Anti-asialo GM1 Research Articles

A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

Establishment of anti-asialo-GM1 rabbit monoclonal antibodies capable of reducing natural killer cell activity in mice.

Rabbit anti-asialo-GM1 (ASGM1) serum or polyclonal antibodies can eliminate mouse splenic natural killer (NK) cell activity in vitro and in vivo. We developed rabbit monoclonal antibodies (mAbs) against ASGM1 using a single-cell analysis and isolation system. Five mAbs (GA109, GA115, GA116, GA131, and GA134) that were reactive to ASGM1 were isolated from the spleen lymphocytes of rabbits immunized with ASGM1. Enzyme-linked immunosorbent assay and thin-layer chromatography immunostaining results showed that the mAbs strongly reacted with ASGM1. Two mAbs (GA116 and GA134) reacted exclusively with ASGM1, whereas three mAbs (GA109, GA115, and GA131) showed slight or considerable cross-reactivity with GM1. The administration of the mAbs (4-20 μg) to BALB/c mice completely abolished NK cell activity in vivo. The anti-ASGM1 rabbit mAbs obtained in this study may provide a useful and reproducible tool for various future studies, such as depleting NK cell activity to enhance xenograft engraftment in mouse models.

Abstract LB336: pH-sensitive polymeric micelles loading IL-12 profoundly inflame the tumor microenvironment to eradicate cold tumors

Abstract Background: Treatment of immunologically cold tumors is a major challenge. IL-12 can activates both innate and adaptive immunity to elicit strong antitumor immunity. Nevertheless, the poor pharmacokinetic and severe side effects of IL-12 limit the application. We have recently developed pH-sensitive polymeric micelles loading IL-12 (IL-12/m) that can selective activate in tumor tissues to invigorate immunotherapy and enhance efficacy. Herein, we elucidated the mechanisms behind the enhanced efficacy of IL-12/m and their ability to synergize with immune checkpoint inhibitors (ICIs). Methods: To determine the contribution of the immune cells on the efficacy on IL-12/m, mice bearing orthotopic 4T1 tumor models were treated with anti-CD4 (for depleting CD4+ cells), anti-CD8 (for depleting CD8+ cells), anti-asialo GM1 (for depleting NK cells). The tumor volume was tracked after treatment with IL-12/m. The immune status of the tumors was studied by histology, flow cytometry and RNA-seq after treatment with free IL-12, IL-12/m and their combination with ICIs. The efficacy of the treatments was assessed by following the tumor growth and mice survival. Results: IL-12 can promote the growth and function of T cells to initiate the enhanced anti-tumoral immune reactions. Flow cytometry analysis IL-12/m significantly enhanced tumor infiltration of CD8+ T cells compared to free IL-12. The immune cell depletion study revealed that the depletion of CD8+ cytotoxic T cells, CD4+ T cells and NK cells significantly lessen efficacy of IL-12/m, which is consistent with the biological function of IL-12. The depletion experiment suggests CD8+ cytotoxic T cells are the most important fraction for IL-12/m efficacy. Moreover, immunohistochemistry analysis of 4T1 revealed IL-12/m promoted high infiltration of CD8+ cells and the upregulation of Granzyme B in CD8 + Cells. IL-12/m also increased PD-L1 levels in tumors. The effects of IL-12/m on the TME was further investigated as a monotherapy and in combination with anti-PD-1 antibodies. The combination of IL-12/m with anti-PD-1antibodies presented high CD45+ cells in tumors. Lymphoid cells and T cells were upregulated by IL-12 and the anti-PD-1 antibody combination. The general CD4+ T cells also showed upregulation in the IL-12/m treated groups. While anti-PD-1 monotherapy did not show any suppression on Tregs in the tumor model, the combination of IL-12/m plus anti-PD-1 depleted Tregs. Moreover, IL-12/m increase the presence of Th cells. We then investigated the activation by transcriptome analysis. The heatmap of global gene expression indicated differentiated gene profiles. GOBP and KEGG analysis showed that IL-12/m upregulated the stimulation of proliferation, differentiation and activation of various effector immune cells, differentiation of T cells into Th cells, and T cell activation and T cell receptor signaling pathway. The enhanced the intratumoral immunity of IL-12/m plus ICIs allowed eradicating 4T1 tumors. Conclusions: Systemically injected IL-12/m ensured strong stimulation of antitumor immunity leading to high safety and strong therapeutic outcomes in cold tumor models. The enhanced immune response of IL-12 synergize with checkpoint blockade to eradicate cold tumors. The approach has potential for developing safer and more effective immunotherapies. Citation Format: Horacio Cabral, Pengwen Chen, Koji Nagaoka, Takuya Miyazaki, Kazunori Kataoka, Kazuhiro Kakimi. pH-sensitive polymeric micelles loading IL-12 profoundly inflame the tumor microenvironment to eradicate cold tumors [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 LB336.

