Increased NK Cell Activity Research Articles

A phase 1 study of interleukin-15 in combination with mogamulizumab in relapsed and refractory T-cell malignancies

IntroductionRecombinant human interleukin-15 (rhIL-15) is an immunotherapeutic which enhances NK-cells to augment the antibody directed cellular cytotoxicity (ADCC) of monoclonal antibodies. Mogamulizumab is a CCR4 directed monoclonal antibodies that exerts cytotoxicity through ADCC and depletes regulatory T-cells within the tumor microenvironment. MethodsWe conducted a phase 1 clinical trial of rhIL-15 combined with mogamulizumab. Patients with relapsed or refractory adult T-cell leukemia/lymphoma (ATLL), mycosis fungoides (MF) and Sezary syndrome (SS) received fixed dose mogamulizumab combined with escalating doses of rhIL-15 to identify the maximum tolerated dose (MTD). ResultsSix patients were enrolled, 4 with ATLL and 2 with MF/SS. The most common adverse events were rash, infection and fever (67%, in all). Two patients (33%) had grade 4 acute kidney injury and 25% of cycles had grade ≥3 anemia. The MTD was dose level 1. One patient with ATLL had a partial response despite receiving only 4 cycles due to grade 4 myositis. Circulating NK cells were increased in all patients during the first cycle and a rapid reduction in tumor cells within the peripheral circulation was noted. Ex vivo assessment demonstrated increased NK cell activation and increased cell lysis in the presence of monoclonal antibodies after only 5 days. DiscussionOur small study suggests that rhIL-15 combined with mogamulizumab leads to effector NK cell activation and regulatory T-cell depletion but has an unfavorable safety profile. Future development of combinations of immunotherapy that target the microenvironment in relapsed or refractory T-cell lymphomas remain rational. NCT04185220

Dabur Chyawankidz Gummies: A novel dosage form for immunomodulation

Across age groups, a tasty eatable product is always cherished and so is immunity on the other hand, equally indispensable throughout lifetime. Optimal and sustained immunity is a contiguous requirement for growth and welfare of the body and goodness of herbs of Chyawanprash is a time-tested formula in Ayurveda for a healthy life. Combining the innovation of a chewable system of drug delivery with goodness of Ayurveda, Dabur India Limited has prepared the Dabur ChyawanKidz Gummies – a palatable, chewable and immune boosting formulation. To assess its efficacy, the active immune boosting formulation of the product were studied for its activity on NK Cell, Cytokines (TNF-α) and phagocytosis by Macrophages; wherein compared to the control groups, the test product showed up to 49% increased NK Cell activity, 66-fold increase in TNF-α (Cytokines) and 55% increased phagocytic activity by macrophages. The result indicates 3x (triple) immunity action of the Dabur ChyawanKidz Gummies, making it a unique choice of chewable immunomodulatory preparation, which appeals across ages, combining taste with immunity.

Functional comparison of Fc-engineering strategies to improve anti-HIV-1 antibody effector functions

Substantial reduction of the intact proviral reservoir is essential towards HIV-1 cure. In vivo administration of broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) trimer can decrease the viral reservoir, through Fc-mediated killing of infected cells. In this study, we compared three commonly used antibody engineering strategies to enhance Fc-mediated effector functions: (i) glyco-engineering, (ii) protein engineering, and (iii) subclass/hinge modifications in a panel of anti-HIV-1 antibodies. We found that antibody-dependent cellular phagocytosis (ADCP) was improved by elongating the hinge domain and switching to an IgG3 constant domain. In addition, potent NK cell activation and ADCC activity was observed for afucosylated antibodies and antibodies bearing the GASDALIE mutations. The combination of these engineering strategies further increased NK cell activation and induced antibody dependent cytotoxicity (ADCC) of infected cells at low antibody concentrations. The bNAb N6 was most effective at killing HIV-1 infected cells, likely due to its high affinity and optimal angle of approach. Overall, the findings of this study are applicable to other antibody formats, and can aid the development of effective immunotherapies and antibody-based treatments for HIV-1 cure strategies.

