NK Cells In Mice Research Articles

Downregulation of NK cell activities in Apolipoprotein C-III-induced hyperlipidemia resulting from lipid-induced metabolic reprogramming and crosstalk with lipid-laden dendritic cells

ObjectiveApolipoprotein C-III (Apoc3) is a key component of triglyceride-rich lipoproteins (TRL). The Apoc3-transgenic mice are characterized by high levels of plasma triglyceride and free fatty acids (FFAs). Apoc3 stimulates human monocytes via activation of the NLRP3 inflammasome. Considering the NK cell downregulation in obese individuals and the possible stimulatory-effects of macrophages, variations of NK cell functions and underlying mechanisms were investigated in mice with Apoc3-induced hyperlipidemia. MethodsVariations of activities and glycolipid metabolism in NK cells of the Apoc3-transgenic mice with hyperlipidemia were detected. Molecular mechanisms of lipid-induced metabolic-reprogramming in NK cells were analyzed based on the transcriptome sequencing. Finally, effects of DCs in mice with hyperlipidemia on NK cell functions were determined. ResultsImpaired number and function of NK cells in Apoc3TG mice was involved with the increased fatty acid oxidation and decreased glycolysis. Increased uptake of FFAs in Apoc3TG-NK cells contributed to the peroxisome proliferator-activated receptor (PPAR) activation and the downstream PTEN–AKT–mTOR/FOXO1 signaling pathway. Inhibition of PPAR or CPT1α only partly reversed the IFN-γ production of Apoc3TG-NK cells, but completely restored IFN-γ secretion by palmitic acid-treated NK cells ex vivo, indicating that other factors contributed to the Apoc3TG-NK cell downregulation. Meanwhile, Apoc3TG-DCs, which contained more lipids in the cytoplasm, depended on reactive oxygen species (ROS) to increase the expressions PD-L1, TGF-β1, and NKG2D ligands and suppress NK cell activities. DCs of the Apoc3TG-CD36−/+ hybrid mice with less intracellular lipids and ROS production could not inhibit NK cells, indicating that intracellular FFAs promoted the immune-modulatory function of DCs. ConclusionsThe downregulation of NK cell activities in individuals with Apoc3-induced hyperlipidemia was due to the increased fatty acid oxidation in NK cells and the bystander suppression caused by lipid-laden DCs. The dual recovery function of NK cells and DCs would improve the prognosis of patients with metabolic syndrome.

Modulation of CXCR1 and CXCR3 expression on NK cells via Tim-3 in a murine model of primary biliary cholangitis

Tim-3, which is expressed on a variety of innate immune cells including NK cells, plays a key role in many autoimmune diseases. However, the immunomodulatory actions of Tim-3 on NK cells in primary biliary cholangitis (PBC) remain uncertain. Using a murine model of PBC we evaluated the expression of Tim-3 and its ligand Gal-9 in peripheral blood, liver, and spleen. Additionally, we studied Tim-3 regulation of chemokine receptors (CXCR1 and CXCR3) in vitro. Flow cytometric analysis indicated large numbers of infiltrating NK cells in the liver which exhibited high expression of Tim-3 and CXCR3. Moreover, we found overexpression of CXCR1 in liver tissue and liver-derived NK cells in PBC mice. We also observed lower levels of soluble Tim-3 in the serum of PBC mice. In vitro experiments with liver-derived NK cells from PBC mice indicated that CXCR3 was up-regulated by treatment with recombinant mouse TIM-3 Fc (rmTim-3 Fc) to activate the Tim-3 pathway. Furthermore, stimulating normal mouse spleen NK cells with poly I:C resulted in elevated expression of CXCR1 and interferon-γ release. Nonetheless, adding rmTim-3 Fc or rmGal-9 significantly down-regulated CXCR1 expression and IFN-γ release in NK cells activated by poly I:C, proposing a means to exploit the Tim-3 pathway to reverse responses in NK cells. In conclusion, our data demonstrate that dysregulation of Tim-3/Gal-9 is involved in modulating the local immune microenvironment in PBC mice. Our findings highlight the potential of Tim-3 pathway to modulate chemokine responses in NK cells during autoimmunity.