Immunostimulating activities and anti-cancer efficacy of rhamnogalacturonan-I rich polysaccharide purified from Panax ginseng leaf

This study aimed to evaluate the anti-cancer effects as well as stimulation of various immune related factors such as complementary system, macrophages, and NK cells of polysaccharides isolated from Panax ginseng (P. ginseng) leaf. The polysaccharide fractions (PGEP, PGEP-I, –II, and –III) isolated from P. ginseng were contained rhamnose (Rha), arabinose (Ara), galactose (Gal), galacturonic acid (GalA), and glucuronic acid (GlcA), all of which are typical sugar components of rhamnogalacturonan (RG)-I. Only PGEP-I reacted with the β-glucosyl Yariv reagent (37.29%), indicating the presence of type-II arabinogalactan. Additionally, methylation analysis indicated the PGEP-I mainly comprised 19 different sugar linkages including 4-linked GalpA, 2,4-linked Rhap, 3,6-linked Galp, 5-linked Araf, terminal GlcpA, and terminal Arap which are characteristics of RG-I. Meanwhile, PGEP-I enhanced the production of cytokines such as interleukin (IL)-6, IL-12, and tumor necrosis factor-α and augmented the complement system. The intravenous administration of PGEP-I activated the natural killer (NK) cells in a dose-dependent manner against YAC-1. Prophylactic intravenous administration of PGEP-I significantly suppressed Colon26-M3.1 carcinoma inoculation-induced lung cancer (48.7%). Moreover, the anti-cancer effects of PGEP-I were significant even though NK cell functions were blocked by anti-asialo GM1. The findings suggest that PGEP-I, an RG-I polysaccharide, not only activates the complement system, macrophages and NK cells, but also has strong anticancer effects that inhibit lung cancer.

Disturbed natural killer cell homeostasis in the salivary gland enhances autoimmune pathology via IFN-γ in a mouse model of primary Sjögren’s syndrome

Innate lymphoid cells (ILCs), including natural killer (NK) cells, ILC1, ILC2, lymphoid tissue-inducer (LTi) cells, and ILC3 cell, play a key role in various immune responses. Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by chronic inflammation of exocrine glands, such as the lacrimal and salivary glands (SGs). The role of NK cells among ILCs in the pathogenesis of pSS is still unclear. In this study, the characteristics and subsets of NK cells in the salivary gland (SG) tissue were analyzed using a murine model of pSS. Multiple phenotypes and cytotoxic signature of the SG NK cells in control and pSS model mice were evaluated by flow cytometric analysis. Intracellular expression of interferon-γ (IFN-γ) among T cells and NK cells from the SG tissues was compared by in vitro experiments. In addition, pathological analysis was performed using anti-asialo-GM1 (ASGM1) antibody (Ab)-injected pSS model mice. The number of conventional NK (cNK) cells in the SG of pSS model mice significantly increased compared with that in control mice at 6 weeks of age. The production level of IFN-γ was significantly higher in SG NK cells than in SG T cells. The depletion of NK cells by ASGM1 Ab altered the ratio of tissue resident NK (rNK) cells to cNK cells, which inhibited the injury to SG cells with the recovery of saliva secretion in pSS model mice. The results indicate that SG cNK cells may enhance the autoreactive response in the target organ by upregulating of IFN-γ, whereas SG rNK cells protect target cells against T cell cytotoxicity. Therefore, the activation process and multiple functions of NK cells in the target organ could be helpful to develop potential markers for determining autoimmune disease activity and target molecules for incurable immune disorders.

Abstract 2047: MUC4 enables immune tumor evasion in HER2+ breast cancer

Abstract HER2+ is a breast cancer (BC) subtype characterized by the overexpression/amplification of HER2. Patients receive trastuzumab (Tz) but many (27-42%) do not achieve an objective response. We demonstrated that the overexpression of TNFɑ induces Tz resistance in tumors by upregulating the membrane glycoprotein MUC4, which masks Tz epitope on HER2, impairing its binding and reducing its therapeutic effects. We have also proved that blocking the soluble TNFɑ isoform with INB03 (DN) reduces MUC4 expression, overcomes Tz resistance and unleashes an antitumor innate immune response characterized by an increase in NK cell-activation and degranulation and a macrophage (Mφ) polarization to the M1 subtype. This study aims to determine the impact of MUC4 expression on Mφ and NK cells antitumor activity in the presence of Tz or Tz+DN, and on human T-lymphocyte recruitment and differentiation. Tz-resistant HER2+ BC human cell lines, JIMT-1 and KPL-4, were genetically modified to stably express a doxycycline-inducible (Dox) MUC4 shRNA (shMUC4) or a scramble one (shControl), and injected s.c. into female nude mice. After tumor establishment, the animals were randomly divided into a control group (-Dox) or an induced group (+Dox), and treated twice a week i.p. with IgG (5 mg/kg) or Tz+DN (5 and 10 mg/kg each, respectively) (-Dox) and with IgG or Tz (+Dox). Chlodronate or anti-asialo GM1 was used to deplete Mφ or NK cells, respectively. Tumor volume was measured routinely and, at the end point, tumors were processed and infiltrating immune cells were analyzed by flow cytometry. In -Dox tumors, both Mφ and NK cells are needed to achieve Tz+DN antitumor effect (p<0.01 and p<0.05, respectively). However, upon MUC4 silencing, only Mφ are required to mediate Tz antitumor effect (p<0.01). To address MUC4 role on T-lymphocyte differentiation, we isolated human T-cells from peripheral blood, activated them with CD3/CD28 beads and exposed them to conditioned mediums obtained from JIMT-1 and KPL-4 cells with or without MUC4 expression. The absence of MUC4 generated a secretome that promoted the differentiation of naïve T-cells to an effector subtype. Finally, we studied the tumor infiltrating lymphocytes (TILs) in a cohort of 90 patients with HER2+ BC and found a negative correlation between the presence of TILs and MUC4 expression (p=0.01). We conclude that Mφ are key players in the Tz-mediated antitumor innate immune response. Moreover, MUC4 expression impairs T-cell effector function and promotes immunologically cold HER2+ tumors, an additional resistance mechanism of this glycoprotein. We propose that women with HER2+MUC4+ BC could benefit from the combined treatment of Tz+DN to enhance innate and adaptive antitumor immune responses and prevent or overcome Tz resistance. Because MUC4 expression can be easily determined from biopsy tissue, early determination of MUC4 status in HER2+ BC could guide therapeutic choices to improve treatment outcomes. Citation Format: Sofia Bruni, Florencia L. Mauro, Maria F. Mercogliano, Cecilia J. Proietti, Carla Adami, Agustina Dupont, Gloria Inurrigarro, Rosalia Cordo Russo, Patricia V. Elizalde, Roxana Schillaci. MUC4 enables immune tumor evasion in HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2047.