Abstract 3574: Cancer testis antigen expression correlates with stromal natural killer cell activation and longer overall survival in small bowel neuroendocrine tumors

Abstract Background: Patients with small bowel neuroendocrine tumors (SBNET) frequently present with metastatic disease and the efficacy of available therapies, including immune checkpoint blockade (ICB), is limited. Toward developing novel immunomodulatory strategies, we interrogated the tumor immune microenvironment of SBNETs using bulk transcriptional and digital spatial profiling (DSP). Methods: Patients with SBNET who underwent resection at MD Anderson Cancer Center from 2003-2016 were retrospectively analyzed. Overall survival (OS) was assessed using the Kaplan-Meier method and the Cox proportional hazards model was used for multivariate analysis (MVA). Bulk transcriptional profiling was performed using the Nanostring PanCancer-Immune Panel. DSP with whole human transcriptome was performed using PanCK to segment tumor and adjacent stroma; 88 distinct regions were analyzed using differential gene expression and Reactome pathway enrichment pipelines. Results: Resected SBNET from 42 patients were selected for transcriptional analysis. Unsupervised clustering of gene expression demonstrated dichotomization into high and low cancer testis antigen (CTA) expression. CTA-high patients (n=12) demonstrated elevated expression of type-I interferons (IFN), and CTAs such as PRAME, GAGE1 and MAGEA3 and had significantly longer OS compared to CTA-low patients (n=30; median OS not reached vs 1975 days, p=0.008). MVA controlling for age, sex, stage, and Ki-67% confirmed CTA-high status as an independent predictor of longer OS (hazard ratio [HR] 0.2, 95% confidence interval [CI] 0.05-0.83, p=0.014). A pilot DSP experiment on 4 CTA-high and 4 CTA-low tumors confirmed differential gene expression in the PanCK+ tumor compartment of these samples (R2=0.09, p=0.001). In particular, epigenetic modification genes (e.g., H2BC8, H4C8, H3C1, p<0.001) and epigenetic pathways (e.g., DNA methylation, histone acetylation) were enriched in CTA-high tumors. CTA-high tumors exhibited increased M2 macrophage presence (p=0.025) and increased NK cell activation within the PanCK- stromal compartment (p=0.001). Conclusions: High CTA and type-I IFN expression in resected SBNET identifies patients with longer survival, agnostic of stage or grade. While CTA expression has been implicated in tumor immunogenicity, this is the first work to identify a clinically relevant signal in SBNET. Increased IFN in CTA-high tumors, corroborated by increased stromal NK cell activation, suggests that enhanced immunogenicity may drive this survival difference. Spatial transcriptomic analysis reveals epigenetic differences in CTA-high tumors and highlights the potential for combination epigenetic modifiers and immunotherapy in future trials. Citation Format: Reed I. Ayabe, Yongwoo D. Seo, Brenda Melendez, Rossana Lazcano, Brittany C. Fields, Khalida Wani, Sarah Johnson, Manoj Chelvanambi, Courtney Hudgens, Sharia D. Hernandez, Nadim J. Ajami, Jennifer A. Wargo, Alexander J. Lazar, Daniel M. Halperin, Jeannelyn S. Estrella, Jessica E. Maxwell. Cancer testis antigen expression correlates with stromal natural killer cell activation and longer overall survival in small bowel neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3574.

KIR-генетические факторы и ответ на терапию ингибиторами тирозинкиназ при хроническом миелоидном лейкозе

The development of tyrosine kinase inhibitors (TKIs) and their introduction into clinical practice considerably improved the prognosis for chronic myeloid leukemia (CML) patients. About 50 % of patients with achieved deep molecular response are eligible for safe TKI discontinuation. Despite these advances, no reliable biomarkers are known to predict a response and sustaining treatment-free remission after TKI withdrawal. As TKIs do not destroy leukemic stem cells, which can be responsible for relapse, critical importance in CML is attached to natural killers (NK-cells) having antitumor activity. Functional activity of NK-cells is evaluated by expression level and repertoire of killer cell immunoglobulin-like receptors (KIR). Current studies demonstrate that a patient’s KIR genotype affects the probability of achieving early and deep molecular responses to first- and second-generation TKIs, progression-free and overall survivals, and sustaining treatment-free remission. On that ground, KIR-genetic factors can be regarded as promising predictors of response to TKI therapy in CML. Early clinical studies, which dealt with monoclonal antibodies blocking the inhibitory KIR in order to increase NK-cell activity, revealed an acceptable safety profile and efficacy in some hematological diseases (such as acute myeloid leukemia, multiple myeloma, Т-cell lymphoma) if used in combination with cytostatic drugs or antitumor monoclonal antibodies. KIR genotype determination can contribute to the development of effective therapies of this malignant hematological tumor.