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.

P74: Effects of different stimulation protocols on the percentage and phenotype of uterine NK cells in mice

P74: Effects of different stimulation protocols on the percentage and phenotype of uterine NK cells in mice

T-box transcription factors Eomes and T-bet regulate primary human NK cell IFNγ response and in vivo persistence

Abstract T-box transcription factors (TF) Eomes and T-bet are vital for NK cell development. However, their role in mature NK cell homeostasis and function remains unclear. Recently we reported that Eomes regulates mouse NK cell cytotoxicity and stage-specific survival using an NK-specific, tamoxifen-inducible Eomes KO model. However, the role of Eomes and T-bet have not been investigated in primary human NK (hNK) cell biology using genetic loss-of-function approaches. We hypothesized that these TFs play a critical role in maintaining NK cell functional molecular programs. Here we used CRISPR-Cas9 to genetically delete Eomes and T-bet expression in primary hNK cells. In vitro stimulation assays revealed that IFNγ response is impaired in CD56bright NK cells only when Eomes is deleted (p<0.01) but not T-bet. This impairment is even more striking when both TFs are deleted, resulting in both CD56bright and CD56dim NK cells having reduced IFNγ (p<0.01 and p<0.05, respectively). Using NSG xenografts, we found that Eomes/T-bet double CRISPR-edited (DKO) hNK cells have impaired persistence/expansion in vivo. After 3 weeks, we recovered in the spleen on average 3E5 control compared to only 6E4 DKO hNK cells. A similar ~80% decrease was also observed in the blood. To determine the mechanism of this, we assessed proliferation and apoptosis of control compared to DKO cells. Ki-67 and dye dilution revealed impaired proliferation of DKO cells in vivo, and we also observed increased apoptosis measured by Annexin V/7-AAD staining (p<0.05). Our data suggest that Eomes and T-bet are critical for the function and homeostasis of mature hNK cells. Understanding their function in mature NK cells will provide insights to improve NK cell therapy for diseases such as cancer.

Short-term IL-15 priming leaves a long-lasting signalling imprint in mouse NK cells independently of a metabolic switch.

IL-15 priming of NK cells is a broadly accepted concept, but the dynamics and underlying molecular mechanisms remain poorly understood. We show that as little as 5 min of IL-15 treatment in vitro, followed by removal of excess cytokines, results in a long-lasting, but reversible, augmentation of NK cell responsiveness upon activating receptor cross-linking. In contrast to long-term stimulation, improved NK cell function after short-term IL-15 priming was not associated with enhanced metabolism but was based on the increased steady-state phosphorylation level of signalling molecules downstream of activating receptors. Inhibition of JAK3 eliminated this priming effect, suggesting a cross talk between the IL-15 receptor and ITAM-dependent activating receptors. Increased signalling molecule phosphorylation levels, calcium flux, and IFN-γ secretion lasted for up to 3 h after IL-15 stimulation before returning to baseline. We conclude that IL-15 rapidly and reversibly primes NK cell function by modulating activating receptor signalling. Our findings suggest a mechanism by which NK cell reactivity can potentially be maintained in vivo based on only brief encounters with IL-15 trans-presenting cells.

Abstract IA09: The IRE1 ER stress sensor activates natural killer cell immunity by regulating c-Myc

Abstract Natural killer (NK) cells are critical mediators of host immunity to pathogens. Here, we demonstrate that the endoplasmic retic- ulum stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box-binding protein 1 (XBP1) drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mammalian target of rapamycin signaling pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, and NK cells with c-Myc haplo- insufficency phenocopied IRE1α-XBP1 deficiency. c-Myc overexpression largely rescued the proliferation defect in IRE1α−/− NK cells. Like c-Myc, IRE1α-XBP1 also promotes oxidative phosphorylation in NK cells. Overall, our study identifies a IRE1α-XBP1- cMyc axis in NK cell immunity, providing insight into host protection against infection and cancer. Citation Format: Laurie H. Glimcher. The IRE1 ER stress sensor activates natural killer cell immunity by regulating c-Myc [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr IA09.

