Augmented Natural Killer Research Articles

R848/TLR7-Mediated Stronger CD8+ T Immunity Is Dependent on DC-NK Cell Interactions

Background: Toll-like receptor (TLR) 7 agonists are effective candidates for Th1 immune adjuvants, which compensate for the insufficient Th1 immune responses induced by traditional adjuvants. This effect is currently dependent on TLR7-mediated induction of dendritic cell (DC) maturation and increased IL-12 production. Methods: In vivo, we intraperitoneally injected TLR agonists with OVA, and LNs were collected for detection. In vitro, Activated DCs, natural killer (NK) cells, and CD8⁺ T cells were tested using flow cytometry for surface expression and enzyme-linked immunosorbent assay for cytokine production. NK cell migration was evaluated using transwell system. All experiments were performed in both C57BL/6 and BALB/C backgrounds. Results: Our findings revealed that the enhanced CD8⁺ T immunity characterized by CD8⁺ T accumulation, proliferation, and IFN-γ⁺CD8⁺ T induction induced by R848 was attributed to DC-dependent NK cell migration and DC-NK interactions. Our results demonstrated that R848 induced CD8⁺ T cell accumulation and IFN-γ⁺CD8⁺ T cells in lymph nodes (LNs) to a greater degree in vivo than TLR4 agonists (lipopolysaccharide) and TLR9 agonists (Class C CPG). R848-activated DCs enhanced CD8⁺ T cell proliferation and increased IFN-γ⁺CD8⁺ T cells with the assistance of NK cells. In contrast, depletion of NK cell decreased IFN-γ⁺CD8⁺ T cell production. Greater NK cell migration to LNs occurred in R848-immunized mice. A similar effect of R848 on NK cell migration was observed in an in vitro transwell study. When co-cultured, NK cells plus R848 could promote DCs maturation, and in turn, DCs in combination of R848 augmented NK cells activation. Further studies demonstrated that among several TLR agonists, R848 produced the largest amount of the chemokine CXCL9 from activated DCs, which is relevant to NK cell migration. CXCL9 blockade reduced the number of migrated NK cells, and the addition of CXCL9 increased the number of NK cells. Discussion: Taken together, R848-mediated stronger CD8⁺ T cell immunity does not depend on DC activation alone, rather that NK cells must also be considered. By increasing our immunological understanding of the effect of R848/TLR7, these findings provide a new perspective for applying R848 in future clinical studies.

Nicotinamide mononucleotide augments the cytotoxic activity of natural killer cells in young and elderly mice.

Nicotinamide mononucleotide (NMN), a key nicotinamide adenine dinucleotide (NAD+) intermediate, has been shown to ameliorate various pathologies in elderly mouse disease models. Natural killer (NK) cells are important innate immune cells; however, their functions decline with aging. In this study, we examined the effect of NMN treatment on NK cells in mice. Intraperitoneal administration of NMN augmented NK cell cytotoxic activity in both young and elderly B6 mice as well as young BALB/c mice. Oral administration of NMN also increased NK cell cytotoxicity in elderly B6 and BALB/c mice. However, the NK cell population was not increased in the mice whose NK cell cytotoxic activity was activated by NMN. Interestingly, NMN administration did not augment NK cell cytotoxic activity in IFN-γ deficient mice. These results suggest that NMN administration augments NK cell cytotoxic activity, but not cell number, in a manner dependent on IFN-γ in both young and elderly mice.

Highly Efficient Generation of Transgenically Augmented CAR NK Cells Overexpressing CXCR4.

Natural killer (NK) cells are a noteworthy lymphocyte subset in cancer adoptive cell therapy. NK cells initiate innate immune responses against infections and malignancies with natural cytotoxicity, which is independent of foreign antigen recognition. Based on these substantive features, genetically modifying NK cells is among the prime goals in immunotherapy but is currently difficult to achieve. Recently, we reported a fully human CAR19 construct (huCAR19) with remarkable function in gene-modified T-cells. Here, we show efficient and stable gene delivery of huCAR19 to primary human NK cells using lentiviral vectors with transduction efficiencies comparable to those achieved with NK cell lines. These huCAR19 NK cells display specific and potent cytotoxic activity against target cells. To improve homing of NK cells to the bone marrow, we augmented huCAR19 NK cells with the human CXCR4 gene, resulting in transgenically augmented CAR NK cells (TRACKs). Compared to conventional CAR NK cells, TRACKs exhibit enhanced migration capacity in response to recombinant SDF-1 or bone marrow stromal cells while retaining functional and cytolytic activity against target cells. Based on these promising findings, TRACKs may become a novel candidate for immunotherapeutic strategies in clinical applications.