Abstract 3366: Novel oncolytic adenovirus for systemic treatment of disseminated lung cancer

Abstract The increased cancer incidence and associated mortality call for the development of novel treatment modalities to address needs of cancer patients, which do not respond to existing therapies. Oncolytic viruses emerge as a new modality in cancer therapy due to their natural ability to infect and kill cancer cells, and to stimulate patient's own immune system to eliminate cancer cells throughout the body. For this study, we used novel engineered oncolytic adenovirus, Ad5/35-3M (AVID-317), which demonstrated greatly-improved safety profile after systemic virus administration due to a set of targeted mutations in the adenovirus capsid (Atasheva et al, Science Translational Medicine, 2020, eabc6659). Systemic administration of AVID-317 to NCr/Nude mice with disseminated lung adenocarcinoma slowed tumor progression and significantly extended survival, compared to buffer-treated control tumor-bearing mice. In AVID-317 treated group, about 25% of mice demonstrated complete rejection of disseminated tumors and the rest of mice showed significantly extended survival. To understand the mechanism of AVID-317-mediated tumor suppression and to design more effective combination therapies, we analyzed disseminated tumors from the lungs in NCr/Nude mice using single cell RNA-seq and Mass Cytometry (Cy-TOF) after virotherapy. Our scRNA-seq analysis showed that in a subset of mice responding poorly to AVID-317 therapy, tumor-associated CSF1R-expressing myeloid cells expressed higher levels of MHC-II, Ctsb, and Arg1. In addition, CD3e+ T cells expressed functional exhaustion marker, PD-1, exclusively in mice that responded poorly to virotherapy. Mass Cytometry analysis with 37 metal isotope-labeled antibodies confirmed elevated expression of activation markers on myeloid cells after virotherapy. Next, we analyzed the efficacy of a combination therapy where in addition to AVID-317 administration, tumor-bearing mice were treated with anti PD-1 antibody to block immune-suppressive signaling in T cells, anti-CSF1R antibody to block CSF1R signaling, and anti-Asialo GM1 antibody to deplete anti-viral NK cells. Our analyses showed that combination therapy regimen significantly improved therapeutic outcomes in mice with disseminated lung cancer, by extending median survival to 95 days from 55-56 days observed in mice treated with either virus alone or combination drugs without the virus. Taken together, our study demonstrated feasibility of using AVID-317 oncolytic adenovirus for systemic therapy of disseminated lung cancer. Our study also showed that a combination of virotherapy with a defined select of immunotherapeutic drugs allows for the significant improvement of therapeutic efficacy in pre-clinical model of disseminated lung cancer. Citation Format: Jia Yao, Svetlana Atasheva, Dmitry Shayakhmetov. Novel oncolytic adenovirus for systemic treatment of disseminated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3366.

Effects of intravenous administration of polysaccharide purified from fermented barley on tumor metastasis inhibition via immunostimulating activities

The present study was aimed that determine the effects of polysaccharide isolated from fermented barley about tumor metastasis inhibition via immune related factors such as complementary system, macrophages, natural killer (NK) cells, and cytotoxic T lymphocyte (CTL). To characterize the tumor inhibition effects through immunostimulation of polysaccharides purified from fermented barley, the active crude polysaccharide (BF-CP) was isolated from fermented barley and further fractionated into three purified polysaccharides (BF-I, –II, and –III) with different molecular weights through gel permeation chromatography. BF-I was mainly composed of four monosaccharides, including xylose (32.90%), arabinose (26.71%), and glucose (33.43%). BF-I augmented the secretion of cytokines, such as interleukin (IL)-6, IL-12, and tumor necrosis factor-α, through peritoneal macrophages and potently activated the complement system. In the experiment for NK cells and CTL activities, intravenous (i.v.) administration of BF-I significantly augmented the cytotoxicity of NK cells and CTL-mediated cytotoxicity against tumor cells. In addition, i.v. administration of BF-I potently inhibited tumor metastasis in a mouse model of lung cancer and tumor metastasis in mice with suppressed NK cell function through anti-asialo GM1. Consequently, BF-I shows potent tumor inhibition effects, and these effects are related to various immune factors such as macrophages, the complement system, NK cells, and CTL. Therefore, BF-I, the polysaccharide purified from fermented barley possess the new possibility for developing functional foods and medical ingredients.

