Natural Killer Activity Research Articles

Response of CD68-Positive Thymic Cells in Rats to Selenium and Carcinogen Administration

Macrophages are the key thymic cells that take part in the antigen-independent differentiation of T-lymphocytes and their further selection. The level of macrophages indicates changes in the functions of the organ under study. As an essential trace element, selenium is part of a variety of proteins and enzymes, which perform cytoprotective, antimutagenic, and anticancerogenic action. Selenium also plays a significant role in the functioning of the immune system, increasing the activity of natural killers, production of interleukins, and stimulation of phagocytosis. This study was aimed at investigating the response of thymic macrophages to the administration of selenium and a chemical carcinogen. During the experiment, an increase in the level of CD68-positive cells was revealed in all experimental animals receiving selenium and a carcinogen, as well as under their combined action. The observed effects were assumed to be related to the immune-stimulating effect of selenium.

Non-Classical HLA Class 1b and Hepatocellular Carcinoma.

A number of studies are underway to gain a better understanding of the role of immunity in the pathogenesis of hepatocellular carcinoma and to identify subgroups of individuals who may benefit the most from systemic therapy according to the etiology of their tumor. Human leukocyte antigens play a key role in antigen presentation to T cells. This is fundamental to the host's defense against pathogens and tumor cells. In addition, HLA-specific interactions with innate lymphoid cell receptors, such those present on natural killer cells and innate lymphoid cell type 2, have been shown to be important activators of immune function in the context of several liver diseases. More recent studies have highlighted the key role of members of the non-classical HLA-Ib and the transcript adjacent to the HLA-F locus, FAT10, in hepatocarcinoma. The present review analyzes the major contribution of these molecules to hepatic viral infection and hepatocellular prognosis. Particular attention has been paid to the association of natural killer and Vδ2 T-cell activation, mediated by specific HLA class Ib molecules, with risk assessment and novel treatment strategies to improve immunotherapy in HCC.

An efficient feeder-free and chemically-defined expansion strategy for highly purified natural killer cells derived from human cord blood

IntroductionNatural killer cells (NKCs) are immune cells that can attack cancer cells through the direct recognition of ligands without prior sensitization. Cord blood-derived NKCs (CBNKCs) represent a promising tool for allogenic NKC-based cancer immunotherapy. Efficient NKC expansion and decreased T cell inclusion are crucial for the success of allogeneic NKC-based immunotherapy without inducing graft-versus-host reactions. We previously established an efficient ex vivo expansion system consisting of highly purified-NKCs derived from human peripheral blood. Herein, we evaluated the performance of the NKC expansion system using CB and characterized the expanded populations. MethodsFrozen CB mononuclear cells (CBMCs), with T cells removed, were cultured with recombinant human interleukin (rhIL)-18 and rhIL-2 under conditions where anti-NKp46 and anti-CD16 antibodies were immobilized. Following 7, 14, and 21 days of expansion, the purity, fold-expansion rates of NKCs, and the expression levels of NK activating and inhibitory receptors were assessed. The ability of these NKCs to inhibit the growth of T98G, a glioblastoma (GBM) cell line sensitive to NK activity, was also examined. ResultsAll expanded T cell-depleted CBMCs were included in over 80%, 98%, and 99% of CD3−CD56+ NKCs at 7, 14, and 21 days of expansion, respectively. The NK activating receptors LFA-1, NKG2D, DNAM-1, NKp30, NKp44, NKp46, FcγRIII and NK inhibitory receptors TIM-3, TIGIT, TACTILE, NKG2A were expressed on the expanded-CBNKCs. Two out of three of the expanded-CBNKCs weakly expressed PD-1, yet gradually expressed PD-1 according to expansion period. One of the three expanded CBNKCs almost lacked PD-1 expression during the expansion period. LAG-3 expression was variable among donors, and no consistent changes were identified during the expansion period. All of the expanded CBNKCs elicited distinct cytotoxicity-mediated growth inhibition on T98G cells. The level of cytotoxicity was gradually decreased based on the prolonged expansion period. ConclusionsOur established feeder-free expansion system yielded large scale highly purified and cytotoxic NKCs derived from human CB. The system provides a stable supply of clinical grade off-the-shelf NKCs and may be feasible for allogeneic NKC-based immunotherapy for cancers, including GBM.