Single-cell RNA sequencing indicates cordycepin remodels the tumor immune microenvironment to enhance TIGIT blockade's anti-tumor effect in colon cancer

Both preclinical and clinical studies have extensively proven the effectiveness of TIGIT inhibitors in tumor immunotherapy. However, it has been discovered that the presence of CD226 on tumor-infiltrating lymphocytes is crucial for the effectiveness of both anti-TIGIT therapy alone and when combined with anti-PD-1 therapy for tumors. In our investigation, we observed that cordycepin therapy significantly augmented the expression of the Cd226 gene. As a result, it was hypothesized that cordycepin therapy could enhance the effectiveness of anti-TIGIT therapy. By employing single-cell RNA sequencing analysis of immune cells in the MC38 tumor model, we discovered that cordycepin combined with anti-TIGIT therapy led to a significant increase in the proportion of NK cells within the tumor immune microenvironment. This increased NK cell activity and decreased the expression of inhibitory receptors and exhaustion marker genes. In the combination therapy group, CD8+ T cells had lower exhaustion state scores and increased cytotoxicity, indicating a better immune response. The combination therapy group increased DCs in the tumor immune microenvironment and promoted cellular interaction with CD4+ T cell and CD8+ T cell populations while decreasing Treg cell interactions. In conclusion, cordycepin with anti-TIGIT therapy in colon cancer could reshape the tumor immune microenvironment and have notable anticancer effects.

Antioxidant and Immune Stimulating Effects of Allium cepa Skin in the RAW 264.7 Cells and in the C57BL/6 Mouse Immunosuppressed by Cyclophosphamide.

Allium cepa L. (onion) has been reported to have various pharmacological effects, such as preventing heart disease, and improving antimicrobial activity and immunological effects. The Republic of Korea produced 1,195,563 tons of onions (2022). The flesh of onion is used as food while the onion skin (OS) is thrown away as an agro-food by-product and is considered to induce environmental pollution. Thus, we hypothesize that increasing usage of OS as functional food material could help protect from the environment pollution. The antioxidant effects and immune-enhancing effects of OS were evaluated as functional activities of OS. In this study, OS showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and xanthine oxidase (XO) inhibitory activity. The antioxidant activities increased in a dose-dependent manner. The IC50 values of DPPH, ABTS radical scavenging activity, and XO inhibitory activity were 954.9 μg/mL, 28.0 μg/mL, and 10.7 μg/mL, respectively. Superoxide dismutase and catalase activities of OS in RAW 264.7 cells were higher than those of the media control. There was no cytotoxicity of OS found in RAW 264.7 cells. Nitric oxide and cytokines (IL-1β, IL-6, IFN-γ, and TNF-α) concentrations in RAW 264.7 cells significantly increased in a dose dependent manner. Immune-stimulating effects of OS were evaluated in immunosuppressed mice induced by cyclophosphamide. White blood cell count and the B cell proliferation of splenocytes were higher in OS100 (OS extract 100 mg/kg body weight) and OS200 (OS extract 200 mg/kg body weight) groups than in the negative control (NC) group. Serum IgG and cytokine (IL-1β and IFN-γ) levels were also higher in OS100 and OS200 groups than in the NC group. OS treatment increased NK cell activity compared with the NC group. The results suggested that OS can improve antioxidant and immune stimulating effects. The use of OS as functional supplement can reduce the agro-food by-product and it may contribute to carbon neutrality.