Abstract 7: Gross thymic extract inhibits tumor growth by modulating the immune response in mice bearing Ehrlich ascites carcinoma cells

Abstract Purpose. Gross thymic extract (GTE) obtained from pathogen-free pigs, a product also known as Thymax has previously been shown to boost various parameters of the immune system in aged mice, as well as activating human dentritic cells and the DC-directed T-cell response in vitro. The current study examines the immunomodulatory and chemopreventive effects of GTE in mice inoculated with Ehrlich ascites carcinoma (EAC) cells. Materials and Methods. Female Swiss albino mice were inoculated with EAC cells in the thigh to develop solid tumor. Oral treatment with GTE (5.45mg/kg body weight) was started either 14 days prior to tumor cell inoculation or 9 days post inoculation for mice bearing palpable tumors. Body weight and tumor growth were monitored until day 30 post-inoculation. The percentage of infiltrated CD4+, and CD8+T cells, natural killer (NK) cell activity and TNF-α expression in tumor cells were analyzed by flow cytometry and RT-PCR. Tumor histopathology was also examined, and blood lymphocytes and safety parameters were evaluated. GTE (Thymax) was kindly provided by YS Nature Company, Tokyo, Japan. Results. Treatment with GTE prior tumor cell inoculation showed tumor incidence in 61.1 % of the animals. Pre-treatment and post-treatment with GTE, markedly delayed tumor development and resulted in tumor weight reduction by -76.7% and -48.7% of untreated control, respectively. GTE, increased blood lymphocytes count by 60% and 53%, elevated the percentage of infiltrating CD4+ cells by144%, 94% and CD8+ T cells by 146%, 53%, for pre- and post-treatment, respectively. Oral intake of GTE, profoundly augmented NK cell cytolytic activity by 5.8 and 3.1 fold and TNF-α expression by 5.5 and 3.2 fold relative to the untreated control for pre-and post-treatment, respectively. Mice treated with GTE maintained normal body, spleen, liver and kidney weights and normal values for liver function enzymes including Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Gamma-glutamyl transferase (GGT). Histopathological studies showed tumor tissue obtained from GTE -treated groups had a marked decrease in tumor cell density with increased vacuolation of their cytoplasm, marked increase of apoptotic cells and expansion of the degenerative areas. Conclusion: GTE is a safe, non-toxic agent, that exhibits a chemopreventive effect in mice bearing solid Ehrlich carcinoma via its ability to induce potent immunomodulation. This may suggest that GTE could be used for the treatment of solid tumors in humans. Citation Format: Azza I. Othman, Nariman K. Badr El-Din, Maggie E. Amer, Mamdooh Ghoneum. Gross thymic extract inhibits tumor growth by modulating the immune response in mice bearing Ehrlich ascites carcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 7.

Pharmacologic inhibition of lysine-specific demethylase 1 as a therapeutic and immune-sensitization strategy in pediatric high-grade glioma