IMPORT-201 (IMP-MEL): A phase 1 first-in-human dose finding/randomized phase 2 study of a novel iNKT agonist IMM60 and pembrolizumab for advanced melanoma and metastatic non-small cell lung cancer (NSCLC).

2575 Background: Invariant natural killer T-cells (iNKTs) share features of NK-like cells and T-cells, playing a role in both innate and adaptive immune responses. The importance of this relatively rare lymphocyte subset has generated interest due to its dual ability to have a direct cytotoxic effect on CD1d expressing tumors as well as to induce long-lasting antitumor CD8 T cell responses mediated by cross-priming and licensing of dendritic cells. While many current clinical approaches involve the use of allogeneic iNKT cells, here we describe initial clinical studies with IMM60, a synthetically derived agonist of iNKT cells formulated in a liposome (PORT-2). In preclinical studies, IMM60 treatment results in maturation of DCs and B cells and a potent stimulation of iNKT cell-derived IFN-g. In murine efficacy studies, IMM60 demonstrated monotherapy activity in multiple PD-1 resistant models (e.g., B16-F10), as well as upregulation of PD-L1 expression on cancer cells and re-sensitization to PD-1 inhibition. Methods: The IMPORT-201 trial is a multicenter, international expansion of an open-label first-in-human phase I/II investigator initiated study (IMP-MEL). Ph 1 is currently enrolling patients with NSCLC or melanoma. IMM60-containing liposomes are administered IV Q3W at 3 escalating dose levels for 6 doses as monotherapy or with pembrolizumab 200mg Q3W. The Ph I portion of the study seeks to establish the recommended Ph 2 dose of IMM60 alone and in combination with pembrolizumab. The Ph 2 evaluates IMM60 monotherapy, PD-1 monotherapy, and the combination. Results: Eight patients with melanoma (n=4) or NSCLC (n=4) have been enrolled in the IMM60 monotherapy dose cohorts. These patients were heavily pretreated, having a median of 3.5 prior therapies (range 2-7). One patient with NSCLC has been enrolled to the IMM60 + pembrolizumab combination cohort. IMM60 was well tolerated with no treatment-related SAEs or G3-5 AEs, and no MTD was determined. The most common related AE is G1-2 fatigue, reported in 2 patients. In the monotherapy cohort, a preliminary review of individual target lesions showed 2 lesions that completely resolved, 1 lesion with 69% decrease in size, 10 lesions that were stable, and 6 lesions with >20% increase in size. Serum biomarker analysis demonstrated evidence of iNKT and NK activation, as well as increases in dendritic and CD86+ B cells. Pharmacokinetics demonstrate dose proportionality with a terminal half-life of ~8 hours. Conclusions: IMM60 is well tolerated as monotherapy and in combination with pembrolizumab at the doses tested. These data show that a single agent iNKT agonist can be safely administered in a heavily pre-treated population and initiate immune activation. The combination with pembrolizumab is ongoing, and updated results of the Ph 1 portion of the study will be reported. Clinical trial information: NCT05709821 .

Recombinant hemagglutinin displaying on yeast reshapes congenital lymphocyte subsets to prompt optimized systemic immune protection against avian influenza infection.