Anti-Asialo GM1 Antibody-Positive Optic Neuritis and Optic Perineuritis in Chronic Inflammatory Demyelinating Polyneuropathy.

Anti-Asialo GM1 Antibody-Positive Optic Neuritis and Optic Perineuritis in Chronic Inflammatory Demyelinating Polyneuropathy.

Abstract P5-13-32: Mucin 4 expression in high risk breast cancer: Predicting and overcoming resistance to immunotherapy

Abstract Background HER2-positive (+) and triple negative breast cancer (TNBC) have the worst survival among BC. BC patients are treated with chemotherapy (CT) and/or radiotherapy (RT), and HER2+ BC patients also receive targeted therapies, such as trastuzumab (Tz). The abundance of tumor infiltrating lymphocytes (TILs), in both HER2+ and TNBC, has a major good prognostic value. Thus, indicating that immunological evasion mechanisms are present in the tumor microenvironment (TME) hampering the efficacy of the treatments. We previously showed that soluble tumor necrosis factor α (sTNF) induces upregulation of mucin 4 (MUC4), which shields Tz epitope on HER2 impairing Tz binding and its effects. In preclinical models of de no5vo Tz-resistant tumors, administration of the sTNF blocking agent INB03 (DN) together with Tz inhibited tumor growth. We proved that MUC4 expression is an independent predictor of poor DFS in patients treated with adjuvant Tz. Our goal is to study whether MUC4 plays a role in tumor immune evasion in HER2+ and TNBC. Methods Untreated primary BC samples were assessed for TILs density (H&E) and MUC4 expression by immunohistochemistry. Tumors with TILs ≥30% and >50%, for TNBC and HER2+ BC respectively, and MUC4 scores 2 and 3 (0-3) were deemed positive. A cohort of 56 TNBC and 90 HER2+BC, stage I-III were retrospectively retrieved from Hospital Fernández and Instituto Henry Moore from 2013-2017, and clinicopathological and treatment characteristics were obtained from electronic records. TNBC were treated with adjuvant (41) or neoadjuvant CT +/- RT (15). HER2+BC patients received adjuvant Tz + CT. The association between MUC4 and OS was assessed by Kaplan Meier and log rank test and between MUC4 and TILs using Chi2. JIMT-1 HER2+ BC, de novo resistant tumors to Tz, containing a doxycycline (Dox)-inducible shRNA MUC4 plasmid (JIMT-1shMUC4) growing in nude mice were treated with IgG, Tz, DN or Tz + DN. Tumor growth was measured and macrophages and NK cells were determined in the TME by flow cytometry. Anti-asialo GM1 and clodronate-encapsulated liposomes were used to deplete NK cells and macrophages, respectively. Results We found an inverse relationship between TILs and MUC4 expression in HER2+ and TNBC (P=0.02 and P= 5 x10-5, respectively). Patients with MUC4+ TNBC have a shorter OS (P=0.03) and MUC4 was an independent predictor of OS [P=0.01; HR 4.9 (95%CI 1.4-17.0)]. To study MUC4 involvement in macrophage and NK cells recruitment in a Tz resistant model, nude mice bearing JIMT-1-shMUC4 tumors were treated or not with Dox to abolish MUC4 expression. Both groups received IgG, Tz, DN or DN + Tz. In control groups (without Dox), only Tz + DN administration was able to inhibit tumor growth (75% inhibition, P<0.0001 vs. IgG), in line with our previous results, and DN treatment reduced MUC4 expression. Knockdown of MUC4 expression by Dox, showed that Tz alone was effective in inhibiting JIMT-shMUC4 tumor growth at similar levels than Tz + DN group. Tumor growth inhibition was accompanied by an increase in NK cells activation and degranulation, and a rise in M1/M2 macrophage ratio. Depletion of macrophages or NK cells totally blunted antitumor effect of Tz + DN in control tumors. In MUC4-silenced tumors only macrophage depletion was able to abolish Tz antitumor effect. Conclusion Our results suggest that i) MUC4 expression is associated with immunologically “cold” HER2+ and TNBC, inducing an immunosuppressive TME that reflects in poor DFS/OS, and it confers resistance to Tz in HER2+ BC; ii) elimination of MUC4 expression reverses resistance to Tz; iii) tumor infiltrating macrophages are critical to the anti-tumor response in HER2+ BC. Patients with MUC4+ HER2+ or MUC4+ TNBC should benefit from sTNF blockade treatment leading to MUC4 downregulation and higher TILs, which would result in a better response to Tz and probably to immune checkpoint inhibitors. Citation Format: Roxana Schillaci, Sofia Bruni, Florencia Mauro, María F Mercogliano, Agustina Roldan-Deamicis, Cecilia J Proietti, Rosalía Cordo-Russo, Gloria Inurrigarro, Agustina Dupont, Carla Adami, Daniel Lopez Della Vecchia, Sabrina Barchuck, Silvina Figurelli, Ernesto Gil Deza, Sandra Ares, Felipe G Gercovich, Patricia V Elizalde. Mucin 4 expression in high risk breast cancer: Predicting and overcoming resistance to immunotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-32.