Diffuse midline gliomas (DMG), including brainstem diffuse intrinsic pontine glioma (DIPG), are incurable pediatric high-grade gliomas (pHGG). Mutations in the H3 histone tail (H3.1/3.3-K27M) are a feature of DIPG, rendering them therapeutically sensitive to small-molecule inhibition of chromatin modifiers. Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) is clinically relevant but has not been carefully investigated in pHGG or DIPG. Patient-derived DIPG cell lines, orthotopic mouse models, and pHGG datasets were used to evaluate effects of LSD1 inhibitors on cytotoxicity and immune gene expression. Immune cell cytotoxicity was assessed in DIPG cells pretreated with LSD1 inhibitors, and informatics platforms were used to determine immune infiltration of pHGG. Selective cytotoxicity and an immunogenic gene signature were established in DIPG cell lines using clinically relevant LSD1 inhibitors. Pediatric HGG patient sequencing data demonstrated survival benefit of this LSD1-dependent gene signature. Pretreatment of DIPG with these inhibitors increased lysis by natural killer (NK) cells. Catalytic LSD1 inhibitors induced tumor regression and augmented NK cell infusion in vivo to reduce tumor burden. CIBERSORT analysis of patient data confirmed NK infiltration is beneficial to patient survival, while CD8 T cells are negatively prognostic. Catalytic LSD1 inhibitors are nonperturbing to NK cells, while scaffolding LSD1 inhibitors are toxic to NK cells and do not induce the gene signature in DIPG cells. LSD1 inhibition using catalytic inhibitors is selectively cytotoxic and promotes an immune gene signature that increases NK cell killing in vitro and in vivo, representing a therapeutic opportunity for pHGG. 1. LSD1 inhibition using several clinically relevant compounds is selectively cytotoxic in DIPG and shows in vivo efficacy as a single agent.2. An LSD1-controlled gene signature predicts survival in pHGG patients and is seen in neural tissue from LSD1 inhibitor-treated mice.3. LSD1 inhibition enhances NK cell cytotoxicity against DIPG in vivo and in vitro with correlative genetic biomarkers.

Natural Killer Cells Dampen the Pathogenic Features of Recall Responses to Influenza Infection.

Despite evidence of augmented Natural Killer (NK) cell responses after influenza vaccination, the role of these cells in vaccine-induced immunity remains unclear. Here, we hypothesized that NK cells might increase viral clearance but possibly at the expense of increased severity of pathology. On the contrary, we found that NK cells serve a homeostatic role during influenza virus infection of vaccinated mice, allowing viral clearance with minimal pathology. Using a diphtheria toxin receptor transgenic mouse model, we were able to specifically deplete NKp46+ NK cells through the administration of diphtheria toxin. Using this model, we assessed the effect of NK cell depletion prior to influenza challenge in vaccinated and unvaccinated mice. NK-depleted, vaccinated animals lost significantly more weight after viral challenge than vaccinated NK intact animals, indicating that NK cells ameliorate disease in vaccinated animals. However, there was also a significant reduction in viral load in NK-depleted, unvaccinated animals indicating that NK cells also constrain viral clearance. Depletion of NK cells after vaccination, but 21 days before infection, did not affect viral clearance or weight loss—indicating that it is the presence of NK cells during the infection itself that promotes homeostasis. Further work is needed to identify the mechanism(s) by which NK cells regulate adaptive immunity in influenza-vaccinated animals to allow efficient and effective virus control whilst simultaneously minimizing inflammation and pathology.

Histidine-rich glycoprotein augments natural killer cell function by modulating PD-1 expression via CLEC-1B.

Augmentation of natural killer (NK) cell cytotoxicity is one of the greatest challenges for cancer immunotherapy. Although histidine‐rich glycoprotein (HRG), a 75‐kDa glycoprotein with various immunomodulatory activities, reportedly elicits antitumor immunity, its effect on NK cell cytotoxicity is unclear. We assessed NK cell cytotoxicity against K562 cells. We also measured concentrations of cytokines and granzyme B in the cell supernatant. The proportion of CD56bright NK cells and NK cell surface PD‐1 expression was assessed with flow cytometry. The neutralizing effects of anti‐C‐type lectin‐like receptor (CLEC) 1B against HRG were also measured. NK cell morphological changes were analyzed via confocal microscopy. HRG significantly increased NK cell cytotoxicity against K562 cell lines. HRG also increased the release of granzyme B and the proportion of CD56bright NK cells. Further, HRG was able to decrease NK cell surface PD‐1 expression. The effects of HRG on NK cells were reversed with anti‐CLEC‐1B antibodies. Additionally, we confirmed NK cell nuclear morphology and F‐actin distribution, which are involved in the regulation of cytotoxic granule secretion. Because both PD‐1 and CLEC‐1B are associated with prognosis during malignancy, HRG incorporates these molecules to exert the antitumor immunity role. These facts indicate the potential of HRG to be a new target for cancer immunotherapy.