Prophylactic vaccination is regarded as the most effective means to control avian flu infection. Currently, there is a need for a universal vaccine that provides broad and long-lasting protection against influenza virus. Meanwhile, although yeast-based vaccines have been used in clinic, studies are still required to further understand the molecular mechanism of yeast-based vaccines under physiological conditions. We generated a yeast-based vaccine against influenza hemagglutinin (HA) of H5, H7 and H9 using surface displaying technology and evaluated the protective efficacy of chickens after exposure to H9N2 influenza virus. Oral yeast vaccine provided less clinical syndrome, reduced viral loading and alleviated airway damage significantly. Compared to the commercial inactivated vaccine, yeast vaccine stimulated the activation of splenic NK and APCs cells and boosted TLR7-IRF7-IFN signaling in spleen. Meanwhile, γδ T cells in the bursa of Fabricius were activated and the innate lymphoid cells (ILCs) in the bursa of Fabricius promoted the CILPs to differentiate to ILC3 cells in oral yeast birds. Moreover, the reshaped gut microbiota and a suppressed Th17-IL17-mediated inflammation in intestine was observed in oral yeast chickens, which might facilitate the recovery of intestinal mucosal immunity upon virus infection. Collectively, our findings suggest that oral yeast based multivalent bird flu vaccines provide an attractive strategy to update host defense function via reshapes of multi-systemic immune homeostasis.

Adenovirotherapy delivering cross-hybrid IgGA Fc engineering PD-L1 inhibitors for enhanced cancer immunotherapy

Adenovirotherapy delivering cross-hybrid IgGA Fc engineering PD-L1 inhibitors for enhanced cancer immunotherapy

Calreticulin exposure orchestrates innate immunosurveillance

Calreticulin (CALR) exposure on the cell surface is known to deliver robust pro-phagocytic signals to myeloid cells. In Nature, Sen Santara etal. demonstrate that surface-exposed CALR also operates as an endogenous activator of natural killer (NK) cells. Collectively, these findings suggest that CALR exposure orchestrates multiple facets of innate immunosurveillance.

Early activation and recruitment of invariant natural killer T cells during liver ischemia-reperfusion: the major role of the alarmin interleukin-33.

Over the past thirty years, the complexity of the αβ-T cell compartment has been enriched by the identification of innate-like T cells (ITCs), which are composed mainly of invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. Based on animal studies using ischemia-reperfusion (IR) models, a key role has been attributed to iNKT cells in close connection with the alarmin/cytokine interleukin (IL)-33, as early sensors of cell-stress in the initiation of acute sterile inflammation. Here we have investigated whether the new concept of a biological axis of circulating iNKT cells and IL-33 applies to humans, and may be extended to other ITC subsets, namely MAIT and γδ-T cells, in the acute sterile inflammation sequence occurring during liver transplant (LT). From a prospective biological collection of recipients, we reported that LT was accompanied by an early and preferential activation of iNKT cells, as attested by almost 40% of cells having acquired the expression of CD69 at the end of LT (i.e. 1-3 hours after portal reperfusion), as opposed to only 3-4% of conventional T cells. Early activation of iNKT cells was positively correlated with the systemic release of the alarmin IL-33 at graft reperfusion. Moreover, in a mouse model of hepatic IR, iNKT cells were activated in the periphery (spleen), and recruited in the liver in WT mice, as early as the first hour after reperfusion, whereas this phenomenon was virtually missing in IL-33-deficient mice. Although to a lesser degree than iNKT cells, MAIT and γδ-T cells also seemed targeted during LT, as attested by 30% and 10% of them acquiring CD69 expression, respectively. Like iNKT cells, and in clear contrast to γδ-T cells, activation of MAIT cells during LT was closely associated with both release of IL-33 immediately after graft reperfusion and severity of liver dysfunction occurring during the first three post-operative days. All in all, this study identifies iNKT and MAIT cells in connection with IL-33 as new key cellular factors and mechanisms of acute sterile inflammation in humans. Further investigations are required to confirm the implication of MAIT and iNKT cell subsets, and to precisely assess their functions, in the clinical course of sterile inflammation accompanying LT.

Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T cells.

Invariant natural killer T (iNKT) cells act at the interface between lipid metabolism and immunity because of their restriction to lipid antigens presented on CD1d by antigen-presenting cells (APCs). How foreign lipid antigens are delivered to APCs remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time to our knowledge, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), isoglobotrihexosylceramide, and OCH, a sphingosine-truncated analog of αGalCer, with VLDL and/or LDL in vitro and in vivo. We demonstrate LDL receptor-mediated (LDLR-mediated) uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a potentially novel mechanism of lipid antigen delivery to APCs and provides further insight into the immunological capacities of circulating lipoproteins.

