthy controls. Untreated, active, and potential CD were associated with an enrichment of activated intestinal T cell populations, including CD4+ follicular T helper (TFH) cells, regulatory T cells (Tregs), and natural CD8+ αβ and γδ T-IELs. Natural CD8+ αβ and γδ T-IELs expressing activating natural killer cell receptors (NKRs) exhibited a distinct TCR repertoire in CD and persisted in patients on a gluten-free diet without intestinal inflammation. Our data further show that NKR-expressing cytotoxic cells, which appear to mediate intestinal damage in CD, arise from a distinct NKR-expressing memory population of T-IELs. After gluten ingestion, both αβ and γδ T cell clones from this memory population of T-IELs circulated systemically along with gluten-specific CD4+ T cells and assumed a cytotoxic and activating NKR-expressing phenotype. Collectively, these findings suggest that cytotoxic T cells in CD are rapidly mobilized in parallel with gluten-specific CD4+ T cells after gluten ingestion.

Chimeric antigen receptor-natural killer cells: a promising sword against insidious tumor cells.

Natural killer (NK) cells are a critical component of innate immunity, particularly in initial cancer recognition and inhibition of additional tumor growth or metastasis propagation. NK cells recognize transformed cells without prior sensitization via stimulatory receptors and rapidly eradicate them. However, the protective tumor microenvironment facilitates tumor escaping via induction of an exhaustion state in immune cells, including NK cells. Hence, genetic manipulation of NK cells for specific identification of tumor-associated antigens or a more robust response against tumor cells is a promising strategy for NK cells' tumoricidal augmentation. Regarding the remarkable achievement of engineered CAR-T cells in treating hematologic malignancies, there is evolving interest in CAR-NK cell recruitment in cancer immunotherapy. Innate functionality of NK cells, higher safety, superior in vivo maintenance, and the off-the-shelf potential move CAR-NK-based therapy superior to CAR-T cells treatment. In this review, we have comprehensively discussed the recent genetic manipulations of CAR-NK cell manufacturing regarding different domains of CAR constructs and their following delivery systems into diverse sources of NK cells. Then highlight the preclinical and clinical investigations of CAR-NK cells and examine the current challenges and prospects as an optimistic remedy in cancer immunotherapy.

Yeast β-glucan modulates macrophages and improves antitumor NK-cell responses in cancer.

As the largest proportion of myeloid immune cells in tumors, macrophages play an important role in tumor growth and regression according to their different phenotypes, thus reprogramming macrophages has become a new research direction for cancer immunotherapy. Yeast-derived whole β-glucan particles (WGPs) can induce M0 macrophages to differentiate into M1 macrophages and convert M2 macrophages and tumor-associated macrophages (TAMs) into M1 macrophages. In vitro, studies have confirmed that WGP-treated macrophages increase the activating receptors in natural killer cells (NK cells) and enhance the cytotoxicity of NK cells. The extracellular regulated protein kinases (ERK) signaling pathway is involved in WGP-mediated regulation of the macrophage phenotype. Further in vivo studies show that oral WGP can significantly delay tumor growth, which is related to the increased proportion of macrophages and NK cells, the macrophage phenotype reversal, and the enhancement of NK cell immune function. NK-cell depletion reduces the therapeutic efficacy of WGP in tumor-bearing mice. These findings revealed that in addition to T cells, NK cells also participate in the antitumor process of WGP. It was confirmed that WGP regulates the macrophage phenotype to regulate NK-cell function.

HLA-DP on Epithelial Cells Enables Tissue Damage by NKp44+ Natural Killer Cells in Ulcerative Colitis

Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic associations of UC and HLA-DP haplotypes and their functional implications were investigated. HLA-DP haplotype and UC risk association analyses were performed (UC: n= 13,927; control: n= 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to determine functional consequences of interactions between HLA-DP and NKp44. These studies identified HLA-DPA1∗01:03-DPB1∗04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1∗01:03-DPB1∗03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301pos IECs. HLA-DP401pos IECs in organoids triggered increased degranulation and tumor necrosis factor production by NKp44+ NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures. We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44+ NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interaction between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44+ NK cell-mediated destruction of the intestinal epithelium in UC.

Largely preserved functionality after the combined loss of NKG2D, NCR1 and CD16 demonstrates the remarkable plasticity of NK cell responsiveness.

