Levels Of Natural Killer Cells Research Articles

DNMT1 silencing induces KIR2DL1/2/3 expression via methylation to alleviate graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Natural killer (NK) cells are the promoters in graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), while demethylation can regulate NK cell function. We explored the mechanism of demethylation regulating NK cell function to affect GVHD after allo-HSCT. BALB/c mice were transfused with C57BL/6 mouse-derived NK and bone marrow cells to establish GVHD models, followed by isolation and in-vitro expansion of NK cells. NK cell purity, cytokine levels, proliferation, and cytokine-producing NK cell levels were measured via flow cytometry. KIR2DL1/2/3 methylation was tested by Methylation-specific polymerase chain reaction (MSP), with determination of mouse survival and GVHD scores. KIR2DL1/2/3 and DNMT1 expression was detected through qRT-PCR and/or western blot. Methylation levels were upregulated and KIR2DL1/2/3 expression was downregulated in GVHD mouse model-derived NK cells following IL-2 stimulation. DNMT1 silencing promoted KIR2DL1/2/3 expression, proliferation, and the secretion of Granzyme, Perforin, and Interferon-γ (IFN-γ) in C57BL/6 mouse-derived NK cells. DNMT1 silencing also enhanced mouse survival, reduced GVHD scores, promoted KIR2DL1/2/3 expression on the NK cell surface, and increased the secretion of Granzyme, Perforin, IFN-γ, and the number of cytokine-producing NK cells in the spleen, liver, and lung tissues of the models. Collectively, DNMT1 silencing induced KIR2DL1/2/3 expression in NK cells through reducing methylation to alleviate GVHD after allo-HSCT.

Single-cell analysis of chemotherapy-resistant microenvironment identifies a chemo-response biomarker for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal, aggressive cancer due to limited response chemotherapy. The tumor microenvironment (TME) has emerged as a key player in the development of chemoresistance and in malignant progression. In this study, we hypothesized that chemotherapy response is predictable by chemotherapy-related cell types and their differentially expressed genes (DEGs). DEGs of chemoresistance cell types were identified via single cell analysis and Wilcoxon test. A chemotherapy response signature was established using a random forest model and validated with public datasets. Bulk cell fraction was analyzed using BayesPrism algorithms. Log-rank test was used to analyze survival of PDAC patients. We found that natural killer (NK) cells, myeloid cells, and erythroid cells were highly infiltrated in chemo-resistant TME. A total of 36 chemoresistance-related DEGs of chemo-resistant cells were identified in chemo-resistant PDAC. Functional enrichment analysis showed that chemoresistance upregulated various inflammation-related pathways, including TGF-β signaling. Based on these features, we constructed a random forest model to predict the response and survival for PDAC patients, which accurately distinguished high-risk and chemoresistant patients with significantly poorer prognosis in both the training and independent validation datasets. Cox regression analysis indicated that predicted labels were an independent prognostic factor in PDAC. Moreover, deconvolution of TME confirmed higher infiltration levels of M2 macrophage and NK cells in predicted chemoresistance. When combined with chemotherapy response related tumor mutations, the model showed excellent ability in predicting chemotherapy response and survival. The TME was closely associated with the chemotherapy response and prognosis of PDAC. Our TME-based random forest model predicted chemotherapy response with complementary knowledge to the response-related genetic mutations.

Retraction: Intralipid supplementation in women with recurrent spontaneous abortion and elevated levels of natural killer cells.

Retraction: D. M. R. Dakhly, Y. A. Bayoumi, M. Sharkawy, S. H. Gad Allah, M. A. Hassan, H. M. Gouda, A. T. Hashem, D. L. Hatem, M. F. Ahmed, W.El-Khayat. "Intralipid supplementation in women with recurrent spontaneous abortion and elevated levels of natural killer cells." International Journal of Gynecology & Obstetrics 135, no. 3 (2016): 324-327. https://doi.org/10.1016/j.ijgo.2016.06.026. The above article, published online on 30 August 2016 in Wiley Online Library (wileyonlinelibrary.com) has been retracted by agreement between the journal's Editor-in-Chief, Michael Geary, International Federation of Gynecology and Obstetrics and John Wiley & Sons Ltd. The retraction has been agreed following an investigation based on concerns raised by a third party. Several discrepancies were found between the information provided during the trial registration and the published article including, the study dates, participant eligibility criteria and the study's focus on natural killer cells. Furthermore, the definition of clinical pregnancy differs between the article and trial registration. Errors were also uncovered in Table2 of the article; the number of spontaneous abortions and ongoing pregnancies do not add up to the total number of clinical pregnancies stated. Additionally, the calculation specified to define implantation rate is not consistent with the values reported in Table2. The authors were unable to provide the original data upon request or provide an explanation for the discrepancies. Given the nature and extent of the discrepancies, the editors have lost confidence in the data and conclusions presented. The author, Waleed El-Khayat, disagrees with the retraction; the other authors were not available for comment.