Inhibition of Ly-49/LILRB-MHC-I interactions by anti-MHC-I augments innate and adaptive immunity.

Abstract Mouse NK cells recognize MHC-I antigens via stochastically expressed inhibitory Ly49 receptors. The loss of MHC-I expression on tumor cells (“missing self”) abrogates inhibitory signals, resulting in NK activation. We have identified an anti-mouse pan MHC-I mAb (M1/42), which blocks Ly49-MHC-I interactions but not TCR-MHC-I interactions. Administration of M1/42 in vivo markedly activated IFNg-producing NK cells that further drove the proliferation of NK cells and memory phenotype (MP) T cells and profoundly augmented adaptive immunity against viral infection and cancer metastasis. In contrast to mouse Ly-49 antigens, human killer cell inhibitory receptors bind to HLA at a site overlapping the TCR binding site and blocking of KIR binding to MHC-I would inhibit TCR recognition. However, two anti-pan-human MHC-I abs (DX17 and W6/32) markedly induced NK cell proliferation and IFNg production in cultures of human PBMC. DX17 potently blocked the interaction of leukocyte Ig-like inhibitory receptors LILRB1, B2, B3 and B5 with human MHC-I but had no effect on TCR-MHC-I interactions. The crystal structure of a DX17 Fab/MHC-I complex reveals that the footprint of DX17 overlaps the LILRB1 binding site on MHC-I. Administration of W6/32 Fab to humanized mice (PBMC reconstituted NSG or NK cell reconstituted NOG-IL-15) induced human NK and MP T cell proliferation. Marked reduction of tumor size was seen when NOG-hIL-15Tg mice were reconstituted with CD3 −cells and then treated with W6/32 Fab. DX17 Fc silenced mAb (DX17 LALAPG) activated human tumor infiltrating lymphocytes and monocytes. These results strongly suggest that inhibition of LILRB-MHC-I interactions by anti-MHC-I in humans may result in marked augmentation of anti-tumor immunity. This work was supported by the Intramural Research Program of NIAID, NIH.

NK-92-CD16A cell externalization of CD107a for detection of antigen-specific antibodies able to support ADCC: sensitive discrimination of antibody a-fucosylation

Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes kills cells infected with enveloped viruses. Antiviral ADCC has three components: antigen-specific IgG1 or IgG3; effector cells with CD16A that recognize the Fc of IgG; and antigenic proteins in the plasma membranes of infected cells. Afucosylated antibodies support ADCC better than fucosylated antibodies; afucosylated fractions vary among individuals. Current assays that detect afucosylated antibodies to specific viruses involve antigen-specific antibody purification and mass spectrometry. We are developing clinical bioassays to detect antibodies that will support ADCC and assess their extents of afucosylation. Our pilot assay uses externalization of CD107a by NK-92-CD16A cells, this’ occurs when NK cells recognize cell-bound antibodies. We used two humanized monoclonal antibodies on CD20, GA101 WT or glycoengineered GE, 10% or ~50% afucosylated, with Raji cells. Cellular CD107a externalization was more sensitive than AF-647 donkey anti-IgG to detect total cell-bound antibody. The effective concentrations (EC50s) of the two antibodies for CD107a externalization differed by >15-fold. The more afucosylated GA101-GE was more detectable, at 0.2 ng/ml (=/< 700 Abs/Raji cell). CD107a externalization increased with excess antibody-bearing cells. Short 37 °C incubation (40 minutes) optimized detection of ADCC over intrinsic NK activity. Controls of GA101-GE alone and 56 °C heated human serum lacked effects on CD107a externalization. The assay appears compatible with formaldehyde treatment before NK cell staining. This sensitive bioassay appears suitable for assess serum samples for antiviral antibodies that will support ADCC. UNR Foundation C.E. Hudig Fund.