Abstract 5660: IGM-7354, an immunocytokine with IL-15 fused to an anti-PD-L1 IgM, induces NK and CD8+ T cell mediated cytotoxicity of PD-L1-positive tumor cells

Abstract Immunostimulatory cytokines are a promising immunotherapy for the treatment of advanced malignancies, but generally have been associated with severe toxicities when administered systemically. The recent development of antibody-cytokine fusion proteins, or immunocytokines, aims to localize cytokine activity to the tumor microenvironment and thus improve their therapeutic index. We have developed IGM-7354, a high affinity, high avidity anti-PD-L1 pentameric IgM antibody with an IL-15Rα chain and IL-15 fused to the joining (J) chain. The IGM-7354 immunocytokine was designed to deliver IL-15-mediated stimulation of NK and CD8+ T cells to PD-L1-expressing tumors and antigen-presenting cells, to enhance anti-tumor immune responses. The multivalent binding of IGM-7354 to PD-L1 provided a stronger binding avidity for human PD-L1 than the monovalent binding of IL-15 to IL-15Rb as confirmed in kinetic binding assays. In vitro IGM-7354 induced the proliferation of a cytotoxic T cell line responsive to IL-15 stimulation and enhanced the proliferation of NK and CD8+ T cells from healthy donor human PBMCs. In cytotoxicity assays with human PBMC and PD-L1+ cancer cell lines, IGM-7354 enhanced cancer cell killing through NK and CD8+ T cell expansion and cytotoxic activity, evidenced by Ki67 and Granzyme B upregulation in these cell populations. Next, in vivo pharmacodynamic studies were performed in two humanized mouse models: non-tumor-bearing BRGSF-HIS mice engrafted with human CD34+ cells, and PD-L1+ MDA-MB-231 tumor-bearing MHC-/- NSG mice engrafted with human PBMCs. In the BRGSF model, IGM-7354 increased NK cell activation and Granzyme B expression as well as NK and CD8+ T cell proliferation. In the tumor-bearing mouse model, IGM-7354 dose-dependently increased NK and CD8+ T cell proliferation in blood and infiltration of lymphocytes into the tumor. This pharmacodynamic activity correlated with IGM-7354 anti-tumor activity in the MDA-MB-231 model. Lastly, IGM-7354 increased the proliferation of NK and CD8+ T cells in cynomolgus monkeys and particularly induced the expansion of effector memory CD8+ T cells in the periphery. In summary, IGM-7354 induces NK and CD8+ T cell proliferation in both in vitro and in vivo preclinical models, resulting in the killing of PD-L1+ tumor cells. The strong avidity of IGM-7354 for PD-L1 may enhance IL-15 delivery to tumors and antigen-presenting cells and thus provide a more favorable safety profile. A Phase 1 clinical trial is planned. Citation Format: Thierry D. Giffon, Melanie Desbois, Poonam Yakkundi, Susan Calhoun, Keerthana Sekar, Carolyn Denson, Tasnim Kothambawala, Alexander Pearson, Sivani Pandey, Deepal Pandya, Rodnie Rosete, Daniel Machado, Pat Raichlen, Dean Ng, Abhinav R. Jain, Roel Funke, Eric Humke, Paul R. Hinton, Beatrice Wang, Bruce A. Keyt, Maya F. Kotturi, Angus M. Sinclair. IGM-7354, an immunocytokine with IL-15 fused to an anti-PD-L1 IgM, induces NK and CD8+ T cell mediated cytotoxicity of PD-L1-positive tumor cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5660.

Abstract 2907: Development of a CAR-NK cellular platform for cancer immunotherapy