Increased Natural Killer (NK)-cell cytotoxicity and Trypanosoma cruzi-specific memory B cells in subjects with discordant serology for Chagas disease

The presence of memory T cell specific for Trypanosoma cruzi in subjects with discordant serology for Chagas disease supports a cleared infection in these subjects. Using high-dimensional flow cytometry, ELISPOT assays and quantitative PCR, antibody-secreting cells and memory B cells specific for T. cruzi, total B-cell phenotypes, innate immune responses and parasite DNA were evaluated in serodiscordant, seropositive and seronegative subjects for T. cruzi infection. T. cruzi-specific memory B cells but no antibody-secreting cells specific for T. cruzi, increased proportion of nonclassical monocytes and increased levels of polyfunctional NK cells were found in serodiscordant compared with seropositive subjects. None of the serodiscordant subjects evaluated showed detectable parasite DNA, most of them did not show cardiac abnormalities and a group of them had had confirmed positive serology for Chagas disease. The unique immune profiles in serodiscordant subjects support that T. cruzi infection was cleared or profoundly controlled in these subjects.

Abstract PO4-03-09: Profile of circulating natural killer cells in stage III triple negative breast cancer before and after neoadjuvant chemotherapy

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. The treatment for locally advanced disease is based on neoadjuvant chemotherapy and immunotherapy, with complete pathological response (pCR) serving as a predictor of disease-free survival. Although the presence of immune cell infiltration in the tumor has a favorable prognostic impact, there is a lack of predictive biomarkers for complete pathological response to neoadjuvant chemotherapy (NAC). Objective: to analyze the profile of circulating Natural Killer (NK) cells and NK subpopulations (NK FAS+ and NK PD1+) in association with the occurrence of complete pathological response after neoadjuvant chemotherapy in women with clinical stage III TNBC. Methods: This was a longitudinal prospective and translational cohort study, including women aged 18 to 60 years with stage III TNBC diagnosed between March 2015 and July 2017, with a follow-up of 24 months. A comparison control group consisted of 30 healthy women aged 18 to 60 years without prior or current cancer diagnosis and no family history of breast cancer. Peripheral blood samples were collected before NAC and before surgery. Flow cytometry was used to analyze the percentage levels of NK cells and NK subpopulations (NK FAS+ and NK PD1+). The Shapiro-Wilk test was initially applied to assess the normality of quantitative variables. The non-parametric Mann-Whitney test was used for comparison between two groups. The statistical significance level was set at p< 0.05. Overall survival (OS) was estimated using the Kaplan-Meier method. Statistical analysis was performed using GraphPad Prism v9.5.1. Results: The median age was 45 years. Stage IIIA was identified in 28% of the patients, while IIIB was observed in 72%. Regarding pathological response, 14 patients (48%) achieved pCR, while 15 patients (52%) had a non-complete pathological response (non-pCR). The 2-year OS was 86%, with no median reached. Higher percentage levels of NK cells, NK FAS+, and NK PD1+ were observed in the patient group compared to the control group, with significant differences. There was a significant increase in the percentage levels of NK cells after NAC compared to pre-NAC levels, and a reduction in NK PD1+ levels. NK cell levels were significantly higher in patients with stage IIIA compared to stage IIIB, while NK FAS+ levels were lower. NK cell and NK FAS+ subpopulation levels were significantly higher in patients who achieved pCR compared to non-pCR patients, with no difference in NK PD1+ cell levels. Conclusions: Elevated levels of circulating NK cells and NK FAS+ subpopulation collected from peripheral blood before NAC may serve as potential biomarkers for pCR in patients with TNBC. Citation Format: Luiz Jose de Barros Batista, Jurema Telles, Candice Santos, Ariani Souza, Marcelo Salgado, Leuridan Torres. Profile of circulating natural killer cells in stage III triple negative breast cancer before and after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-03-09.

Effects of Splenectomy on Natural Killer Cell Levels in β-Thalassemia Major Patients.

In this study, we investigated how splenectomy affects natural killer (NK) cell levels in patients with β-thalassemia major (β-TM). Seventy patients with β-TM (38 splenectomized and 32 nonsplenectomized) and 25 healthy controls were included in this study. The hemogram parameters, ferritin, T lymphocyte, T-helper cell, T-suppressor cell, and NK cell numbers, were measured. The T lymphocyte (CD3+) level was found to be significantly higher in the patient group (p < 0.05). CD3+/CD4+ T lymphocytes were detected to be significantly higher in the patient group (p < 0.05). Although the CD3+/CD4+ T lymphocyte level was significantly higher in the nonsplenectomy group (p < 0.05), this was not the case in the splenectomy group. When the patient and control groups were compared, no significant difference was detected regarding CD3+/CD8+ T lymphocyte levels. CD3-/CD16+CD56+ NK cell level was found to be significantly lower only in the splenectomy group than in the control group (p < 0.05). We found that there was a significant negative correlation between serum ferritin levels and both total lymphocyte (r = -0.617) and CD3+ lymphocyte (r = -0.718) levels in the control group (p < 0.05). A significant negative correlation was detected between serum ferritin levels and CD3-/CD16+CD56+ NK cell levels in the patient group (r = -0.410) (p < 0.05). Splenectomy reduces NK cell levels in patients with β-TM. The negative relationship between ferritin levels and NK cells indicates that ferritin levels should be kept under control in patients with β-TM.