Evaluation of antitumor metastasis via immunostimulating activities of pectic polysaccharides isolated from radish leaves

We investigated the antimetastatic effects of polysaccharides (RWP) isolated from radish leaves by hot water extraction and ethanol precipitation. From the sugar composition analysis, RWP was estimated to be a pectic polysaccharide. Peritoneal macrophages stimulated with RWP significantly augmented the production of various cytokines. In an assay for natural killer (NK) cell activity, both intravenous (i.v.) and oral administration of RWP significantly enhanced NK cells cytotoxicity against YAC-1 lymphoma. In an experiment on lung metastasis of Colon26-M3.1 carcinoma, prophylactic i.v. and oral administration of RWP inhibited metastasis by 77.2% and 55.0%, respectively, at 1000 μg/mouse. Furthermore, pretreatment with anti-asialo GM1 for NK cells depletion partly suppressed the inhibitory effect of RWP on lung metastasis. Thus, our study results suggest that RWP has superior antimetastatic activity, and this effect is related to the activation of various immune factors.

CX3CL1 Recruits NK Cells Into the Central Nervous System and Aggravates Brain Injury of Mice Caused by Angiostrongylus cantonensis Infection.

Background Angiostrongylus cantonensis (A. cantonensis), is a food-borne zoonotic parasite that can cause central nervous system (CNS) injury characterized by eosinophilic meningitis. However, the pathogenesis of angiostrongylosis remains elusive. Natural killer cells (NK cells) are unique innate lymphocytes important in early defense against pathogens. The aim of this study was to investigate the role of NK cells in A. cantonensis infection and to elucidate the key factors that recruit NK cells into the CNS.MethodsMouse model of A. cantonensis infection was established by intragastric administration of third-stage larvae. The expression of cytokines and chemokines at gene and protein levels was analyzed by qRT-PCR and ELISA. Distribution of NK cells was observed by immunohistochemistry and flow cytometry. NK cell-mediated cytotoxicity against YAC-1 cells was detected by LDH release assay. The ability of NK cells to secrete cytokines was determined by intracellular flow cytometry and ELISA. Depletion and adoptive transfer of NK cells in vivo was induced by tail vein injection of anti-asialo GM1 rabbit serum and purified splenic NK cells, respectively. CX3CL1 neutralization experiment was performed by intraperitoneal injection of anti-CX3CL1 rat IgG.ResultsThe infiltration of NK cells in the CNS of A. cantonensis-infected mice was observed from 14 dpi and reached the peak on 18 and 22 dpi. Compared with uninfected splenic NK cells, the CNS-infiltrated NK cells of infected mice showed enhanced cytotoxicity and increased IFN-γ and TNF-α production ability. Depletion of NK cells alleviated brain injury, whereas adoptive transfer of NK cells exacerbated brain damage in A. cantonensis-infected mice. The expression of CX3CL1 in the brain tissue and its receptor CX3CR1 on the CNS-infiltrated NK cells were both elevated after A. cantonensis infection. CX3CL1 neutralization reduced the percentage and absolute number of the CNS-infiltrated NK cells and relieved brain damage caused by A. cantonensis infection.ConclusionsOur results demonstrate that the up-regulated CX3CL1 in the brain tissue recruits NK cells into the CNS and aggravates brain damage caused by A. cantonensis infection. The findings improve the understanding of the pathogenesis of angiostrongyliasis and expand the therapeutic intervention in CNS disease.

Natural Killer Cells are Essential for Allograft Vasculopathy in a Model of CD4+ T Cell-Mediated Chronic Rejection