Immune-boosting Effects of Chrysanthemum zawadskii Herbich var. latilobum Kitamura Extract on Cyclophosphamide-induced Immunosuppressed Sprague-Dawley Animal Model

Background: Chrysanthemum zawadskii Herbich var. latilobum Kitamura extracts (CZE) is used to treat of various inflammatory and chronic diseases. Although commonly adminstered, no reports suggest its potential usage as an immune-booster.BRMethods and Results: In the cyclophosphamide (CP, 5 ㎎/㎏)-induced immunosuppression model, the immune-boosting effect of CZE (oral administration for 2 weeks with CP) was evaluated by investigating cell proliferation, activity of natural killer (NK) cells, cytotoxic T lymphocytes, and production of CP-repressed cytokines [such as tumor necrosis factor-α, interferon-γ, interluekin (IL)-2, and IL-12] in isolated splenocytes. In Vitro, CZE treatment enhanced cell proliferation, activity of NK cells and cytotoxic T lymphocytes activity, and production of CP-repressed cytokines. In Vivo, CZE treatment promoted the production of white blood cells, lymphocytes, medium-sized cells, and granulocytes and proliferation of NK cells in CP-induced immunosuppressed mice. Additionally, the CZE treatment restored the TNF-α and IL-12 reduced by CP to normal levels and prevented spleen tissue damage.BRConclusions: CZE could be an effective immune-booster and potentially be added to functional foods to enhance immunity.

Evaluation of Euphorbia tirucalli L. Immunomodulatory and Antitumor Activity

Euphorbia tirucalli L. is commonly used to treat various pathologies, including cancer. The objective of this study was to evaluate the effect of a crude extract of E. tirucalli on the development of Ehrlich solid tumors and immune function. We prepared an extract by macerating the aerial parts of the plant with 80% ethanol (1:10 g/v) for 15 days. Its effects on tumor development were assessed in male Swiss mice (n=10). The mice were injected subcutaneously with 10⁶ tumor cells and then treated by gavage daily with the extract (33.33, 67, and 133.34 mg kg-1) or saline (0.5% ethanol) for 30 days. The treatment had no toxic effect and did not reduce tumor growth. However, the weight of tumor mass was lowest in the mice treated with extract at 67 mg kg-1. Immunomodulatory activity was evaluated in mice (n=8), with and without tumors, treated with the extract (67 mg kg-1), under the same conditions as described above for 7 and 14 days. Lymphoproliferation, NK activity and IL-12, TNF-a, IFN-y, and IL-10 levels did not differ significantly between the groups. However, IL-4 levels were reduced in the E. tirucalli treated groups at 7 and 14 days when compared to the controls. We concluded that the extract is not toxic and cannot inhibit Ehrlich solid tumor development. Its immunomodulatory activity involves its ability to modulate IL-4 levels.

Vitamin A deficiency and vitamin A supplementation affect innate and T cell immune responses to rotavirus A infection in a conventional sow model.