Highlight of 2023: From fundamental studies to clinical trials, the importance of NK cells against cancer.

In this article for the Highlights of 2023 Series, we discuss how various factors affect the ability of natural killer (NK) cells to fight tumors. For instance, tumor cells can hinder NK cell function by reducing surface protrusions or increasing HLA-E expression via platelets. Lower UTX protein levels in male NK cells also decrease their cytotoxicity compared with females. Fortunately, recent advancements in therapeutic approaches have emerged, including the development of a comprehensive atlas of NK cell heterogeneity within the tumor microenvironment, as well as a trispecific engager molecule that has shown promise in enhancing the anti-tumor functions of NK cells.

Peripheral NK cells identified as the predictor of response in extensive-stage small cell lung cancer patients treated with first-line immunotherapy plus chemotherapy.

Although immunotherapy improves outcomes in extensive-stage small-cell lung cancer (ES-SCLC), the search for biomarkers predicting treatment success is crucial. Natural killer (NK) cells are potential indicators in various cancers, however, their precise role in ES-SCLC prognosis remains unclear. In this retrospective study, 33 patients with ES-SCLC treated with first-line immuno-chemotherapy were enrolled. The peripheral NK cell percentage and its longitudinal dynamics were analyzed using flow cytometry. Progression-free survival (PFS) and overall survival (OS) were calculated as hazard ratio (HR) and compared statistically. The median PFS was better in the group with normal baseline NK cell levels than the low group (7.0 vs. 4.6months; HR = 0.17; 95% CI 0.07-0.41; P < 0.0001), but there was no association with OS (14.9 vs. 10.3months; HR = 0.55; 95% CI 0.23-1.31; P = 0.171). Furthermore, the NK cell% for 95.0% of patients increased after immunochemotherapy in the clinical response group (P = 0.0047), which led to a better median PFS (6.3 vs. 2.1months; HR = 0.23; 95% CI 0.05-0.98; P < 0.0001) and OS (14.9 vs. 5.9months; HR = 0.20; 95% CI 0.04-1.02; P < 0.0001). Similar trends were observed with NK cell% changes up to disease progression, improving PFS (6.5 vs. 4.3; HR = 0.41; 95% CI 0.12-0.92; P = 0.0049) and OS (17.4 vs. 9.7; HR = 0.42; 95% CI 0.17-1.02; P < 0.0001). In patients with ES-SCLC, the percentage and changes in peripheral NK cells can predict the response to combined immunotherapy and chemotherapy.

Activin A, a Novel Chemokine, Induces Mouse NK Cell Migration via AKT and Calcium Signaling.

Natural killer (NK) cells can migrate quickly to the tumor site to exert cytotoxic effects on tumors, and some chemokines, including CXCL8, CXCL10 or and CXCL12, can regulate the migration of NK cells. Activin A, a member of the transforming growth factor β (TGF-β) superfamily, is highly expressed in tumor tissues and involved in tumor development and immune cell activation. In this study, we focus on the effects of activin A on NK cell migration. In vitro, activin A induced NK cell migration and invasion, promoted cell polarization and inhibited cell adhesion. Moreover, activin A increased Ca2+, p-SMAD3 and p-AKT levels in NK cells. An AKT inhibitor and Ca2+ chelator partially blocked activin A-induced NK cell migration. In vivo, exogenous activin A increased tumor-infiltrating NK cells in NS-1 cell solid tumors and inhibited tumor growth, and blocking endogenous activin A with anti-activin A antibody reduced tumor-infiltrating NK cells in 4T-1 cell solid tumors. These results suggest that activin A induces NK cell migration through AKT signaling and calcium signaling and may enhance the antitumor effect of NK cells by increasing tumor-infiltrating NK cells.

Trade-offs shaping transmission of sylvatic dengue and Zika viruses in monkey hosts

Mosquito-borne dengue (DENV) and Zika (ZIKV) viruses originated in Old World sylvatic (forest) cycles involving monkeys and canopy-living Aedes mosquitoes. Both viruses spilled over into human transmission and were translocated to the Americas, opening a path for spillback into Neotropical sylvatic cycles. Studies of the trade-offs that shape within-host dynamics and transmission of these viruses are lacking, hampering efforts to predict spillover and spillback. We infected a native, Asian host species (cynomolgus macaque) and a novel, American host species (squirrel monkey) with sylvatic strains of DENV-2 or ZIKV via mosquito bite. We then monitored aspects of viral replication (viremia), innate and adaptive immune response (natural killer (NK) cells and neutralizing antibodies, respectively), and transmission to mosquitoes. In both hosts, ZIKV reached high titers that translated into high transmission to mosquitoes; in contrast DENV-2 replicated to low levels and, unexpectedly, transmission occurred only when serum viremia was below or near the limit of detection. Our data reveal evidence of an immunologically-mediated trade-off between duration and magnitude of virus replication, as higher peak ZIKV titers are associated with shorter durations of viremia, and higher NK cell levels are associated with lower peak ZIKV titers and lower anti-DENV-2 antibody levels. Furthermore, patterns of transmission of each virus from a Neotropical monkey suggest that ZIKV has greater potential than DENV-2 to establish a sylvatic transmission cycle in the Americas.