Abstract Although Chimeric Antigen Receptor (CAR)-T cell therapy has proven to be successful in treating multiple hematological malignancies, their use is limited due to the potential life-threatening adverse events, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). CAR natural killer (CAR-NK) cells are rapidly emerging as a promising alternative cellular therapeutic with improved efficacy and reduced adverse effects. Moreover, due to the intrinsic characteristics of NK cells, whereby they lack antigen-specific cell surface receptors, CAR-NK cells offer the additional advantage of being an “off-the-shelf” product, thus satisfying the requirement for large-scale production for cancer immunotherapy, giving it the potential to reach more patients at a reduced cost. Here we outline the method to generate, expand and enrich human primary CD19 CAR-NK cells, and demonstrate their efficacy in-vitro against CD19-expressing target cancer cells. Human primary NK cells were isolated from healthy donors and transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV, as an empty vector transduced control. Transduction efficiency greater than 10% or 20% was achieved following transduction and expansion over a 7-day period in response to CD19 CAR LV or GFP LV respectively. Following sufficient expansion, cytotoxicity of the CD19 CAR-NK cells was tested in-vitro in a Tumor Killing Assay (TKA) against CD19+ (Raji) or CD19- (SKOV-3) target cells. As expected, CD19 CAR-NK cells displayed increased cytotoxicity towards CD19+ target cells when compared to non-transduced or GFP transduced controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting specific CD19 CAR mediated killing. Moreover, CD19 CAR-NK cells showed increased degranulation and IFNꝩ secretion when compared to both non-transduced and GFP-transduced controls, suggesting increased NK cell activation and cytotoxicity. Additionally, when degranulation of CD19 CAR positive and CD19 CAR negative NK populations was directly compared, the level of NK cell degranulation was analogous between the two populations, suggesting that CD19 CAR NK cells can enhance the cytotoxic activity of neighboring non-transduced cells, resulting in enhanced killing of CD19+ target cells by the total NK population present. Taken together these data demonstrate the generation, expansion and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This culture system provides the scientific community a platform to screen novel CAR-constructs and to test novel immunomodulators which could enhance NK cell expansion or functional potential to further develop new, more widely available cellular therapeutics to treat cancer. Citation Format: Karina Di Gregoli, Ravindra Mode, Kaitlin Mitchell, David Cobeta-Lopez, Sabrina de Munnik, Rene McLaughlin, Gemma Moiset, Louise Brackenbury, Lauren Schewitz-Bowers, Robert Nunan. Development of a CAR-NK cellular platform for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2907.

Natural Killer Cell Activity Test Helps to Suspect Aggressive Natural Killer Cell Leukemia - Diagnostic Challenge.

Aggressive natural killer cell leukemia (ANKL) is a rare disease with an aggressive clinical course. We aimed to assess the clinicopathological characteristics of the difficult to diagnose ANKL. During ten years, nine patients with ANKL were diagnosed. All the patients exhibited aggressive clinical course and underwent the BM study to rule out lymphoma and hemophagocytic lymphohistiocytosis (HLH). BM examination showed varying degrees of infiltration of neoplastic cells, which were mainly positive for CD2, CD56, cytoplasmic CD3 and EBV in situ hybridization. Five BM aspirates showed histiocytic proliferation with active heomphagocytosis. Normal or increased NK cell activity test results were obtained from 3 patients who were available for testing. Four had multiple BM studies until diagnosis. An aggressive clinical course and positive EBV in situ hybridization, often with associated secondary HLH, should raise the suspicion of an ANKL. Conducting additional supplementary tests such as NK cell activity and NK cell proportion would be helpful for the diagnosis of ANKL.

Biodegradable nanoparticles induce cGAS/STING-dependent reprogramming of myeloid cells to promote tumor immunotherapy.

Cancer treatment utilizing infusion therapies to enhance the patient’s own immune response against the tumor have shown significant functionality in a small subpopulation of patients. Additionally, advances have been made in the utilization of nanotechnology for the treatment of disease. We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic-co-glycolic acid) (PLGA) nanoparticles (IMPs; named ONP-302) for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

Siplizumab Induces NK Cell Fratricide Through Antibody-Dependent Cell-Mediated Cytotoxicity.

The glycoprotein CD2 is expressed on T and NK cells and contributes to cell-cell conjugation, agonistic signaling and actin cytoskeleton rearrangement. CD2 has previously been shown to have an important function in natural NK cell cytotoxicity but to be expendable in antibody-mediated cytotoxicity. Siplizumab is a monoclonal anti-CD2 IgG1 antibody that is currently undergoing clinical trials in the field of transplantation. This study investigated the effect of CD2 binding and Fc γ receptor binding by siplizumab (Fc-active) and Fc-silent anti-CD2 monoclonal antibodies in allogeneic mixed lymphocyte reaction and autologous lymphocyte culture. Further, induction of NK cell fratricide and inhibition of natural cytotoxicity as well as antibody-dependent cytotoxicity by these agents were assessed. Blockade of CD2 via monoclonal antibodies in the absence of Fc γ receptor binding inhibited NK cell activation in allogeneic mixed lymphocyte reaction. In contrast, siplizumab increased NK cell activation in both mixed lymphocyte reaction and autologous lymphocyte culture due to FcγRIIIA binding. However, experiments using purified NK cells did not show an inhibitory effect of CD2 blockade on natural cytotoxicity or antibody-dependent cytotoxicity. Lastly, it was shown that siplizumab induces NK cell fratricide. Concluding, siplizumab is a promising biopharmaceutical drug candidate for depletion of T and NK cells with minimal off-target effects.