Natural killer (NK) cells play an important role in the early defense against tumors and virally infected cells. Their function is thought to be controlled by the balance between activating and inhibitory receptors, which often compete for the same ligands. Several activating receptors expressed on virtually all NK cells lack an inhibitory partner, most notably CD16, NCR1 and NKG2D. We therefore hypothesized that a signal through at least one of these receptors is always required for full NK cell activation. We generated animals lacking all three receptors (TKO) and analyzed their NK cells. In vitro, TKO NK cells did not show reduced ability to kill tumor targets but displayed hyperresponsiveness to NK1.1 stimulation. In vivo, TKO animals had a minor reduction in their ability to control non-hematopoietic tumors and cytomegalovirus infection, which was the result of reduced NK cell activity. Together, our findings show that activating NK cell receptors without an inhibitory partner do not provide a 'master' signal but are integrated in the cumulative balance of activating and inhibitory signals. Their activity is controlled through regulation of the responsiveness and expression of other activating receptors. Our findings may be important for future development of NK cell-based cancer immunotherapy.

Impact of HIV-1 Vpu-mediated downregulation of CD48 on NK-cell-mediated antibody-dependent cellular cytotoxicity.

HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.

Nonviral technologies can pave the way for CAR-NK cell therapy.

Natural killer cells are a promising platform for cancer immunotherapy. Natural killer cells have high intrinsic killing capability, and the insertion of a chimeric antigen receptor can further enhance their antitumor potential. In first-in-human trials, chimeric antigen receptor-natural killer cells demonstrated strong clinical activity without therapy-induced side effects. The applicability of natural killer cells as an "off-the-shelf" product makes them highly attractive for gene-engineered cell therapies. Traditionally, viral transduction has been used for gene editing; however, the use of viral vectors remains a safety concern and is associated with high costs and regulatory requirements. Here, we review the current landscape of nonviral approaches for chimeric antigen receptor-natural killer cell generation. This includes transfection of vector particles and electroporation of mRNA and DNA vectors, resulting in transient modification and chimeric antigen receptor expression. In addition, using nonviral transposon technologies, natural killer cells can be stably modified ensuring long-lasting chimeric antigen receptor expression. Finally, we discuss CRISPR/Cas9 tools to edit key genes for natural killer cell functionality.

Establishing NK-Cell Receptor Restriction by Flow Cytometry and Detecting Potential NK-Cell Clones of Uncertain Significance

Natural killer (NK) cells develop a complex inhibitory and/or activating NK-cell receptor system, including killer cell immunoglobulin-like receptors (KIRs or CD158) and CD94/NKG2 dimers, which are variably combined to generate the individual’s NK-cell receptor repertoire. Establishing NK-cell receptor restriction by flow cytometric immunophenotyping is an important step in diagnosing NK-cell neoplasms, but reference interval (RI) data for interpreting these studies are lacking. Specimens from 145 donors and 63 patients with NK-cell neoplasms were used to identify discriminatory rules based on 95% and 99% nonparametric RIs for CD158a+, CD158b+, CD158e+, KIR-negative, and NKG2A+ NK-cell populations to establish NK-cell receptor restriction. These 99% upper RI limits (NKG2a >88% or CD158a >53% or CD158b >72% or CD158e >54% or KIR-negative >72%) provided optimal discrimination between NK-cell neoplasm cases and healthy donor controls with an accuracy of 100% compared with the clinicopathologic diagnosis. The selected rules were applied to 62 consecutive samples received in our flow cytometry laboratory that were reflexed to an NK-cell panel due to an expanded NK-cell percentage (exceeding 40% of total lymphocytes). Twenty-two (35%) of 62 samples were found to harbor a very small NK-cell population with restricted NK-cell receptor expression based on the rule combination, suggestive of NK-cell clonality. A thorough clinicopathologic evaluation for the 62 patients did not reveal diagnostic features of NK-cell neoplasms; therefore, these potential clonal populations of NK cells were designated as NK-cell clones of uncertain significance (NK-CUS). In this study, we established decision rules for NK-cell receptor restriction from the largest published cohorts of healthy donors and NK-cell neoplasms. The presence of small NK-cell populations with restricted NK-cell receptors does not appear to be an uncommon finding, and its significance requires further exploration.

AATF/Che-1 RNA polymerase II binding protein overexpression reduces the anti-tumor NK-cell cytotoxicity through activating receptors modulation.

AATF/Che-1 over-expression in different tumors is well known and its effect on tumorigenicity is mainly due to its central role demonstrated in the oncogenic pathways of solid tumors, where it controls proliferation and viability. The effect exerted by tumors overexpressing Che-1 on the immune response has not yet been investigated. Starting from ChIP-sequencing data we confirmed Che-1 enrichment on Nectin-1 promoter. Several co-cultures experiments between NK-cells and tumor cells transduced by lentiviral vectors carrying Che-1-interfering sequence, analyzed by flow-cytometry have allowed a detailed characterization of NK receptors and tumor ligands expression. Here, we show that Che-1 is able to modulate the expression of Nectin-1 ligand at the transcriptional level, leading to the impairment of killing activity of NK-cells. Nectin-1 down-modulation induces a modification in NK-cell ligands expression able to interact with activating receptors and to stimulate NK-cell function. In addition, NK-cells from Che-1 transgenic mice, confirming a reduced expression of activating receptors, exhibit impaired activation and a preferential immature status. The critical equilibrium between NK-cell ligand expression on tumor cells and the interaction with NK cell receptors is affected by Che-1 over-expression and partially restored by Che-1 interference. The evidence of a new role for Che-1 as regulator of anti-tumor immunity supports the necessity to develop approaches able to target this molecule which shows a dual tumorigenic function as cancer promoter and immune response modulator.