Single-Cell Transcriptome Reveals Potential Mechanisms for Coronary Artery Lesions in Kawasaki Disease.

Coronary artery lesions (CALs) are the most common and major complication of Kawasaki disease (KD) in developed countries. However, the underlying immunologic mechanisms of CAL development in KD remain unclear. Here, we conducted single-cell transcriptome analyses of 212 210 peripheral blood mononuclear cells collected from a cross-sectional cohort of 16 children, including 4 patients with KD with CALs, 5 patients with KD without CALs, 4 healthy controls, and 3 febrile controls. KD altered the proportion of peripheral blood mononuclear cells, including an increasing trend in inflammatory cells (megakaryocytes and monocytes) and a decreasing trend in lymphocytes (eg, CD4+ T, CD8+ T, mucosal-associated invariant T, natural killer, and γδ T cells), highlighting the potential presence of lymphopenia phenomenon in KD. Our data indicated the presence of inflammatory cytokine storm in patients with KD with CALs, caused by systemic upregulation of TNFSF13B (tumor necrosis factor superfamily member 13b), CXCL16 (C-X-C motif chemokine ligand 16), TNFSF10 (tumor necrosis factor superfamily member 10), and IL1RN (interleukin 1 receptor antagonist), mainly produced by monocytes (especially for the Mono_CD14-CD16 cluster) and megakaryocytes. We also found that myeloid cells of patients with KD, particularly in those with CALs, might play a role in vascular injury (eg, increased MMP [matrix metalloproteinase] 9, MMP17, and MMP25) and immune cell recruitment. The immune landscape of patients with KD with CALs was featured by lower exhaustion levels in natural killer cells, a high cytotoxic state in the CD8_Pro cluster, and activation of the complement system in monocytes. Additionally, the activation of B cells was more pronounced in the early stage of KD. Collectively, this study provides a comprehensive understanding of the roles of various immune cells and inflammatory cytokine storms in the development of CALs in KD and offers a valuable resource for identifying novel therapeutic targets for patients with KD with CALs.

Downregulation of Hypoxia Inducible Factor-1α in Primary Human Natural Killer Cells Using Small Interfering RNA Delivery with ExPERT ATx by MaxCyte.

Natural killer (NK) cells are innate cytokine-producing and cytolytic effector lymphocytes. Their function is responsive to environmental factors, e.g., hypoxia, a frequent feature of inflamed tissues. Such responses require that the NK cells up-regulate HIF-1α (hypoxia inducible factor-1α), the major mediator of cellular responses to hypoxia that affects cell survival as well as immune responses. Thus, a major approach to the study of NK cell effector function under hypoxic conditions involves the ability to regulate HIF-1α levels in primary human NK cells. One difficulty with this approach, however, is that NK cells are difficult-to-transfect cells and common transfection methods, including electroporation or lipofection, suffer from variable transfection efficiency and cell viability. Moreover, the detection of HIF-1α is technically challenging because of the rapid degradation of the protein under normoxic conditions. Here, using the commercially available ExPERT ATx by MaxCyte, we report a workflow for the reliable delivery of small interfering RNA (siRNA) for targeting HIF-1α expression in primary human NK cells. We further provide a protocol for the detection of HIF-1α by immunoblot analysis demonstrating its efficient downregulation by siRNA. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of natural killer cells from human peripheral blood mononuclear cells Basic Protocol 2: Delivery of non-coding small interfering RNA and HIF-1α targeting siRNA into natural killer cells using ExPERT ATx Basic Protocol 3: Assessing the downregulation of HIF-1α protein using immunoblot analysis Support Protocol 1: Exemplary assessment of transfection efficiency using fluorescently labeled non-targeting siRNA Support Protocol 2: Exemplary assessment of NK cell viability 20 hr post-transfection Support Protocol 3: Exemplary assessment of HIF-1α knockdown using immunoblot analysis.

DOES HIGH-INTENSITY RESISTANCE TRAINING AFFECT AN ATHLETE'S IMMUNE FUNCTIONS?

This research aims to comprehensively examine the latest literature on the effects of high-intensity resistance training on the immune system's long-term performance. Many athletes and active individuals believe that moderate physical activity can enhance resistance to minor illnesses like upper respiratory tract infections (URTI), while intense exercise may have the opposite effect. The study involved 90 athletes who regularly train at Ahly Sporting Club and were divided into three groups: Group 1 included 30 athletes at rest, Group 2 comprised 30 athletes after normal training, and Group 3 had 30 athletes after intense training. Athletes who undergo intense training have lower total white blood cell counts than those who train at normal or resting levels. However, their neutrophil numbers slightly increase after heavy training, while lymphocyte and natural killer cell levels decrease. Additionally, those who undergo heavy training have higher PHA-induced proliferation levels but show a decline in serum immunoglobulin levels, mucosal immunoglobulin concentrations, and plasma glutamine levels. To prevent upper respiratory tract infections in athletes, it is essential to avoid over training and provide sufficient rest and recovery during and after training and competition. It is currently uncertain whether moderate exercise training can prevent infectious illness among the general population.