Rotavirus A (RVA) causes ~200,000 diarrheal deaths annually in children <5yrs, mostly in low- and middle-income countries. Risk factors include nutritional status, social factors, breastfeeding status, and immunodeficiency. We evaluated the effects of vitamin A (VA) deficiency/VA supplementation and RVA exposure (anamnestic) on innate and T cell immune responses in RVA seropositive pregnant and lactating sows and passive protection of their piglets post-RVA challenge. Sows were fed VA deficient (VAD) or sufficient (VAS) diets starting at gestation day (GD)30. A subset of VAD sows received VA supplementation from GD|76 (30,000IU/day, VAD+VA). Sows (6 groups) were inoculated with porcine RVA G5P[7] (OSU strain) or Minimal Essential Medium (mock) at GD~90: VAD+RVA; VAS+RVA; VAD+VA+RVA; VAD-mock; VAS-mock; and VAD+VA-mock. Blood, milk, and gut-associated tissues were collected from sows at several time points to examine innate [natural killer (NK), dendritic (DC) cells], T cell responses and changes in genes involved in the gut-mammary gland (MG)-immunological axis trafficking. Clinical signs of RVA were evaluated post inoculation of sows and post-challenge of piglets. We observed decreased frequencies of NK cells, total and MHCII+ plasmacytoid DCs, conventional DCs, CD103+ DCs and CD4+/CD8+ and T regulatory cells (Tregs) and NK cell activity in VAD+RVA sows. Polymeric Ig receptor and retinoic acid receptor alpha (RARα) genes were downregulated in mesenteric lymph nodes and ileum of VAD+RVA sows. Interestingly, RVA-specific IFN-γ producing CD4+/CD8+ T cells were increased in VAD-Mock sows, coinciding with increased IL-22 suggesting inflammation in these sows. VA supplementation to VAD+RVA sows restored frequencies of NK cells and pDCs, and NK activity, but not tissue cDCs and blood Tregs. In conclusion, similar to our recent observations of decreased B cell responses in VAD sows that led to decreased passive immune protection of their piglets, VAD impaired innate and T cell responses in sows, while VA supplementation to VAD sows restored some, but not all responses. Our data reiterate the importance of maintaining adequate VA levels and RVA immunization in pregnant and lactating mothers to achieve optimal immune responses, efficient function of the gut-MG-immune cell-axis and to improve passive protection of their piglets.

Abstract LB220: S095029: a novel clinical-stage Fc-silenced NKG2A-blocking antibody with best-in-class potential

Abstract NKG2A and its ligand HLA-E are increasingly recognized as an important immune checkpoint restraining multiple lymphocyte populations involved in cancer immunity. Initially described as an NK cell inhibitory receptor, NKG2A’s role in regulating T-cell functions has recently come to the fore and is increasingly viewed as a key component of its immune suppressive functions. In addition, clinical studies have also provided proof-of-concept evidence for the superiority of dual NKG2A/PD-L1 blockade over anti-PD-L1 monotherapy. As of now clinical safety and efficacy results are only available for a single NKG2A-blocking antibody. This is in contrast with other immune checkpoint receptors (e.g., PD-1) where multiple blocking agents with different pharmacological properties have been assessed in the clinic, thereby providing a broad assessment of the target therapeutic potential and benefit to patients. Here, we report the generation and preclinical characterization of a novel monoclonal antibody (mAb) disrupting the NKG2A/HLA-E interaction. The Fc-attenuated IgG1 mAb S095029 binds NKG2A with a nM-range affinity and reverses the inhibitory effects of the NKG2A/HLA-E interaction in several experimental models, both in vitro and in vivo. As a single agent S095029 enhanced the killing activity and cytokine secretion of NK and γδ T-cells in co-culture with cancer cell lines of multiple tissue origins. We also show that S095029 increased the antibody-dependent cellular cytotoxicity (ADCC) mediated by Fc-competent mAbs in different antigen systems with high HLA-E expression. Of note, these immune-activating properties of S095029 compared favorably in benchmarking experiments with other clinical-stage anti-NKG2A mAbs. Finally, S095029 combination with PD-1 blocking agents, with or without an additional ADCC mAb component, conferred superior anti-tumor activity compared to treatments without NKG2A blockade. Overall, our in vitro and in vivo data support the clinical development of S095029 in tumor settings with adequate HLA-E expression and suggest potential differentiating characteristics compared to other anti-NKG2A mAbs. S095029 is currently being evaluated in phase 1 dose escalation studies as a single agent or in combination with anti-PD-1 therapy (NCT05162755). Citation Format: Maria Melander, Bruno Laugel. S095029: a novel clinical-stage Fc-silenced NKG2A-blocking antibody with best-in-class potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB220.