Purpose In transplantation, recipient CD4+ T cells have two potential means of MHC class II-restricted donor antigen detection: (1) the direct recognition of allogeneic MHC class II expressed by donor antigen-presenting cells (APCs) and (2) the indirect recognition of donor antigens processed and presented by recipient APCs. As donor-derived APCs are gradually depleted over time, the indirect pathway emerges as the dominant mode of allorecognition in the long-term. A clear role of direct CD4+ T cells as effector cells has been shown in acute rejection, however, the role of indirect CD4+ T cells in chronic rejection is less clear. The function of NK cells in solid organ transplantation is multifaceted, but they have been implicated in several different animal models of chronic rejection. Our study sought to test the hypothesis that NK cells are the key effectors in sustaining an anti-donor response in a model of indirect CD4+ T cell mediated chronic allograft vasculopathy (CAV). Methods We utilized a mouse model of cardiac transplantation with MHC class II-deficient B6.C2D (H-2b) hearts engrafted into immune-deficient BALB/c.rag-/- (H-2d) recipients. Recipients had no functional adaptive immunity but an intact innate immune system. After transplantation, the recipients received purified polyclonal BALB/c CD4+ T cells with or without concurrent administration of an NK cell depleting antibody (anti-Asialo GM1). Allografts and recipient spleens were subsequently assessed after 30 days. Results All allografts demonstrated cardiac contraction, as detected by abdominal palpation, for 30 days both with and without CD4+ T cell reconstitution. Only 1/9 (11%) of allografts from un-reconstituted recipients demonstrated evidence of CAV, while 5/7 (72%) of allografts from recipients that received adoptively transferred CD4+ T cells showed disease (p Conclusion In the context of immunodeficient hosts, CD4+ T cells are sufficient to mediate chronic allograft vasculopathy, independent of recipient CD8+ T cell and B cell responses. Recipient NK cells are necessary for the development of this form of chronic rejection.

Epithelial membrane protein 2 (Emp2) modulates innate immune cell population recruitment at the maternal-fetal interface

Epithelial membrane protein 2 (EMP2) is a tetraspan membrane protein that has been revealed in cancer and placental models to mediate a number of vascular responses. Recently, Emp2 modulation has been shown to have an immunologic effect on uterine NK cell recruitment in the mouse placenta. Given the importance of immune cell populations on both placental vascularization and maternal immune tolerance of the developing fetus, we wanted to better characterize the immunologic effects of Emp2 at the placental-fetal interface. We performed flow cytometry of WT and Emp2 KO C57Bl/6 mouse uterine horns at GD12.5 to characterize immune cell populations localized to the various components of the maternal-fetal interface. We found that Emp2 KO decidua and placenta showed an elevated overall percentage of CD45+ cells compared to WT. Characterization of CD45+ cells in the decidua of Emp2 KO dams revealed an increase in NK cells, whereas in the placenta, Emp2 KO dams showed an increased percentage of M1 macrophages (with an increased ratio of M1/M2 macrophages). Given the differences detected in uNK cell populations in the decidua, we further characterized the interaction between Emp2 genetic KO and NK cell deletion via anti-asialo GM1 antibody injections. While the double knock-out of Emp2 and NK cells did not alter individual pup birthweight, it significantly reduced total litter weight and size by ∼50 %. In conclusion, Emp2 appears to regulate uNK and macrophage cell populations in pregnancy.

In Vivo Cytotoxicity by α-GalCer-transactivated NK Cells.

Invariant natural killer T (iNKT) cells are innate-like, lipid-reactive T lymphocytes known for their potent immunomodulatory properties. In addition to expressing and utilizing cytolytic effector molecules of their own against certain target cells, iNKT cells can be stimulated with α-galactosylceramide (α-GalCer) to augment the cytotoxic capacity of natural killer (NK) cells. Herein, we describe a flow cytometry-based in vivo killing assay that enables examination of α-GalCer-promoted cytotoxicity against β2 microglobulin knockout (β2M-/-) target cells, which mimic tumor and virus-infected cells displaying little to no MHC class I molecules on their surface. Using an anti-asialo GM1 antibody, which depletes NK cells but not iNKT cells, we confirmed that the increased clearance of β2M-/- cells in α-GalCer-primed recipients was mediated by NK cells. The protocol detailed here can be leveraged to assess the functional fitness of iNKT cells and their crosstalk with NK cells and to further our understanding of α-GalCer-promoted cytotoxicity in preclinical immunotherapeutic applications.

Depletion of NK Cells Resistant to Ionizing Radiation Increases Mutations in Mice After Whole-body Irradiation.

Ionizing radiation is a very powerful genetic mutagenic agent. Although immune cells are very sensitive to radiation, their sensitivity varies between different types of immune cell. We hypothesized that radiation-resistant immune cells survive after irradiation and then play a role in removing mutant cells. Splenic lymphocytes and mice were irradiated with γ-rays. Cell populations were analyzed using flow cytometry after dyeing with antibodies and expression of B-cell lymphoma 2 (BCL2) was measured by western blot analysis. To deplete natural killer (NK) cells, anti-asialo GM1 antiserum was used. Micronuclei in polychromatic erythrocytes were measured by May-Grunwald/Giemsa staining. H-2Kb loss variant in T-cells induced by irradiation of B6C3F1 mice were detected by flow cytometry. When splenic lymphocytes were irradiated in vitro, B cells notably died, while NK cells did not. In vivo, on the third day after whole-body irradiation, the total number of lymphocytes in the spleen decreased rapidly, but the proportion of NK cells was approximately three times higher than that of the normal control group. In addition, it was confirmed that high expression of BCL2 in NK cells was maintained after irradiation, whereas that of B-cells was not. Removal of NK cells by injection with anti-asialo GM1 antiserum immediately after irradiation increased the micronuclei of polychromatic erythrocytes in the bone marrow and the variant fraction with H-2kb loss in the spleen. These results provide important evidence that radioresistant NK cells apparently survive by escaping apoptosis in the early stages after irradiation, and work to eliminate mutant cells resulting from γ-ray irradiation. Future studies are needed to reveal why NK cells are resistant to radiation and the in-depth mechanisms involved in the elimination of radiation-induced mutant cells.