C–C chemokine receptor 5 is essential for conventional NK cell trafficking and liver injury in a murine hepatitis virus-induced fulminant hepatic failure model

BackgroundPrevious studies have demonstrated that natural killer (NK) cells migrated into the liver from peripheral organs and exerted cytotoxic effects on hepatocytes in virus-induced liver failure.AimThis study aimed to investigate the potential therapeutic role of chemokine receptors in the migration of NK cells in a murine hepatitis virus strain 3 (MHV-3)-induced fulminant hepatic failure (MHV-3-FHF) model and its mechanism.ResultsBy gene array analysis, chemokine (C–C motif) receptor 5 (CCR5) was found to have remarkably elevated expression levels in hepatic NK cells after MHV-3 infection. The number of hepatic CCR5+ conventional NK (cNK) cells increased and peaked at 48 h after MHV-3 infection, while the number of hepatic resident NK (rNK) cells steadily declined. Moreover, the expression of CCR5-related chemokines, including macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated on activation, normal T-cell expressed and secreted (RANTES) was significantly upregulated in MHV-3-infected hepatocytes. In an in vitro Transwell migration assay, CCR5-blocked splenic cNK cells showed decreased migration towards MHV-3-infected hepatocytes, and inhibition of MIP-1β or RANTES but not MIP-1α decreased cNK cell migration. Moreover, CCR5 knockout (KO) mice displayed reduced infiltration of hepatic cNK cells after MHV-3 infection, accompanied by attenuated liver injury and improved mouse survival time. Adoptive transfer of cNK cells from wild-type mice into CCR5 KO mice resulted in the abundant accumulation of hepatic cNK cells and aggravated liver injury. Moreover, pharmacological inhibition of CCR5 by maraviroc reduced cNK cell infiltration in the liver and liver injury in the MHV-3-FHF model.ConclusionThe CCR5-MIP-1β/RANTES axis played a critical role in the recruitment of cNK cells to the liver during MHV-3-induced liver injury. Targeted inhibition of CCR5 provides a therapeutic approach to ameliorate liver damage during virus-induced acute liver injury.

Azacitidine, Venetoclax and Allogeneic NK Cells in Newly Diagnosed Acute Myeloid Leukemia (ADVENT-AML): An Investigator-Initiated Multicenter Phase Ib Trial

BACKGROUND AND SIGNIFICANCE : Older/unfit patients with acute myeloid leukemia (AML) have limited treatment options. Outcomes in adverse-risk subgroups of AML are modest with CR/CRi rates of 45% to 60% and median overall survival (OS) of 5 to 12 months with current standard frontline therapy of hypomethylating agent with venetoclax (HMA-VEN).While allogeneic stem cell transplantation (allo-SCT) can significantly improve OS in AML, only 10 to 20% of older pts are able to undergo allo-SCT (Pratz et al. Blood 2019, Maiti et al. Blood 2022) Natural killer (NK) cells bridge the innate and adaptive immune system and play a major role in graft-vs-leukemia (GVL) effect responsible for the clinical benefit noted with allo-SCT. NK cell-based adoptive cellular therapies have shown good safety profiles and promising activity in AML with responses noted in 25% to 88% of patients. Furthermore, NK cells are less likely to induce graft-versus-host disease (GVHD), severe cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). We hypothesize that the combination of azacitidine, venetoclax and allogeneic NK cells will be safe and improve cure rates in AML compared to either Aza/Ven or NK cells alone. Improved clinical activity may be mediated through multiple synergistic mechanisms including reduction of disease burden by Aza/Ven, upregulation of epigenetically silenced NKG2D ligands, dual activation of extrinsic and intrinsic apoptotic cascades, mitochondrial priming of leukemia cells by venetoclax, and bypassing significant morbidity and mortality associated allo-SCT. Significance: This will be the first trial to evaluate 1) cellular therapy in the frontline setting in AML; 2) the synergy of venetoclax with adoptive NK cell therapy; and 3) Aza/Ven as a lymphodepletion strategy. The results will have significant impact on the development of cellular therapies for hematological and solid tumors. STUDY DESIGN AND METHODS: This investigator-initiated, multicenter, open-label, phase Ib study will evaluate the combination of azacitidine, venetoclax and ex vivo-expanded, off-the-shelf cryopreserved allogeneic NK cells in patients with AML (NCT05834244). Eligibility criteria: The dose escalation phase will enroll adults with relapsed or refractory AML or MDS/AML, and the dose expansion phase will enroll older/unfit pts with newly diagnosed adverse-risk AML. Eligible Pts will be ≥ 75 years, or ≥ 18 years with at least one protocol-defined comorbidity. Patients with favorable risk AML, poor end-organ function, needing immunosuppression, uncontrolled infection or CNS leukemia will be excluded. Trial design: The dose escalation phase will enroll a maximum of 12 patients at 2 dose levels to establish a recommended phase 2 dose (RP2D) using a BOIN design. The dose expansion will enroll up to 20 patients at the RP2D ( Fig. 1). Study treatment: Patients will receive Aza/Ven as standard. Two dose levels of NK cells including 0.5 x10 9 and 1 x10 9 flat dose on day 8 and 15 of the first 4 cycles will be evaluated. Subsequently, pts will continue Aza/Ven indefinitely. Objectives: The primary objective is to determine the safety of this novel triplet combination in terms of dose-limiting toxicities including acute GVHD, CRS or ICANS. Secondary objectives include overall response rate, CR/CRi by 4 cycles, rate of negative measurable residual disease, OS and relapse-free survival. Exploratory objectives include additional response and survival endpoints, the persistence of NK cells using SNP-NGS chimerism, kinetics of allorejection, remodeling of host immune microenvironment and impact on AML stem/progenitor cell hierarchies using single cell CyTOF to understand response/resistance mechanisms. Status: Trial approved by the FDA and institutional IRB. Activation planned for 2H 2023. Funding: Gateway for Cancer Research, MDACC PRIME Award, Chimeric Therapeutics, NCI Cancer Center Support Grant.