A water-soluble derivative of propolis augments the cytotoxic activity of natural killer cells

Ethnopharmacological relevancePropolis, a resinous material collected from numerous plants by honeybees, has historically been used as a health-promoting food. Recently, due to its potential anti-tumor effects, use of propolis has been proposed as an adjuvant therapy to chemotherapy; however, the effects of propolis on immune responses remain unclear. Aim of the studyIn this study, we examined the effects of the oral ingestion of propolis on natural killer (NK) cell activity, which is important in immune surveillance against cancer and viral infections. In addition, we assessed the effects of the major components of the water-soluble powder derivative of propolis (WPP). Materials and methodsC57BL/6 (B6) wild-type (WT) and RAG 2-deficient (RAG-/-) mice and BALB/c WT, interferon (IFN)-γ-deficient (IFN-γ–/–), IFN-γ receptor-deficient (IFN-γR-/-) and RAG-/- mice were orally administered WPP or its major components. NK cell populations and cytotoxic activity were then examined by flow cytometry and 51Cr release assay, respectively. ResultsWhile the cytotoxic activity of NK cells was increased following administration of 100 mg/kg/day of WPP for 7 days or 200 or 500 mg/kg/day of WPP for 4 days in WT mice, the proportions of NK cell populations were unaltered. Similar activation of NK cell cytotoxicity was observed when RAG-/-, but not IFN-γ–/– or IFN-γR-/-, mice were orally administered 200 mg/kg/day of WPP for 4 days. Oral ingestion of artepillin C or p-coumaric acid, but not drupanin, augmented NK cell cytotoxicity in a manner similar to WPP and to the mixture of these three components. ConclusionThese results suggest that oral ingestion of WPP enhances NK cell cytotoxic activity, but not proliferation, in a manner dependent on IFN-γ and without the contribution of acquired immune responses. Further, artepillin C or p-coumaric acid, but not drupanin, may be the components responsible for this augmentation of NK cell cytotoxicity. These findings suggest the possible utility of WPP as a therapeutic for prevention of cancer development and against viral infection through NK cell activation.

Abstract LB-195: Bortezomib sensitizes glioblastoma for NK cell immunotherapy

Abstract Introduction: Glioblastoma (GBM) is the most aggressive and lethal brain cancer in adults where best available therapy combining surgery and concomitant chemo radiation prolongs patient survival from 12.1 to 14.6 months. Approximately half of the patients benefit from addition of Temozolomide chemotherapy (those with methylated promoter of the DNA repair gene O6 methylguanine DNA methyltransferase). Immunotherapy has demonstrated unprecedented efficacy in solid tumours and kill targets in unique ways to the conventional therapies. Natural Killer (NK) cells are attractive effectors not only because of their ability to recognize and kill malignant and virus infected cells, but also their potential for modifying the tumor microenvironment to pro-inflammatory state. We have previously demonstrated NK-cell cytotoxicity against GBM. Here we hypothesize that misfolded protein response induced by pretreatment with proteasome inhibitors will sensitize GBM cells to NK cell therapy. Methods: IC50 doses of Bortezomib, Carfilzomib and MG132 proteasome inhibitors were determined by viability assays in P3, 2012-018 and P22 patient-derived GBM cells, as well as in normal human astrocytes (NHA). Phenotyping, time and dose dependent cytotoxicity/apoptosis and degranulation after combination therapy with Bortezomib and NK cells from healthy donors was conducted with flow cytometry and western blotting used to confirm protein signaling. Results: We demonstrated that Bortezomib is the most suitable proteasome inhibitor as it selectively killed GBM cells at low doses (IC50 39-42nM) while requiring higher doses (70nM) for NHA cells. In contrast, Carfilzomib and MG132 killed better the NHA than GBM cells. Bortezomib was more effective in combination treatment with NK cells, than monotherapy of Bortezomib or NK cells. The IC50 dose of Bortezomib was reduced by 40% from 42nM at 48hrs to 25nM at 24hrs when combined with effector:target (E:T) ratio 5:1 NK cells (p&lt0.05). E:T ratio of NK cells was also reduced by 75% in combination therapy, NK cell monotherapy killed only 45% of GBM cells at 20:1 ratio. The killing efficacy of combination treatment was not mediated by increased stress ligand expression, but Bortezomib attenuated autophagy and induced late apoptotic GBM cell death. Although Bortezomib did not increase degranulation or kill NK cells, importantly, it rather induced NK cell differentiation to mature CD56dimCD16-NKG2D+ subsets previously associated with increased natural cytotoxicity. Conclusion: Bortezomib was the best proteasome inhibitor against GBM that synergistically enhanced NK cell killing. Current work is investigating the mechanisms regulating the augmented differentiation and NK cell cytotoxicity during the combination therapy in vitro and in vivo in mice. Citation Format: Andrea Gras Navarro, Aminur Rahman, Marzieh Bahador, Martha Chekenya Enger. Bortezomib sensitizes glioblastoma for NK cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-195. doi:10.1158/1538-7445.AM2017-LB-195