The Anti-B-Cell Maturation Antigen (BCMA) Antibody-α-Amanitin Conjugate Hdp-101 Induces Immunogenic Cell Death and Immunologic Memory in Models of Multiple Myeloma

Background: Patients with relapsed/refractory myeloma continue to represent an unmet medical need, and this is especially true for those with deletion (del) 17p, who have an inferior prognosis. We previously reported the ability of HDP-101 to induce anti-proliferative and pro-apoptotic effects in myeloma cell lines and primary patient samples. HDP-101 showed enhanced efficacy against del 17p models, which are characterized by concurrent haploinsufficiency of RNA polymerase II subunit A (POLR2A), which is the target of amanitin. This occurred in conjunction with activation of the endoplasmic reticulum stress, and at least two arms of the unfolded protein response. Also, a single dose of HDP-101 was sufficient to induce cures in murine models of myeloma both with and without TP53 and/or POLR2A. Therefore, we sought to better understand the mechanisms of action of HDP-101 and its potential immunologic sequelae. Methods: Pre-clinical studies were performed using HDP-101, the unconjugated BCMA antibody, free α-Amanitin, and a non-targeting control Amanitin antibody drug conjugate in myeloma cell line models and in vivo. These included H929, MM1.S, and MOLP-8 TP53 wild-type (WT) lines and isogenic cells in which TP53 had been knocked out (KO) using genome editing techniques. To further model del 17p and POLR2A haploinsufficiency, POLR2A expression was knocked down using sequence-specific shRNAs. Tumor rechallenge experiments were performed after 100 days of tumor free survival in NOD.CB17-Prkdcscid/J mice with longitudinal monitoring of tumor growth kinetics. Results: HDP-101 induced immunogenic cell death (ICD) in myeloma cell lines as determined by increased expression of Calreticulin (CRT), Heat shock protein (HSP)-70, and release of High mobility group box 1 (HMGB1) by flow cytometry and Western blotting. Microscopic analysis of ICD upon treatment with HDP-101 in MM1.S cells showed a speckled arrangement of both CRT and HMGB1 on the cell surface. In addition, an increased ADP/ATP ratio was observed across three different cell lines in a TP53-independent manner suggestive of an operational ICD pathway. Using our current knowledge of ER stress activation and induction of ICD, we evaluated the combination of bortezomib and HDP-101 in H929, MM1.S, and MOLP-8 cells and primary samples. A significant loss of cellular viability (p<0.01) was observed upon combination treatment across the cell lines, and primary samples also showed a similar effect but only in CD138+ve populations while sparing the CD138-ve fraction. NOD.CB17-Prkdcscid/J mice injected with MM1.S luciferase-transfected cells uniformly developed systemic disease, and one dose of HDP-101 induced cures with no evidence of relapse out to 100 days. These same mice were then rechallenged with MM1.S cells but none developed recurrent disease, suggesting the generation of immunologic memory. Natural killer (NK) cells isolated from these mice showed ex vivo activity against myeloma cell lines which was greatest against MM1.S cells. Finally, cured mice were treated with the anti-asialo GM1 antibody that depletes NK cells or control serum first, and then rechallenged with MM1.S cells. While MM1.S-based tumors could not become established in control serum treated mice, pre-treatment with the anti-asialo GM1 antibody did allow tumors to develop. Studies to compare the activity of HDP-101 with other antibody drug conjugates are currently ongoing. Conclusions: Our results support the statement that HDP-101 is a novel anti-BCMA antibody drug conjugate that shows potent activity against drug-naïve and -resistant models of myeloma, and has the potential to show enhanced anti-tumor activity against del 17 p myeloma. Moreover, its mechanism of action involves both a direct cytotoxic effect, as well as the induction of immunogenic cell death, with the latter potentially leading to immunologic memory. These studies were supported by a Leukemia & Lymphoma Society Specialized Center of Research (SCOR-12206-17). Disclosures Jones: Asylia Therapeutics, Inc.: Current equity holder in private company, Patents & Royalties. Lee:Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Genentech: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Sanofi: Consultancy; Daiichi Sankyo: Research Funding; Regeneron: Research Funding; Genentech: Consultancy. Manasanch:Sanofi: Honoraria, Research Funding; Novartis: Research Funding; JW Pharma: Research Funding; Merck: Research Funding; Quest Diagnostics: Research Funding; Takeda: Honoraria; BMS: Honoraria; Glaxo Smith Kline: Honoraria; Adaptive Biotechnologies: Honoraria. Pahl:Heidelberg Pharma AG: Current Employment. Orlowski:Sanofi-Aventis, Servier, Takeda Pharmaceuticals North America, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., AstraZeneca, BMS, Celgene, EcoR1 Capital LLC, Forma Therapeutics, Genzyme, GSK Biologicals, Ionis Pharmaceuticals, Inc., Janssen Biotech, Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners, Regeneron Pharmaceuticals, Inc.,: Honoraria, Membership on an entity's Board of Directors or advisory committees; Laboratory research funding from BioTheryX, and clinical research funding from CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Research Funding; STATinMED Research: Consultancy; Founder of Asylia Therapeutics, Inc., with associated patents and an equity interest, though this technology does not bear on the current submission.: Current equity holder in private company, Patents & Royalties.