Association of recurrent implantation failure and recurrent pregnancy loss with peripheral blood natural killer cells and interferon-gamma level.

Fetal uterine survival depends on maintaining an immune balance between the mother and fetus. This study aimed to investigate the correlation of blood peripheral natural killer (NK) cells and interferon-gamma (IFN-γ) with recurrent recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL). In this case-control study, peripheral blood samples were obtained from three groups of RPL, RIF, and parous women without a history of abortion or infertility problems and analyzed by lymphocyte-based flow cytometry. Afterward, the levels of NK cells and IFN-γ were determined. All data were analyzed using one-way analysis of variance and nonparametric Kruskal-Wallis tests. The level of IFN-γ in the RPL group was significantly higher than that in parous women and the RIF group (P<0.05), whereas its level in the RIF group was not significantly different from the control group (P>0.05). A significant correlation was found between the levels of IFN-γ and NK cells in the RPL group (r=0.481; P=0.02). However, no significant correlation was found between the levels of IFN-γ and the active NK cells in the RPL group (P=0.08). Moreover, no significant correlation was found between the levels of NK cells (whether activated or not) and IFN-γ in the RIF patients (P>0.05). Immune dysfunction may not be involved in implantation failure during IVF but may be involved in recurrent miscarriage, probably by increasing IFN-γ levels.

Human Cytomegalovirus Infection Promotes the Formation of NKG2C Hi Memory NK Cells That Express Elevated Levels of IL-7R and Cyto-CD3ε

Introduction: Human cytomegalovirus (HCMV) is a b-herpesvirus that is highly prevalent in the adult population and has the ability to establish lifelong latency in healthy individuals. The innate and adaptive immune systems work closely to control viral replication, leading to a dynamic interaction between the virus and the host immune system. This interplay leads to the development of distinct immune-cell repertoires in HCMV seropositive (HCMV +) individuals. Natural killer (NK) cells are cytotoxic innate lymphocytes that are required to manage and control HCMV infections. NK cells utilize their NKG2C/CD94 receptor complex to mount a response to infected cells that present HLA-E molecules loaded with the HCMV UL40-derived peptide. As a result, HCMV + individuals possess higher levels of NKG2C + NK cells, and within this population, there is a distinct subset referred to as “memory” due to their adaptive-like characteristics. The developmental origins and molecular mechanisms involved in the maintenance and persistence of these cells remain largely unknown. In our current study, we investigate the origins and transcriptional signatures of persistent memory NKG2C + NK cells obtained from human donor spleens. Methods &amp; Results: Eight healthy adult human spleens were obtained from four HCMV + and four HCMV seronegative (HCMV -) donors. Donor median age was 59 [IQR 48.5-56.5], 50% (n=4) were identified as female, 50% (n=2) of females were HCMV + and 50% (n=2) of females were HCMV -. Spleens were provided by the Versiti Organ Donor Center of Wisconsin and were processed to a single cell suspension. In line with previous findings in the peripheral blood of HCMV + individuals, we observed significantly elevated levels of NKG2C + NK cells in the spleens of HCMV + donors ( Fig. 1A). This observed significance of higher NKG2C + NK cells was consistent across all HCMV + donors when compared to our HCMV - donors ( Fig. 1A). To investigate the molecular mechanisms involved in the maintenance and persistence of memory NKG2C + NK cells, we performed single-cell RNA sequencing (scRNA-seq), using the sorted NK cells from all eight donors. Using unbiased clustering analysis, we identified and characterized four distinct NKG2C + splenic NK cell subsets ( Fig. 1B). Our findings indicated that the relative composition of these subsets was highly influenced by the HCMV status of the donors. Specifically, we observed that HCMV + donors had significantly higher levels of NKG2C Hi memory NK cells ( Fig. 1B). This NKG2C Hi memory NK subset had significantly higher expression of NKG2C, CD52, CD3 e, and IL7R ( Fig. 1C) and significantly lower expression of FCER1G, KLRC1, CD247, ZBTB16, SYK, and SH2D1B ( Fig. 1C). These findings suggest NKG2C Hi memory NK cells possess unique transcriptional and molecular mechanisms that may contribute to their ability to persist over time. To explore the developmental cell fate of NKG2C Hi memory NK, we utilized both the Monocle 2 and Monocle 3 software's to track how NKG2C + cells transition between transcriptomic states. Monocle 3 analysis yielded a simple early-to-late cell fate trajectory that was projected onto our UMAP plot ( Fig. 2A). We found that NKG2A Hi NKG2C + subset served as the early timepoint in the developmental trajectory, ultimately leading to the development of NKG2C Hi memory NK ( Fig. 2A). The Monocle 2 analysis produced unique developmental trajectory plots, allowing us to visualize distinct developmental pathways and transitions ( Fig. 2B). Interestingly, we observed a unique branch-point exclusive to the HCMV + donors ( Fig. 2B) that expressed significantly higher levels of CD3 e and IL7R. These observations could indicate a specific developmental pathway unique to NKG2C Hi memory NK. Conclusion: Here, we demonstrate that HCMV infection can induce the formation of NKG2C Hi memory NK cell subset that displays a unique transcriptional and developmental profile. These findings can influence the future isolation and application of memory NK cells in cellular immunotherapies.