Abstract LB337: Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells

Abstract The E3 ubiquitin ligase CBL-B is a master regulator of effector T cells through its downstream regulation of T-cell receptor and co-stimulatory stimulation, but it has also been shown to play a negative feedback role in NK cells. CBL-B depletion enhances the production of IFNγ and the cytotoxicity of activated NK cells. Similar to the effect of depleting CD8 T cells in mice, depletion of NK cells abolishes the tumor growth delay observed in Cbl-b-/- mice. With emerging data supporting the potential role of exogenous NK cells in anti-tumor immunity, inhibition of CBL-B represents a promising approach to enhance both adaptive and innate immunity for cancer immunotherapy. Using one of our small molecules, allosteric CBL-B inhibitors, HOT-A, we demonstrated that CBL-B inhibition enhanced NK activation, IFNγ production and granzyme B secretion in response to the various stimulation conditions, including cytokines (such as IL-15 and IL12 + IL-18) and stimulatory receptors (such as NKp30 and NKG2D + CD244). NK cell proliferation was also enhanced in the presence of HOT-A. Single cell analysis further suggested that HOT-A increased NK polyfunctional cytokine expression during IL-12 + IL-18 activation. In a coculture system of NK cells and K562 target cells, HOT-A dose-dependently promoted primary human NK cell activation, cytokine production and cytotoxicity in the K562 cells. In the in vivo CT-26 tumor model, granzyme B+ NK cells in the TIL were significantly increased in the HOT-A treated tumor bearing mice and NK function gene signature was also increased by Nanostring analysis, suggesting an enhanced NK function in the tumor microenvironment. Taken together, the pre-clinical data presented here demonstrate that our CBL-B inhibitor enhances NK cell function in vitro and in vivo, suggesting that its enhancement of anti-tumor immunity may be driven through both effector T cell and NK cell-driven biology. Citation Format: Yilin Qi, Jun Kuai, Yingzhi Bi, David Greco, Samira Jaeger, Ken Carson, Hadi Danaee, Timothy Reilly, Geraldine Harriman, Fang Wang. Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB337.

Immune profiling of SARS-CoV-2 infection during pregnancy reveals NK cell and γδ T cell perturbations.

Pregnancy poses a greater risk for severe COVID-19; however, underlying immunological changes associated with SARS-CoV-2 during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in unvaccinated pregnant and nonpregnant women with acute and convalescent COVID-19, quantifying 217 immunological parameters. Humoral responses to SARS-CoV-2 were similar in pregnant and nonpregnant women, although our systems serology approach revealed distinct antibody and FcγR profiles between pregnant and nonpregnant women. Cellular analyses demonstrated marked differences in NK cell and unconventional T cell activation dynamics in pregnant women. Healthy pregnant women displayed preactivated NK cells and γδ T cells when compared with healthy nonpregnant women, which remained unchanged during acute and convalescent COVID-19. Conversely, nonpregnant women had prototypical activation of NK and γδ T cells. Activation of CD4+ and CD8+ T cells and T follicular helper cells was similar in SARS-CoV-2-infected pregnant and nonpregnant women, while antibody-secreting B cells were increased in pregnant women during acute COVID-19. Elevated levels of IL-8, IL-10, and IL-18 were found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, we demonstrate perturbations in NK cell and γδ T cell activation in unvaccinated pregnant women with COVID-19, which may impact disease progression and severity during pregnancy.

Engineering a HER2-CAR-NK Cell Secreting Soluble Programmed Cell Death Protein with Superior Antitumor Efficacy