Expression and function of NKp46 W32R: the human homologous protein of mouse NKp46 W32R (No\xe9)

Natural killer (NK) cells eradicate infected cells and tumors following the triggering of activating receptors, like the Natural Cytotoxicity Receptors (NCRs), which include NKp30, NKp44 and NKp46. NKp46 is the only NCR expressed in mice (mNKp46), and except for some Innate Lymphoid Cell (ILC) populations (ILC1/3 subsets), its expression is restricted to NK cells. Previously, a mouse named Noé was generated in which a random point mutation (W32R) impaired the cell surface expression of mNKp46. Interestingly, the Noé mice NK cells expressed twice as much of the transcription factor Helios, and displayed general non-NKp46 specific hyperactivity. We recently showed that the mNKp46 W32R (Noé) protein was expressed on the surface of various cells; albeit slowly and unstably, that it is aberrantly glycosylated and accumulates in the ER. Interestingly, the Tryptophan (Trp) residue in position 32 is conserved between humans and mice. Therefore, we studied here the human orthologue protein of mNKp46 W32R, the human NKp46 W32R. We demonstrated that NKp46 W32R is aberrantly glycosylated, accumulates in the ER, and is unstable on the cell surface. Furthermore, we showed that overexpression of NKp46 W32R or Helios resulted in augmented NK cell activation, which may be applied to boost NK activity for therapeutic applications.

Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma with fully annotated clinical follow-up

Lung adenocarcinomas (LUADs) lead to the majority of deaths attributable to lung cancer. We performed whole-exome sequencing (WES) and immune profiling analyses of a unique set of clinically annotated early-stage LUADs to better understand the pathogenesis of this disease and identify clinically relevant molecular markers. We performed WES of 108 paired stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Ten immune markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry (IHC) in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fisher's exact test. Cox proportional hazards regression models were used for multivariate analysis of clinical outcome. LUADs in this cohort exhibited an average of 243 coding mutations. We identified 28 genes with significant enrichment for mutation. SETD2-mutated LUADs exhibited relatively poor recurrence- free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS among KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis revealed that LUADs in smokers and with relatively high mutation burdens exhibited increased levels of immune markers. Analysis of immunophenotypes revealed that LUADs with STK11 mutations exhibited relatively low levels of infiltrating CD4+/CD8+ T-cells indicative of a muted immune response. Tumoral PD-L1 was significantly elevated in TP53 mutant LUADs whereas PIK3CA mutant LUADs exhibited markedly down-regulated PD-L1 expression. LUADs with TP53 or KEAP1 mutations displayed relatively increased CD57 and Granzyme B levels indicative of augmented natural killer (NK) cell infiltration. Our study highlights molecular and immune phenotypes that warrant further analysis for their roles in clinical outcomes and personalized immune-based therapy of LUAD.

Augmentation of Cytolytic Activity in Murine Natural Killer Cells and Inhibition of Tumor Growth by the Ethanol Fraction of Oyster Extract.

A reduced number and/or reduced activity of natural killer (NK) cells, which are important for defense against a variety of cancers and viral infections, occur under various stress conditions and in patients with various diseases. In this article, we report that the 30% to 50% ethanol precipitate of oyster extract (EPOE50) dose-dependently enhanced the activity of mouse spleen NK cells in vitro and in vivo. The activity of EPOE50 was eluted with a molecular weight of about 2000 by gel filtration and was inactivated by periodate but not by proteinase K. The activity of highly purified NK cells was also augmented by EPOE50 but not by oligodeoxyribonucleotide 1585, which mimics bacterial DNA. Administration of EPOE50 to mice stimulated splenic NK cell activity without a change in splenic NK cell populations. Although the proliferation of B16 tumor cells in vitro was slightly stimulated by EPOE50, the growth of B16 melanoma in vivo was dose-dependently suppressed by administration of EPOE50. Taken together, our results indicate that EPOE50 augmented NK cell activity and that its administration to mice inhibited tumor growth presumably through the activation of NK cells and also suggest that the active substance is a sugar-containing oligomer or polymer and is not of bacterial origin.