CYT-338 NK Cell Engager Mutispecific Antibody Engagement of NKp46 Activation Receptor Overcomes Anti-CD38 Mab Mediated Fratricide of NK Cells and Accords Enhanced Cancer Immunotherapy

Background and Significance: Despite the clinical success of the first anti-CD38 targeted monoclonal antibody, daratumumab, approved for the treatment of multiple myeloma (MM), significant challenges remain such as CD38 antigen loss/internalization and/or natural killer (NK) cell fratricide resulting in resistance to treatment over time. Study Design and Methods: To overcome the above-mentioned challenges, Cytovia has utilized its proprietary multifunctional tetravalent Flex Engager TM bispecific antibody IgG scaffold platform that contains a flexible linker for simultaneous engagement of effector immune cells and tumors targets. Through this, we generated CYT-338, an NK cell engager mutispecific antibody that targets CD38 on MM tumors and NKp46 and CD16 activation receptors on NK cells to mediate potent NK cell redirected killing of MM tumor cells more efficaciously than daratumumab. In serial killing assays against MM tumors, CYT-338 reversed dysfunctional NK cells to its original activation state and prevented exhaustion of killing activity of NK cells over time. In addition, we have also previously reported that CYT-338 has a higher potency over daratumumab for antibody dependent cellular phagocytosis (ADCP) of MM tumors. Preclinically,CYT-338 shows significantly reduced NK cell fratricide compared to daratumumab but the reason for this was unknown. We hypothesized that CYT-338 could overcome anti-CD38 mAb mediated fratricide of NK cells by engaging NKp46 activation receptor. To this end we incubated peripheral blood NK cells (PBNK) with either anti-CD38 mAb, daratumumab, CYT-338 or human IgG1 isotype control either in the presence or absence of blocking anti-NKp46 mAb (IgG1) for 24 hrs at 37 oC and stained with Annexin V apoptosis and Zombie NIR viability dye and analyzed NK fratricide by flow cytometry. We also evaluated the ability of anti-NKp46 mAb to inhibit daratumumab-mediated fratricide of NK cells under the same conditions. Current Status: Through functional preclinical investigations, we have been able to deconvolute the novel molecular mechanisms whereby NKp46 and CD16 co-engagement signals by CYT-338 on NK cells are able to overcome the CD38 apoptotic fratricide signals induced by the single anti-CD38 mAb. Specifically, we have found that blockade of CYT-338 binding to NKp46 by anti-NKp46 IgG1 antibody reversed the fratricide protection provided by CYT-338, indicating that the fratricide protection was mediated by NKp46 activation signals. In addition, the single anti-NKp46 mAb was able to inhibit daratumumab-mediated NK cell fratricide providing direct evidence that daratumumabmediated fratricide can also be reversed by engagement of NKp46 activation receptor. Conclusions: The unique NKp46 activation mechanism provided by CYT-338 for reducing NK cell fratricide and maintaining NK cell levels together with the enhanced potency including reversal of NK cell dysfunction makes it an attractive best-in-class anti-CD38 therapeutic against MM compared to daratumumab. These results support the development of CYT-338 in both monotherapy and combination with other complementary immunotherapy agents being developed or approved for MM. A first-in-human Phase 1 study targeting patients with relapsed/refractory MM is in development in the U.S.