Lipid emulsion therapy in women with recurrent pregnancy loss and repeated implantation failure: The role of abnormal natural killer cell activity.

Altered immune and/or inflammatory response plays an important role in cases of recurrent pregnancy loss (RPL) and repeated implantation failure (RIF). Exacerbation of the maternal immune response through increased NK cell activity and inflammatory cytokines can cause embryo rejection leading to abortion or embryo implantation failure. Immunosuppressors or immunomodulators can help or prevent this condition. Currently, lipid emulsion therapy (LET) has emerged as a treatment for RPL and RIF in women with abnormal NK cell activity, by decreasing the exacerbated immune response of the maternal uterus and providing a more receptive environment for the embryo. However, the mechanisms by which the intralipid acts to reduce NK cell activity are still unclear. In this review, we focus on the studies that conducted LET to treat patients with RPL and RIF with abnormal NK cell activity. We find that although some authors recommend LET as an effective intervention, more studies are necessary to confirm its effectiveness in restoring NK cell activity to normal levels and to comprehend the underlying mechanisms of the lipids action in ameliorating the maternal environment and improving the pregnancy rate.

Dynamics of the NK cell cytolytic activity in acute myocardial infarction: the role of circulating immune complexes

NK cells play an important role in the pathogenesis of atherosclerosis, using their cytotoxic mechanisms that promote the initiation, progression, and rupture of atherosclerotic plaques that leads to acute myocardial infarction. However the data about their cytolytic activity in post-infarction period remain controversial. The aim of this work was to study the dynamics of changes in the NK cell cytolytic activity within three weeks after acute myocardial infarction in connection with the level of circulating immune complexes in the peripheral blood. Material and methods. 16 healthy donors and 35 patients after the development of acute myocardial infarction (AMI) on the 1st, 7th and 21st days was studied for the NK cell cytolytic activity of the mononuclear fraction of peripheral blood, evaluated by the cytolysis of erythroleukemia K-562 cells in vitro and levels of serum circulating immune complexes (CIC) using the method of precipitation with polyethylene glycol-6000. Results and discussion. In the most patients, AMI led to functional anergy of NK cells, but in the smaller part of patients, the increase in their cytotoxicity was observed. The post-infarction period was characterized by an increased content of CIC, the level of which positively correlated with the development of functional anergy of NK cells. Possible mechanisms for the development of both functional anergy and an increase in NK cell activity in the post-infarction period are discussed. Keywords: аcute myocardial infarction, NK cells, circulating immune complexes.

PVRIG is a novel natural killer cell immune checkpoint receptor in acute myeloid leukemia

This study explored the novel immune checkpoint poliovirus receptor- related immunoglobulin domain-containing (PVRIG) in acute myeloid leukemia (AML). We showed that AML patient blasts consistently expressed the PVRIG ligand (poliovirus receptor-related 2, PVRL2). Furthermore, PVRIG blockade significantly enhanced naural killer (NK)-cell killing of PVRL2+, poliovirus receptor (PVR)lo AML cell lines, and significantly increased NK-cell activation and degranulation in the context of patient primary AML blasts. However, in AML patient bone marrow, NK-cell PVRIG expression levels were not increased. In order to understand how PVRIG blockade might potentially be exploited therapeutically, we investigated the biology of PVRIG and revealed that NK-cell activation resulted in reduced PVRIG expression on the cell surface. This occurred whether NK cells were activated by tumor cell recognition, cytokines (interleukin 2 [IL-2] and IL-12) or activating receptor stimulation (CD16 and NKp46). PVRIG was present at higher levels in the cytoplasm than on the cell surface, particularly on CD56bright NK cells, which further increased cytoplasmic PVRIG levels following IL-2 and IL-12 activation. PVRIG was continually transported to the cell surface via the endoplasmic reticulum and Golgi in both unstimulated and activated NK cells. Taken together, our findings suggest that anti-PVRIG blocking antibody functions by binding to surface-bound PVRIG, which undergoes rapid turnover in both unstimulated and activated NK cells. We conclude that the PVRIG-PVRL2 immune checkpoint axis can feasibly be targeted with PVRIG blocking antibody for NK-mediated immunotherapy of PVRL2+ AML.