P-339 Activities and changes in the number of natural killer cells in endometriosis: systematic review and meta-analysis

Abstract Study question What are the differences in peripheral, uterine, and peritoneal NK (uNK, pNK, pfNK) cell counts/percentages, and activities in women with endometriosis compared controls Summary answer The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference in women with endometriosis compared controls What is known already NK cells play an important role in the pathogenesis of endometriosis Study design, size, duration This systematic review and meta-analysis included 36 experimental studies (case control and cross sectional). Narrative review is also conducted on the NK cell activity, cytokine expression, regulations and receptors. Participants/materials, setting, methods Women suffering with endometriosis confirmed with laparoscopy and/or pathology was the cases whereas, women having different pelvic pathology such as myoma, ovarian cyst, healthy woman and verified not having endometriosis with laparoscopy and/or pathology was considered as controls for this study. Databases (PubMed, Web of Science, Scopus, Google scholar, and EMBASE through OVID) were used to search for the available studies. RevMan 5.4 and STATA 14 were used to analyze the data. Main results and the role of chance The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference (standard mean difference SMD 0.13, 95%CI -0.35, 0.62; P = 0.59, I2 74%; total 393 women, 6 studies), (SMD 0.27, 95%CI -0.07, 0.61; P = 0.12, I2 80%; total 825 women, 11 studies), and (SMD 0.31, 95%CI -0.68, 01.29; P = 0.59, I2 94%; total 404 women,7 studies) in endometriosis patients compared with controls respectively. The pooled NK (uNK, pNK, pfNK) cell cytotoxicity/activity level is significantly higher in controls compared with women with endometriosis (MD 5.43, 95%CI 2.29, 8.57; P &amp;lt; 0.007, I2 50%; total 323 women, 7 studies). The NK cytotoxicity/activity level in early stages of endometriosis has no significant difference compared with advanced stages of endometriosis (MD 2.29, 95%CI –1.98-6.57; P = 0.29, I2 87%; total 263 women, 7 studies). There are variations in studies conducted in NK cell activities in endometriosis: broadly categorized as NK cell cytotoxicity, cytokine expression and NK cell regulation and receptors (Inhibition and activation). The cytotoxic activity of NK cells decreased in women with endometriosis and correlated with the severity of the disease, however the cytokine expression and inhibitor receptors are equivocal across studies. Limitations, reasons for caution We unable to conduct sub-group analysis to investigate the cytotoxic activity of NK cell in women with endometriosis and its stages in different samples such as pNK, uNK, and pfNK due to the lack of studies for the analysis. Wider implications of the findings This study revealed that the activity of NK cells in women with endometriosis is significantly lower than controls.Though the mean difference of NK cell count and percentage has no significant difference, the cytotoxicity activity of NK cell is higher in controls compared with women with endometriosis Trial registration number CRD42022384060

Generation and Functional Characterization of Anti-CD19 Chimeric Antigen Receptor-Natural Killer Cells from Human Induced Pluripotent Stem Cells.

Natural killer (NK) cells are a part of innate immunity that can be activated rapidly in response to malignant transformed cells without prior sensitization. Engineering NK cells to express chimeric antigen receptors (CARs) allows them to be directed against corresponding target tumor antigens. CAR-NK cells are regarded as a promising candidate for cellular immunotherapy alternatives to conventional CAR-T cells, due to the relatively low risk of graft-versus-host disease and safer clinical profile. Human induced pluripotent stem cells (iPSCs) are a promising renewable cell source of clinical NK cells. In the present study, we successfully introduced a third-generation CAR targeting CD19, which was validated to have effective signaling domains suitable for NK cells, into umbilical cord blood NK-derived iPSCs, followed by a single-cell clone selection and thorough iPSC characterization. The established single-cell clone of CAR19-NK/iPSCs, which is highly desirable for clinical application, can be differentiated using serum- and feeder-free protocols into functional CAR19-iNK-like cells with improved anti-tumor activity against CD19-positive hematologic cancer cells when compared with wild-type (WT)-iNK-like cells. With the feasibility of being an alternative source for off-the-shelf CAR-NK cells, a library of single-cell clones of CAR-engineered NK/iPSCs targeting different tumor antigens may be created for future clinical application.