Decrease in peripheral natural killer cell level during early pregnancy predicts live birth among women with unexplained recurrent pregnancy loss: a prospective cohort study

BACKGROUNDPrevious studies have suggested that the trophoblast cells inhibit the proliferation of peripheral natural killer (pNK) cells and the level of pNK cells decrease in middle and late pregnancy stage among healthy women. The change of pNK cells level during early pregnancy and the relationship between the change of pNK level and pregnancy outcomes in women with unexplained recurrent pregnancy loss (URPL) has not been sufficiently explored. OBJECTIVEThis study aims to characterize the level of pre-pregnancy pNK to early pregnancy among women with URPL and whether the change in the level of pNK cells in early pregnancy from pre-pregnancy can predict pregnancy outcomes. STUDY DESIGNIn this prospective cohort study, 1758 women with recurrent pregnancy loss were recruited between January 2017 and December 2021, of which 252 URPL women had pre-pregnancy and early pregnancy (4-6 weeks gestation) pNK measurements. These 252 women were divided into two groups: those with a lower gestational pNK levels (Group 1) compared to pre pregnancy, and those without (Group 2). Their respective outcomes on live birth and pregnancy loss were comparatively analyzed using Chi-square and Student’s t test. Candidate influence factors for live birth were selected using the Akaike information criterion (AIC). Then the participates were randomly divided into training and testing groups. Multivariable logistic regression model was performed, and nomogram was calculated to assess the possibility of live birth. Predictive accuracy was discriminated by area under the receiver operating characteristic curve (ROC), validated by plotting the predicted probabilities and the observed probabilities. Hosmer-Lemeshow test was used to assess the goodsess of fit. RESULTSWhen early gestational pNK cells levels were compared with pre pregnancy pNK cells levels, 61.5% (154) women had a comparatively lower early gestational pNK cells levels versus 38.9% (98) women with increase or no change of their pNK cells levels. The live birth rate in Group 1 was 89.0% (137/154), which was significantly higher than 49.0% (48/98) of Group 2 (p<0.001). Decrease of pNK cells level (odds ratio [OR]=1.36, 95% CI 1.22-1.55, p<0.001) and anti-Muellerian hormone (AMH) (OR=1.41, 95% CI 1.14-1.81, p=0.003) were important predicting factors for higher live birth. Female BMI (OR=0.97, 95% CI 0.82-1.15, p=0.763) and parity (OR=1.61, 95% CI 0.71-4.12, p=0.287) were also predicting factors. Furthermore, the area under the ROC curve of the model to diagnose of live birth was 0.853 with a sensitivity of 81.6% and specificity of 78.0% using the training dataset. And the Hosmer−Lemeshow test showed that the model was a good fit (p = 6.068). CONCLUSIONWe report a comparative decrease in pNK cells levels in over 60% URPL women at 4-6 weeks of gestation, when compared to their pre-pregnancy pNK cells level. Compared to pre pregnancy pNK cells levels, a decrease pNK cells level during early pregnancy might be a useful predictor for LBR in women with URPL.

Development and validation of diagnostic and activity-assessing models for relapsing polychondritis based on laboratory parameters.

Relapsing polychondritis (RP) as a rare autoimmune disease is characterized by recurrent inflammation of the organs containing cartilage. Currently, no biomarkers have been integrated into clinical practice. This study aimed to construct and evaluate models based on laboratory parameters to aid in RP diagnosis, assess activity assessment, and explore associations with the pathological process. RP patients and healthy controls (HCs) were recruited at the Peking Union Medical College Hospital from July 2017 to July 2023. Clinical data including Relapsing Polychondritis Disease Activity Index (RPDAI) score and laboratory tests were collected. Differences in laboratory data between RP patients and HCs and active and inactive patients were analyzed. The discovery cohort (cohort 1) consisted of 78 RP patients and 94 HCs. A model based on monocyte counts and neutrophil to lymphocyte ratio (NLR) could effectively distinguish RP patients from HCs with an AUC of 0.845. Active RP patients exhibited increased erythrocyte sedimentation rate, complement 3, platelet to lymphocyte ratio (PLR), NLR, and C-reactive protein to albumin ratio (CAR) compared with stable patients, which were also positively correlated with RPDAI. Notably, CAR emerged as an independent risk factor of disease activity (OR = 4.422) and could identify active patients with an AUC of 0.758. To confirm the reliability and stability of the aforementioned models, a replication cohort (cohort 2) was enrolled, including 79 RP patients and 94 HCs. The monocyte-combined NLR and CAR showed a sensitivity of 0.886 and 0.577 and a specificity of 0.830 and 0.833 in RP diagnosis and activity prediction, respectively. Furthermore, lower natural killer cell levels in RP patients and higher B-cell levels in active patients may contribute to elucidating the pathological mechanisms of disease occurrence and exacerbation. The utilization of laboratory parameters provides cost-effective and valuable markers that can assist in RP diagnosis, identify disease activity, and elucidate pathogenic mechanisms.