Mangiferin induces immune responses and evaluates the survival rate in WEHI-3 cell generated mouse leukemia in vivo.

Mangiferin is a naturally occurring polyphenol, widely distributed in Thymeraceae families, and presents pharmacological activity, including anti-cancer activities in many human cancer cell lines. Mangiferin has also been reported to affect immune responses; however, no available information concerning the effects of mangiferin on immune reactions in leukemia mice in vivo. In the present study, we investigated the effects of mangiferin on leukemia WEHI-3 cell generated leukemia BLAB/c mice. Overall, the experiments were divided into two parts, one part was immune responses experiment and the other was the survival rate experiment. The immune responses and survival rate study, 40 mice for each part, were randomly separated into five groups (N = 8): Group I was normal animals and groups II-V WEHI-3 cell generated leukemia mice. Group II mice were fed normal diet as a positive control; group III, IV, and V mice received mangiferin at 40, 80, and 120 mg/kg, respectively, by intraperitoneal injection every 2 days for 20 days. Leukocytes cell population, macrophage phagocytosis, and NK cell activities were analyzed by flow cytometry. Isolated splenocytes stimulated with lipopolysaccharide (LPS) and concanavalin A (Con A) were used to determine the proliferation of B and T cells, respectively, and subsequently were analyzed by flow cytometry. Results indicated that mangiferin significantly increased body weight, decreased the liver and spleen weights of leukemia mice. Mangiferin also increased CD3 T-cell and CD19 B cell population but decreased Mac-3 macrophage and CD11b monocyte. Furthermore, mangiferin decreased phagocytosis of macrophages from PBMC and peritoneal cavity at 40, 80, and 120 mg/kg treatment. However, it also increased NK cell activity at 40 and 120 mg/kg treatment. There were no effects on T and B cell proliferation at three examined doses. In survival rate studies, mangiferin significantly elevated survival rate at 40 and 120 mg/kg treatment of leukemia mice in vivo.

Prevention of liver metastases through perioperative acute CpG-C immune stimulation.

Following excision of colorectal tumors, metastatic disease is prevalent, primarily occurs in the liver, and is highly predictive of poor prognosis. The perioperative period is now recognized as critical in determining the incidence of postoperative metastases and long-term cancer outcomes. Thus, various perioperative prophylactic interventions are currently studied during this time frame. However, immune stimulation during the perioperative period has rarely been attempted due to specific contraindications to surgery and various adverse effects. Here, to prevent liver metastases, we perioperatively employed a TLR-9 agonist, CpG-C, which exhibits minimal pyrogenic and other adverse effects in patients. We found that marginating-hepatic (MH) cells in BALB/c mice contained high percentage of NK cells, but exhibited negligible NK cytotoxicity, as previously reported in humans. However, a single CpG-C administration (25-100µg/mouse) doubled MH-NK cell numbers, increased NK cell activation and maturation markers (NKp46, CD11b), decreased the inhibitory NKG2A ligand, and dramatically increased MH-NK-cell cytotoxicity against the syngeneic CT26 colon cancer line. Moreover, in operated mice, this innocuous intervention also markedly improved resistance to CT26 and MC38 hepatic metastases in BALB/c and C57BL/6 mice, respectively. Beneficial effects of CpG-C were mediated through activation of MH-NK cells, as indicated by an in vivo NK depletion study. Last, CpG-C protected against surgery-induced suppression of MH-NK cytotoxicity and improved their activation indices. Thus, we suggest that systemic perioperative CpG-C treatment should be considered and studied as a novel therapeutic approach to improve long-term cancer outcomes in colorectal cancer patients.

NK Cells Regulate CD8+ T Cell Mediated Autoimmunity.