Biological sex, not reproductive cycle, influences peripheral blood immune cell prevalence in mice.

Traditionally the female sex, compared with the male sex, has been perceived as having greater variability in many physiological traits, including within the immune system. We investigated effects of biological sex and the female reproductive cycle on numbers of circulating leukocytes in C57BL/6J mice. We show that biological sex, but not female reproductive cyclicity, has a significant effect on peripheral blood immune cell prevalence and variability, and that sex differences were not consistent amongst common inbred laboratory mouse strains. We found that male C57BL/6J mice, compared with female mice, have greater variability in peripheral blood immunophenotype, and that this was influenced by body weight. We created summary tables for researchers to facilitate experiment planning and sample size calculations for peripheral immune cells that consider the effects of biological sex. Immunophenotyping (i.e. quantifying the number and types of circulating leukocytes) is used to characterize immune changes during health and disease, and in response to pharmacological and other interventions. Despite the importance of biological sex in immune function, there is considerable uncertainty amongst researchers as to the extent to which biological sex or the female reproductive cycle influence blood immunophenotype. We quantified circulating leukocytes by multicolour flow cytometry in young C57BL/6J mice and assessed the effects of the reproductive cycle, biological sex, and other experimental and biological factors on data variability. We found that there are no significant effects of the female reproductive cycle on the prevalence of peripheral blood B cells, NK cells, CD4+ T cells, CD8+ T cells, monocytes, or neutrophils. Immunophenotype composition and variability do not significantly change between stages of the female reproductive cycle. There are, however, sex-specific differences in immune cell prevalence, with fewer monocytes, neutrophils, and NK cells in female mice. Surprisingly, immunophenotype is more variable in male mice, and weight is a significant contributing factor. We provide tools for researchers to perform a priori sample size calculations for two-group and factorial analyses. We show that immunophenotype varies between inbred mouse strains, and that using equal sample sizes of male and female mice is not always appropriate for within-sex evaluations of immune cell populations in peripheral blood.

Protopanaxadiol improves endometriosis associated infertility and miscarriage in sex hormones receptors-dependent and independent manners.

Background: Patients with endometriosis (EMs) have high risks of infertility and spontaneous abortion. How to remodel the fertility of patients with EMs has always been the hot spot and difficulty in the field of reproductive medicine. As an aglycone of ginsenosides, protopanaxadiol (PPD) possesses pleiotropic biological functions and has high medicinal values. We aimed to investigate the effect and potential mechanism of PPD in the treatment of EMs-associated infertility and spontaneous abortion.Methods: The EMs mice models were constructed by allotransplantation. The pregnancy rates, embryo implantation numbers and embryo resorption rates of control and EMs were counted. RNA sequencing, qRT-PCR, enzyme linked immunosorbent assay (ELISA) and FCM analysis were performed to screen and confirm the expression of endometrial receptivity/decidualization-related molecules, inflammation cytokines and NK cell function-related molecules in vitro and/or in vivo. The SWISS Target Prediction, STRING and Cytoscape were carried out to predict the potential cellular sensory proteins, the protein-protein interaction (PPI) network between sensory proteins and fertility-related molecules, respectively. Micro-CT detection, liver and kidney function tests were used to evaluate the safety.Results: Here, we observe that PPD significantly up-regulates endometrial receptivity-related molecules (e.g., Lif, Igfbp1, Mmps, collagens) and restricts pelvic inflammatory response (low levels of IL-12 and IFN-γ) of macrophage, and further remodel and improve the fertility of EMs mice. Additionally, PPD increases the expression of decidualization-related genes and Collagens, and promotes the proliferation, residence, immune tolerance and anagogic functions of decidual NK cells (low levels of CD16 and NKp30, high levels of Ki67, VEGF, TGF-β) in pregnant EMs mice, and further triggers decidualization, decidual NK cell-mediated maternal-fetal immune tolerance and angiogenesis, preventing pregnant EMs mice from miscarriage. Mechanically, these effects should be dependent on ESRs, PGR and other sensory proteins (e.g., AR). Compared with GnRHa (the clinic first-line drug for EMs), PPD does not lead to the decline of serum estrogen and bone loss.Conclusion: These data suggest that PPD prevents EMs-associated infertility and miscarriage in sex hormones receptors-dependent and independent manners possibly, and provides a potential therapeutic strategy with high efficiency and low side effects to remodels the fertility of patients with EMs.