CD69 Deficiency Enhances the Host Response to Vaccinia Virus Infection through Altered NK Cell Homeostasis.

During the host response to viral infection, the transmembrane CD69 protein is highly upregulated in all immune cells. We have studied the role of CD69 in the murine immune response to vaccinia virus (VACV) infection, and we report that the absence of CD69 enhances protection against VACV at both short and long times postinfection in immunocompetent and immunodeficient mice. Natural killer (NK) cells were implicated in the increased infection control, since the differences were greatly diminished when NK cells were depleted. This role of NK cells was not based on an altered NK cell reactivity, since CD69 did not affect the NK cell activation threshold in response to major histocompatibility complex class I NK cell targets or protein kinase C activation. Instead, NK cell numbers were increased in the spleen and peritoneum of CD69-deficient infected mice. That was not just secondary to better infection control in CD69-deficient mice, since NK cell numbers in the spleens and the blood of uninfected CD69(-/-) mice were already augmented. CD69-deficient NK cells from infected mice did not have an altered proliferation capacity. However, a lower spontaneous cell death rate was observed for CD69(-/-) lymphocytes. Thus, our results suggest that CD69 limits the innate immune response to VACV infection at least in part through cell homeostatic survival. We show that increased natural killer (NK) cell numbers augment the host response and survival after infection with vaccinia virus. This phenotype is found in the absence of CD69 in immunocompetent and immunodeficient hosts. As part of the innate immune system, NK lymphocytes are activated and participate in the defense against infection. Several studies have focused on the contribution of NK cells to protection against infection with vaccinia virus. In this study, it was demonstrated that the augmented early NK cell response in the absence of CD69 is responsible for the increased protection seen during infection with vaccinia virus even at late times of infection. This work indicates that the CD69 molecule may be a target of therapy to augment the response to poxvirus infection.

Enhanced natural killer cell activation by exopolysaccharides derived from yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1

Yogurt is generally recognized as a beneficial food for our health, but research into its physiological effects has focused mainly on intestinal dysfunctions such as constipation and diarrhea. We previously found yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (hereafter OLL1073R-1) could reduce risks of catching the common cold and flu in human trials. It was assumed that immunostimulatory exopolysaccharide (EPS) produced from OLL1073R-1 play an important role in this context. However, few studies have examined the immunostimulatory effects of traditional Bulgarian yogurts fermented with different strains of lactobacilli and their metabolites. Therefore, we screened 139 L. delbrueckii ssp. bulgaricus strains and identified OLL1073R-1 as the most robust producer of EPS. This strain was also the only strain that induced the production of IFN-γ in vitro. Oral administration of the EPS or yogurt fermented with OLL1073R-1 and Streptococcus thermophilus OLS3059 (OLL1073R-1 yogurt) augmented natural killer (NK) cell activity and induced IFN-γ production in spleen cells in mice, whereas 2 other yogurts fermented with other strains had no effect on NK cell activity. Cellular preparations of the OLL1073R-1 strain also slightly augmented NK cell activity, but were less effective than EPS itself. The EPS-dependent stimulation of NK cell activity was abrogated in IFN-γ knockout mice and in myeloid differentiation factor 88 knockout mice. Furthermore, IFN-γ production from spleen cells stimulated with EPS was completely blocked with both anti-IL-12 and anti-IL-18 antibodies in vitro. These findings suggest that NK cell activation by OLL1073R-1 yogurt is EPS-dependent, occurs via IL-12- and IL-18-mediated IFN-γ production, and requires myeloid differentiation factor 88. We showed that traditional Bulgarian yogurt could exert immunostimulatory effects by selecting starter strains and part of the mechanisms depend on IFN-γ inducible EPS produced from L. delbrueckii ssp. bulgaricus. Further investigations on processes of fermentation to increase of the EPS may lead to the development of new functional foods that keep our immune functions stable.