Elucidating key factors that regulate immune-mediated pathology in vivo is critical for developing improved strategies to treat autoimmune disease and cancer. NK cells can exhibit regulatory functions against CD8+ T cells following viral infection. Here we show that while low doses of lymphocytic choriomeningitis virus (LCMV-WE) can readily induce strong CD8+ T cell responses and diabetes in mice expressing the LCMV glycoprotein on β-islet cells (RIP-GP mice), hyperglycemia does not occur after infection with higher doses of LCMV. High-dose LCMV infection induced an impaired CD8+ T cell response, which coincided with increased NK cell activity during early time points following infection. Notably, we observed increased NKp46 expression on NK cells during infection with higher doses, which resulted in an NK cell dependent suppression of T cells. Accordingly, depletion with antibodies specific for NK1.1 as well as NKp46 deficiency (Ncr1gfp/gfp mice) could restore CD8+ T cell immunity and permitted the induction of diabetes even following infection of RIP-GP mice with high-dose LCMV. Therefore, we identify conditions where innate lymphoid cells can play a regulatory role and interfere with CD8+ T cell mediated tissue specific pathology using an NKp46 dependent mechanism.

Reactivation of Epstein-Barr Virus Hepatitis in T/Natural Killer (NK) Cells Mimicking Liver T/NK-Cell Lymphoma

Diagnosis of Epstein-Barr virus (EBV)-associated hepatitis, chronic active EBV infection, and EBV-associated lymphoproliferative diseases, is always challenging due to the overlapping symptoms and lack of diagnostic criteria. We report such a case of a 40-year-old man with unremarkable past medical history. He presented with fever of unknown origin for 1 month with jaundice for 2 days. Physical exams were unremarkable with body temperature at 98.6 °F. His liver function tests were elevated with alanine transaminase (ALT) 559 U/L, aspartate transaminase (AST) 892 U/L, alkaline phosphatase 319 U/L and total bilirubin 4.4 mg/dL. Computed tomography of his chest, abdomen and pelvis did not show lymphadenopathy or hepatosplenomegaly. A liver biopsy showed moderately acute hepatitis with hemophagocytosis, positive Epstein-Barr virus encoding RNA (EBER) in situ hybridization in CD3 and CD4-positive T cells and CD56-positive natural killer (NK) cells. CD20 was negative. The pathology diagnosis was consistent with reactivation of EBV hepatitis but NK-cell lymphoma needs to be excluded. Steatohepatitis with mild activity was present. His blood EBV DNA was 846,000 copies/mL and continued to increase to 2,000,000 copies/mL. Flow cytometric analysis of his bone marrow revealed an increased NK-cell activity but no T/NK-cell lymphoma was identified. Initial treatment with rituxan, etoposide and/or ruxolitinib/acyclovir failed or only had limited effect. However, subsequent valganciclovir greatly improved his conditions. In his 3 months follow-up, the patient was doing well with almost normal liver function tests except mildly elevated ALT (95 U/L) that was due to mild steatohepatitis. EBV DNA PCR was 2,009 copies/mL. To the best of our knowledge, this is the first documented case with reactivation of EBV hepatitis mimicking NK-cell lymphoma in the English literature. With appropriate anti-EBV viral treatments, the patient eventually became asymptomatic and was able to return to his routine life.

A Gene Signature Predicting Natural Killer Cell Infiltration and Improved Survival in Melanoma Patients.

Natural killer (NK) cell activity is essential for initiating antitumor responses and may be linked to immunotherapy success. NK cells and other innate immune components could be exploitable for cancer treatment, which drives the need for tools and methods that identify therapeutic avenues. Here, we extend our gene-set scoring method singscore to investigate NK cell infiltration by applying RNA-seq analysis to samples from bulk tumors. Computational methods have been developed for the deconvolution of immune cell types within solid tumors. We have taken the NK cell gene signatures from several such tools, then curated the gene list using a comparative analysis of tumors and immune cell types. Using a gene-set scoring method to investigate RNA-seq data from The Cancer Genome Atlas (TCGA), we show that patients with metastatic cutaneous melanoma have an improved survival rate if their tumor shows evidence of NK cell infiltration. Furthermore, these survival effects are enhanced in tumors that show higher expression of genes that encode NK cell stimuli such as the cytokine IL15 Using this signature, we then examine transcriptomic data to identify tumor and stromal components that may influence the penetrance of NK cells into solid tumors. Our results provide evidence that NK cells play a role in the regulation of human tumors and highlight potential survival effects associated with increased NK cell activity. Our computational analysis identifies putative gene targets that may be of therapeutic value for boosting NK cell antitumor immunity.