Single-cell profiling reveals the trajectories of natural killer cell differentiation in bone marrow and a stress signature induced by acute myeloid leukemia

SummaryNatural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56bright NK cells, NK0 cells, as the precursor of both circulating CD56dim NK1-like NK cells and CD56bright NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160high group had a significantly higher survival rate.

Abstract 959: Cellular dynamics of productive anti-tumor response mediating long-term rejection of disseminated lung tumors after systemic therapy with oncolytic adenovirus

Abstract Currently, some of the most advanced and clinically effective solid cancer treatment approaches are based on counteracting immune checkpoint inhibitory pathways (ICI) to activate cytotoxic activity of tumor antigen-specific CD8+ T cells. Unfortunately, the use of ICI targeting drugs provides clinical benefits only to a minority of cancer patients. Although major efforts are put forth to improve understanding of CD8+ T cell-centric adaptive immune mechanisms to increase the the proportion of cancer patients responding to this therapy, the effector functions of innate immunity in mediating tumor rejection remain poorly understood. Using a CD8+ T cell-deficient mouse model and a disseminated human lung cancer model, allowing for a productive replication of human oncolytic adenovirus, AVID-317, in this study we analyzed the recruitment and activation states of hematopoietic and stromal cells that enter the lungs of tumor-bearing mice after systemic oncolytic virus therapy and their correlation with the efficacy of anti-tumor response. Intravenous administration of AVID-317 resulted in a significant suppression of tumor growth and extension of survival of mice with disseminated tumors in the lungs. However, the longitudinal analyses showed that two distinct cohorts of mice could be identified within 21 days after the beginning of the therapy - one where mice responded by a complete rejection of disseminated lung tumors (complete responders) and the other, where mice exhibited unimpeded tumor progression (non-responders). Multi-dimensional flow cytometry analysis revealed that after oncolytic virus treatment, there were quantitative and qualitative differences in tumor-infiltrating cells recovered from the lungs of responder and non-responder mice. After oncolytic virotherapy, the tumor infiltrating cells in non-responder mice were significantly enriched for inflammatory myeloid Ly-6G-, Ly6C-, and CD11b-expressing cells, which also expressed high levels of CD80/CD86 and MHC class II proteins, indicating their highly activated state. In contrast, in responder mice, the tumor infiltrating cells were significantly enriched for B cells, NKT cells, and Ly-6C- and Sca-1-positive stromal cells. The influx of B cells, NK, and NKT cells in the lungs after systemic AVID-317 therapy significantly correlated with tumor burden reduction. Although the relative abundance of NK cells was not different between responder and non-responder mice, the single cell transcriptomics analysis showed that NK cells in responder mice expressed higher levels of KLRG1 and KLRC1 terminal effector markers and perforin. Taken together, our data provides evidence that a systemic therapy with oncolytic adenovirus promotes significant alteration of the tumor microenvironment, leading to the recruitment of lymphocytes, namely B cells, NK cells, and NKT cells, with effector functions, which mediate long-lasting rejection of disseminated lung tumors via innate immune mechanisms. Citation Format: Jia Yao, Svetlana Atasheva, Cedrick B. Young, Dmitry M. Shayakhmetov. Cellular dynamics of productive anti-tumor response mediating long-term rejection of disseminated lung tumors after systemic therapy with oncolytic adenovirus [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 959.