A new therapy strategy for relapsing patients who have received trastuzumab treatment urgently needs to be explored. HER2-specific chimeric antigen receptor (CAR)-expressing NK cells are being rapidly developed for solid tumor therapy, as they have many advantages over HER2-CAR-T cells. Endogenous soluble PD-1 (sPD-1) from the PD-1 extracellular domain blocks PD-1/PD-L1 interaction to promote cancer immunology. Herein, we engineered a new HER2-CAR-NK cell that co-expresses sPD-1 (designed as sPD-1-CAR-NK cells) and assessed its cytotoxic activities toward various cancer cells, activation of immunity and sPD-1 release in vitro and in mouse models bearing breast cancer cells with high HER2 expression, with or without trastuzumab resistance. We demonstrated that sPD-1-CAR-NK cells were able to release bioactive sPD-1, thereby enhancing the cytolytic activities of HER2-CAR-NK cells against HER2 and PD-L1 highly expressing target cells accompanied by increases in the secretion of perforin, granzyme B and IFN-γ. In vivo, sPD-1-CAR-NK cells had superior immunological anticancer efficacy compared to HER2-CAR-NK cells, and they had advantages over HER2-CAR-NK cells in the intraperitoneal injection of sPD-1. Moreover, the infiltration and activation of NK and T cells into tumor tissue were increased in mice with sPD-1-CAR-NK cells. There was no significant change in the body temperature, organ tissue and body weight in all groups except for the group with the PD-1 injection. Together, these data indicate that HER2-specific sPD-1-CAR-NK cells can transport sPD-1 into cancer tissues with high HER2 expression, further improving the efficacy of HER-CAR-NK cells without obvious side effects. sPD-1-CAR-NK is a promising cytotherapeutic agent for patients bearing HER2-positive breast cancer, including those with trastuzumab resistance.

Elevated exosome-transferrable lncRNA EPB41L4A-AS1 in CD56bright NK cells is responsible for the impaired NK function in neuroblastoma patients by suppressing cell glycolysis

NK cells are one of key immune components in neuroblastoma (NB) surveillance and eradication. Glucose metabolism as a major source of fuel for NK activation is exquisitely regulated. Our data revealed a diminished NK activation and a disproportionally augmented CD56bright subset in NB. Further study showed that NK cells in NB presented with an arrested glycolysis accompanied by an elevated expression of the long noncoding RNA (lncRNA) EPB41L4A-AS1, a known crucial participant in glycolysis regulation, in the CD56bright NK subset. The inhibitory function of lncRNA EPB41L4A-AS1 was recapitulated. Interestingly, our study demonstrated that exosomal lncRNA EPB41L4A-AS1 was transferrable from CD56bright NK to CD56dim NK and was able to quench the glycolysis of target NK. Our data demonstrated that an arrested glycolysis in patient NK cells was associated with an elevated lncRNA in CD56bright NK subset and a cross-talk between heterogeneous NK subsets was achieved by transferring metabolic inhibitory lncRNA through exosomes.

Abstract 3970: NCG-hIL15 humanized mice: an ideal model for human immune reconstitution of NK cells

Abstract Introduction: IL-15 is a four α-helix bundle cytokine which is produced by dendritic cells, monocytes, and epithelial cells. IL-15 is necessary for the development, survival, and activation of natural killer (NK) cells. NK cells are unable to persist in immunodeficient NCG mice reconsituted with human NK cells, but after knocking in human IL-15 (hIL-15), NCG-hIL15 mice can support the reconstitution and survival of human NK cells. Results: Compared with NCG mice, the level of hIL-15 was significantly increased in NCG-hIL15 mice. After transplanting human PBMC (huPBMC) or NK (huNK) cells into NCG-hIL15 mice, the peripheral blood level of NK cells in huNK-NCG-hIL15 mice was much higher than in huPBMC-NCG-hIL15 mice. The expression of perforin in peripheral blood of huNK-NCG-hIL15 mice was significantly higher than that in huPBMC-NCG-hIL15 mice. Considering the obvious ADCC of rituximab acting on Raji cells in vitro, we evaluated the efficacy of rituximab in huNK-NCG-hIL15 mice subcutaneously engrafted with Raji cells. Rituximab significantly inhibited the tumor growth in huNK-NCG-hIL15 mice. Conclusion: NCG-hIL15 is a great mouse model to evaluate the anti-tumor efficacy of drugs targeting human NK cells without the interference of T cells. Citation Format: Xing Liu, Meirong Wu, Huiyi Wang, Weiwei Yu, Jianming Xu, Hongyan Sun, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. NCG-hIL15 humanized mice: an ideal model for human immune reconstitution of NK 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 3970.