NK cell-derived exosomes improved lung injury in mouse model of Pseudomonas aeruginosa lung infection

BackgroundPseudomonas aeruginosa (PA) is one of the most common bacteria that causes lung infection in hospital. The aim of our study is to explore the role and action mechanism of NK cells in lung PA infection.MethodsIn this present study, 2.5 × 108 CFU/mouse PA was injected into murine trachea to make lung PA infection mouse model. Anti-asialo GM1 was used to inhibit NK cell. The percentage of NK cells was ensured by flow cytometry, and the M1- and M2-polarized macrophages were determined by flow cytometry, qRT-PCR, and ELISA assay. Besides, H&E staining was performed to ensure the pathological changes in lung tissues. Transmission electron microscopy and western blot were carried out to identify the exosome.ResultsHere, in the mouse model of PA lung infection, NK cell depletion caused M2 polarization of lung macrophage, and exacerbated PA-induced lung injury. Next, our data shown that M2 macrophage polarization was enhanced when the generation of NK cell-derived exosome was blocked in the co-culture system of NK cells and macrophages. Subsequently, we demonstrated that NK cells promoted M1 macrophage polarization both in PA-infected macrophage and the mouse model of PA lung infection, and attenuated lung injury through exosome.ConclusionOverall, our data proved that NK cell may improve PA-induced lung injury through promoting M1 lung macrophage polarization by secreting exosome. Our results provide a new idea for the treatment of PA lung infection.

Asialo GM1-positive liver-resident CD8 T cells that express CD44 and LFA-1 are essential for immune clearance of hepatitis B virus.

Persistent hepatitis B virus (HBV) infection results in chronic liver diseases that may progress to chronic hepatitis, liver cirrhosis, and subsequent hepatocellular carcinoma. Previous studies demonstrated that adaptive immunity, in particular CD8 T cells, is critical in HBV elimination. Recent studies have revealed a distinct tissue-localized T cell lineage, tissue-resident memory (TRM) cells, that is crucial for protective immunity in peripheral tissues. In this study, we showed that treatment with an anti-asialo GM1 (ASGM1) antibody (Ab), which depletes NK cells, led to impairment of HBV clearance in a mouse animal model. Unexpectedly, the ability to clear HBV was not significantly impaired in NFIL3 KO mice, which are deficient in NK cells, implying that other non-NK ASGM1-positive immune cells mediate HBV clearance. We isolated intrahepatic ASGM1-positive cells from NFIL3 KO mice and analyzed the immune phenotype of these cells. Our results demonstrated a distinct population of CD44+ LFA-1hi CD8 T cells that were the major intrahepatic ASGM1-positive immune cells in NFIL3 KO mice. Importantly, transcriptome analysis revealed that these ASGM1-positive CD8 T cells had distinct gene profiles and shared a similar core gene signature with TRM cells. In addition to both transcriptional and phenotypic liver residency characteristics, ASGM1-positive CD8 T cells were able to home to and be retained in the liver after adoptive transfer. Taken together, our study results indicate that these ASGM1-positive liver-resident CD8 T cells are the major effector immune cells mediating anti-HBV immunity.

Regulatory Role for NK Cells in a Mouse Model of Systemic Juvenile Idiopathic Arthritis.

Mice deficient in IFN-γ (IFN-γ knockout [KO] mice) develop a systemic inflammatory syndrome in response to CFA, in contrast to CFA-challenged wild-type (WT) mice who only develop a mild inflammation. Symptoms in CFA-challenged IFN-γ KO resemble systemic juvenile idiopathic arthritis (sJIA), a childhood immune disorder of unknown cause. Dysregulation of innate immune cells is considered to be important in the disease pathogenesis. In this study, we used this murine model to investigate the role of NK cells in the pathogenesis of sJIA. NK cells of CFA-challenged IFN-γ KO mice displayed an aberrant balance of activating and inhibitory NK cell receptors, lower expression of cytotoxic proteins, and a defective NK cell cytotoxicity. Depletion of NK cells (via anti-IL-2Rβ and anti-Asialo-GM1 Abs) or blockade of the NK cell activating receptor NKG2D in CFA-challenged WT mice resulted in increased severity of systemic inflammation and appearance of sJIA-like symptoms. NK cells of CFA-challenged IFN-γ KO mice and from anti-NKG2D-treated mice showed defective degranulation capacities toward autologous activated immune cells, predominantly monocytes. This is in line with the increased numbers of activated inflammatory monocytes in these mice which was particularly reflected in the expression of CCR2, a chemokine receptor, and in the expression of Rae-1, a ligand for NKG2D. In conclusion, NK cells are defective in a mouse model of sJIA and impede disease development in CFA-challenged WT mice. Our findings point toward a regulatory role for NK cells in CFA-induced systemic inflammation via a NKG2D-dependent control of activated immune cells.