Abstract CT053: Use of CRISPR knock-out screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy

Abstract Natural killer (NK) cells play a key role in tumor immune surveillance by their ability to recognize and kill both hematological and solid tumor cells. NK cell-based immunotherapy is emerging as a powerful treatment against certain malignancies. However, intrinsic and extrinsic mechanisms can lead tumor cells to develop resistance mechanisms to escape NK cell-mediated killing, including up or down-regulation of genes involved in the interaction between tumor and immune cells. Here, we use a “two cell type” (TCT) screening system to identify novel molecular targets regulating NK cell-mediated killing of glioblastoma stem cells (GSC), meningioma cells, as well as head and neck squamous cells carcinomas (HNSCC). By using a whole genome-scale CRISPR-Cas9 library, we induced mutations in tumor cells to mimic loss of function mutations involved in resistance or sensitivity to NK cells. After a round of selection with human peripheral blood NK cells, we profiled the genes that impaired the killing activity of NK cells or increased the sensitivity of tumor cells to NK cells. The most common genes lost in GSC and meningioma cells that lead to increased sensitivity to NK cells are involved in the ER-phagosome pathway, antigen presentation and cellular localization. We then used GSC to validate our top genetic hits identified by the screening. One gene of interest, CHMP2A is a component of ESCRT-III, a complex involved in multi vesicular bodies and exosomes formation known to be involved in intercellular communications, mediating numerous physiological processes among which immunology and tumor biology. We confirmed that impaired expression of CHMP2A in GSC lines increases their sensitivity to NK cell-mediated killing. RNA sequencing demonstrates knock-out (KO) of CHMP2A leads to increased expression of immune response genes, including certain chemokines. Indeed, NK cells demonstrated increased migration towards KO GSC corresponding to higher concentrations of CXCL10, CXCL12 and IL-8 in GSC-KO conditioned media. Since HNSCC are among the most highly immune-infiltrated cancer types, including a high degree of infiltrating NK cells, we next tested CHMP2A-KO-Cal27 cells as a model of HNSCC. Again, we observed an increased sensitivity to NK cells, higher levels of CXCL10 and increased migration of NK cells, demonstrating these mechanisms regulate NK cell-mediated killing of diverse tumor types. Additionally, in vivo studies using a Cal27 xenograft model and adoptive transfer of human NK cells in immunodeficient mice demonstrate increased NK cell-mediated killing of CHMP2A-KO-Cal27 cells compared to wild type Cal27 cells. Together, these studies demonstrate the utility of a TCT screen to identify new mechanisms that mediate resistance of diverse tumor types to NK cell-mediated killing and allow us to now identify novel strategies to further improve this immunotherapeutic approach. Citation Format: Davide Bernareggi, Qi Xie, Briana Prager, Li Xiqing, Silvio Gutkind, Dan S. Kaufman. Use of CRISPR knock-out screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy [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 CT053.