ER-Stress-Induced Suppression of HLA-E on Bortezomib-Evading Malignant Plasma Cells Dramatically Enhances Their Susceptibility to NK Cell Killing: Identification of an Achilles Heel in Myeloma Cells That Can be Utilized to Prevent Disease Relapse Following Bortezomib Treatment

Although the proteasome inhibitor bortezomib has significantly improved the survival of patients with multiple myeloma (MM), most patients treated with this drug eventually develop progressive disease. Recent data indicate that MM cells can evade bortezomib-induced cell death by undergoing autophagy as a consequence of endoplasmatic reticulum (ER)-stress triggered by proteasome inhibition. Here we show that bortezomib-evading MM cells become highly sensitized to killing by natural killer (NK) cells via ER-stress-induced reduction of the NK cell inhibitory molecule HLA-E that is normally expressed at high levels on the surface of MM cells. High-resolution flow cytometry-based assays revealed augmented NK cell recognition and degranulation against bortezomib-exposed MM cells (3 fold higher compared to untreated MM controls) was restricted to NK cells exclusively controlled by the HLA-E-binding inhibitory receptor NKG2A (NKG2ASP NK cells) (Figure 1). In contrast, due to unchanged high expression of other HLA class I molecules on the surface of bortezomib-exposed MM cells there was no augmentation in degranulation by NK cells controlled by other inhibitory HLA class I-binding receptors, such as killer immunoglobulin-like receptors (KIRs). Compared to their non-expanded counterparts, ex vivo expanded NK cells have previously been shown to have an increased proportion of NKG2ASP NK cells (50% vs 25%, p<0.01) and bolstered cytotoxic function against tumor cells. Using NK cells expanded in accordance with our ongoing FDA-approved clinical trial evaluating adoptive infusion of autologous ex vivo expanded NK cells in patients with refractory cancers at the NIH, we established these highly cytotoxic NKG2ASP dominant NK cells induced substantially higher lysis of bortezomib-exposed MM cells compared to non-expanded matched control NK cells (38% vs 18%, p<0.05) (Figure 2). Based on these findings, we hypothesize that adoptive infusions of ex vivo expanded autologous NK cells following treatment with bortezomib could eradicate the fraction of MM cells that would normally evade killing through proteasome inhibition alone. The increased vulnerability of bortezomib-evading cells to NK cell cytotoxicity identifies a critical Achilles heel in myeloma cells and suggests adoptive NK cell infusions following bortezomib therapy could potentially be utilized as a strategy to improve long-term survival in MM patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cooperative therapeutic anti-tumor effect of IL-15 agonist ALT-803 and co-targeting soluble NKG2D ligand sMIC.

Shedding of the human NKG2D ligand MIC (MHC class I-chain-related molecule) from tumor cell surfaces correlates with progression of many epithelial cancers. Shedding-derived soluble MIC (sMIC) enables tumor immune escape through multiple immune suppressive mechanisms, such as disturbing natural killer (NK) cell homeostatic maintenance, impairing NKG2D expression on NK cells and effector T cells, and facilitating the expansion of arginase I+ myeloid suppressor cells. Our recent study has demonstrated that sMIC is an effective cancer therapeutic target. Whether targeting tumor-derived sMIC would enhance current active immunotherapy is not known. Here, we determined the in vivo therapeutic effect of an antibody co-targeting sMIC with the immunostimulatory IL-15 superagonist complex, ALT-803, using genetically engineered transplantable syngeneic sMIC+ tumor models. We demonstrate that combined therapy of a nonblocking antibody neutralizing sMIC and ALT-803 improved the survival of animals bearing sMIC+ tumors in comparison to monotherapy. We further demonstrate that the enhanced therapeutic effect with combined therapy is through concurrent augmentation of NK and CD8 T cell anti-tumor responses. In particular, expression of activation-induced surface molecules and increased functional potential by cytokine secretion are improved greatly by the administration of combined therapy. Depletion of NK cells abolished the cooperative therapeutic effect. Our findings suggest that administration of the sMIC-neutralizing antibody can enhance the anti-tumor effects of ALT-803. With ALT-803 currently in clinical trials to treat progressive solid tumors, the majority of which are sMIC+, our findings provide a rationale for co-targeting sMIC to enhance the therapeutic efficacy of ALT-803 or other IL-15 agonists.

Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.

The Adaptor SAP Controls NK Cell Activation by Regulating the Enzymes Vav-1 and SHIP-1 and by Enhancing Conjugates with Target Cells

The adaptor SAP, mutated in X-linked lymphoproliferative disease, has critical roles in multiple immune cell types. Among these, SAP is essential for the ability of natural killer (NK) cells to eliminate abnormal hematopoietic cells. Herein, we elucidated the molecular and cellular bases of this activity. SAP enhanced NK cell responsiveness by a dual molecular mechanism. It coupled SLAM family receptors to the kinase Fyn, which triggered the exchange factor Vav-1 and augmented NK cell activation. SAP also prevented the inhibitory function of SLAM family receptors. This effect was Fyn independent and correlated with uncoupling of SLAM family receptors from the lipid phosphatase SHIP-1. Both mechanisms cooperated to enable conjugate formation with target cells and to stimulate cytotoxicity and cytokine secretion by NK cells. These data showed that SAP secures NK cell activation by a dichotomous molecular mechanism, which is required for conjugate formation. These findings may have implications for the role of SAP in other immune cell types.

Application of paclitaxel in low non-cytotoxic doses supports vaccination with melanoma antigens in normal mice

Chemotherapeutic agents such as paclitaxel applied in ultra-low, non-cytotoxic doses were previously shown to stimulate dendritic cell activity and anti-tumor immune responses upon vaccination in mouse transplantable tumor models. However, the mechanisms of these alterations-termed chemoimmunomodulation or chemomodulation-are still not clear. This study investigated the effect of paclitaxel applied in ultra-low, non-cytotoxic doses on the efficiency of immunization of healthy C57BL/6 mice with the peptide derived from tyrosinase related protein (TRP)-2 as a model melanoma antigen. Using an IFNγ ELISPOT assay, it was found that administration of 1 mg paclitaxel/kg in combination with the peptide vaccination strongly increased the frequencies of TRP-2 specific spleen T-cells as compared to levels due to the vaccination alone. This was associated with a significant decrease in the levels of regulatory T-cells (Treg) and immature myeloid cells (known as a counterpart of myeloid derived suppressor cells [MDSC] in healthy mice). Such impairments of potential immunosuppressive cells were found to correlate with a strong increase in the amount of effector CD8+ and CD4+ T-cells in the bone marrow and spleen. Furthermore, in paclitaxel-treated mice, a significant augmentation of natural killer (NK) cell numbers in the bone marrow and their ability to produce IFNγ were observed. In addition, the level of NK-T-cells in the lymph nodes was also increased. It is suggested that paclitaxel applied in ultra-low, non-cytotoxic doses may potentially enhance the efficacy of anti-tumor vaccinations by neutralizing immunosuppressive Treg and MDSC populations in tumor-bearing hosts.

Dietary Supplementation with an Extract of North American Ginseng in Adult and Juvenile Mice Increases Natural Killer Cells

Cells belonging to the innate immune system are referred to as natural killer (NK) cells. We recently demonstrated that normal, pre-weaned infant mice, injected with a proprietary extract of ginseng (CVT-E002) had augmented NK cell numbers vs. sham-injected mice. In the present study, we extended these observations into juvenile and adult mice. Thus, young adult (age: 8–9 wk) C3H mice were given daily dietary CVT-E002 for 4 wk followed by untreated chow for the following 2 months, then euthanized (age: 20–21 wk). Other C3H mice (juvenile: 4-wk-old) were given CVT-E002 under the same protocol and sampled at 18 wk of age. In spite of withdrawing the extract 2 months earlier, the absolute numbers of NK cells in the young adults, remained significantly (p < 0.01), and slightly, elevated in the spleen and bone marrow (BM), respectively. The relative numbers (%) of NK cells in the blood also remained elevated (p < 0.05). In juvenile mice fed CVT-E002, the absolute numbers (spleen, BM) and % (blood) of NK cells were all elevated (p<0.01 – p<0.05). The mechanisms responsible for these super-normal numbers of NK cells long after withdrawal of CVT-E002, is as yet unknown.