Abstract 2205: TG-1701, a novel irreversible Bruton's kinase (BTK) inhibitor, cooperates with ublituximab-driven ADCC and ADCP in in vitro and in vivo models of ibrutinib-resistant mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a rare subtype of aggressive B-cell non-Hodgkin lymphoma (NHL) that remains incurable with standard therapy. The first-in-class Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has proven to be an effective agent for patients with relapsed/refractory MCL, although development of a cysteine to serine mutation at the BTK catalytic site (BTKC481S) or over-activation of the NF-kB pathway can impair MCL response to most BTK inhibitors (BTKis). TG-1701 is a novel irreversible inhibitor highly specific to BTK, with improved selectivity when compared to ibrutinib, currently being evaluated in a phase 1 clinical trial in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) patients. Here we show that, when compared to irreversible and reversible BTKis including ibrutinib, acalabrutinib, and ARQ-531, TG-1701 showed similar cytotoxic activity either in ibrutinib-sensitive or in ibrutinib-resistant MCL cells. We then evaluated the activity of TG-1701 and ibrutinib on anti-CD20 antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), as ibrutinib has previously been show to block rituximab-mediated ADCC due to off-target inhibition of ITK (Kohrt et al., Blood 2014). In sharp contrast with ibrutinib, which partially antagonized rituximab activity, we found that TG-1701 did not impair the anti-CD20-mediated ADCC and ADCP in a multicellular co-culture system that associated MCL to effector cells. In another set of cell-based assays, we showed that the treatment with TG-1701 led to a strong decrease in IL-10 expression, together with a significant increase in IL-2, IL-6 and IL1RN gene transcription, thus associating TG-1701 exposure with the promotion of an immune response within the tumor microenvironment (TME). Accordingly, TG-1701 cooperated with the novel anti-CD20 antibody ublituximab and the novel PI3Kδ-CK1ϵ dual inhibitor umbralisib in reducing the tumor growth in both ibrutinib-sensitive (REC1) and ibrutinib-resistant (UPN-1res) mouse models of MCL. In accordance with in vitro data, the antitumor activity of the drug combination was associated with increased production of IL-2, IL-6 and TNFα, and with an increased infiltration of mouse macrophages and NK cells. Altogether, these data warrant further clinical investigation of TG-1701-mediated engagement of TME-related anti-tumor effect, and clinical evaluation of the triple combination of TG-1701, ublituximab, and umbralisib, is ongoing. Citation Format: Marcelo L. Ribeiro, Diana Reyes-Garau, Meritxell Vinyoles, Marc Armengol, Hari Miskin, Francesc Bosch, Pablo Menendez, Emmanuel Normant, Gael Roue. TG-1701, a novel irreversible Bruton's kinase (BTK) inhibitor, cooperates with ublituximab-driven ADCC and ADCP in in vitro and in vivo models of ibrutinib-resistant mantle cell lymphoma [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 2205.

Depletion of NK Cells Improves Cognitive Function in the Alzheimer Disease Mouse Model

Despite mounting evidence suggesting the involvement of the immune system in regulating brain function, the specific role of immune and inflammatory cells in neurodegenerative diseases remain poorly understood. In this study, we report that depletion of NK cells, a type of innate lymphocytes, alleviates neuroinflammation, stimulates neurogenesis, and improves cognitive function in a triple-transgenic Alzheimer disease (AD) mouse model. NK cells in the brains of triple-transgenic AD mouse model (3xTg-AD) mice exhibited an enhanced proinflammatory profile. Depletion of NK cells by anti-NK1.1 Abs drastically improved cognitive function of 3xTg-AD mice. NK cell depletion did not affect amyloid β concentrations but enhanced neurogenesis and reduced neuroinflammation. Notably, in 3xTg-AD mice depleted of NK cells, microglia demonstrated a homeostatic-like morphology, decreased proliferative response and reduced expression of neurodestructive proinflammatory cytokines. Together, our results suggest a proinflammatory role for NK cells in 3xTg-AD mice and indicate that targeting NK cells might unlock novel strategies to combat AD.

Abstract PR03: Use of CRISPR knockout screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy

Abstract Natural killer (NK) cells play a key role in tumor immune surveillance by their ability to recognize and kill both hematologic and solid tumor cells. NK cell-based immunotherapy is emerging as a powerful treatment against certain malignancies. However, intrinsic and extrinsic mechanisms can lead tumor cells to develop resistance mechanisms to escape NK cell-mediated killing, including up- or downregulation of genes involved in the interaction between tumor and immune cells. Here, we use a “two cell type” (TCT) screening system to identify novel molecular targets regulating NK cell-mediated killing of glioblastoma stem cells (GSC), meningioma cells, as well as head and neck squamous cell carcinomas (HNSCC). By using a whole genome-scale CRISPR-Cas9 library, we induced mutations in tumor cells to mimic loss of function mutations involved in resistance or sensitivity to NK cells. After a round of selection with human peripheral blood NK cells, we profiled the genes that impaired the killing activity of NK cells or increased the sensitivity of tumor cells to NK cells. The most common genes lost in GSC and meningioma cells that lead to increased sensitivity to NK cells are involved in the ER-phagosome pathway, antigen presentation, and cellular localization. We then used GSC to validate our top genetic hits identified by the screening. One gene of interest, CHMP2A, is a component of ESCRT-III, a complex involved in multivesicular bodies and exosomes formation known to be involved in intercellular communications, mediating numerous physiologic processes among which are immunology and tumor biology. We confirmed that impaired expression of CHMP2A in GSC lines increases their sensitivity to NK cell-mediated killing. RNA sequencing demonstrates knockout (KO) of CHMP2A leads to increased expression of immune response genes, including certain chemokines. Indeed, NK cells demonstrated increased migration towards KO GSC corresponding to higher concentrations of CXCL10, CXCL12, and IL-8 in GSC-KO conditioned media. Since HNSCC are among the most highly immune-infiltrated cancer types, including a high degree of infiltrating NK cells, we next tested CHMP2A-KO-Cal27 cells as a model of HNSCC. Again, we observed an increased sensitivity to NK cells, higher levels of CXCL10, and increased migration of NK cells, demonstrating these mechanisms regulate NK cell-mediated killing of diverse tumor types. Additionally, in vivo studies using a Cal27 xenograft model and adoptive transfer of human NK cells in immunodeficient mice demonstrate increased NK cell-mediated killing of CHMP2A-KO-Cal27 cells compared to wild-type Cal27 cells. Together, these studies demonstrate the utility of a TCT screen to identify new mechanisms that mediate resistance of diverse tumor types to NK cell-mediated killing and allow us to now identify novel strategies to further improve this immunotherapeutic approach. This abstract is also being presented as Poster A25. Citation Format: Davide Bernareggi, Qi Xie, Briana Prager, Li Xiqing, Silvio Gutkind, Dan S. Kaufman. Use of CRISPR knockout screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR03.

Minimal PD-1 expression in mouse and human NK cells under diverse conditions.

PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.

Phenotypically Friendly Allospecific NK Cells Differentially Suppress Allospecific T cell Responses in Prenatal Hematopoietic Chimeras

Abstract Prenatal allospecific tolerance hinges on the developmental selection of NK cells expressing a friendly phenotype that is not cytotoxic to allogeneic donor cells. We wondered if prenatally educated friendly NK cells influenced allospecific T cell responses as an additional mechanism supporting prenatal tolerance. Therefore, we hypothesized that prenatally educated friendly NK cells exert suppressive effects on the induction of allospecific T cell responses. To challenge this hypothesis, we examined the suppression potential of prenatally educated friendly NK cells through gain and loss of function experiments in stable prenatal Balb/c into B6.Thy1.2 chimeras (engrafter mice). We first depleted allospecific NK cells in host engrafter mice and then adoptively transferred Thy1.1+ naïve responder T cells along with CFSE-labeled allogenic or syngeneic target cells into the engrafter hosts. We found that the depletion of allospecific Ly49D+ NK cells in engrafter mice increased Thy1.1+ T cell alloimmunity in vivo. Conversely, adoptive transfer of friendly NK cells suppressed the induction of allospecific T cell responses in host mice. Furthermore, cultured friendly NK cells from engrafter mice showed stronger suppression on the induction of T cell alloimmunity in vitro than that of naïve mice. Lasltly, NK cell-mediated suppression of allospecific T cells required cell contact. From these results, we conclude that prenatally educated friendly NK cell exert enhanced suppressive effects on T cell alloimmunity in vivo and in vitro. Depending on the context of their prenatal education, NK cells can differentially influence T cell responses in prenatal chimeras.

Optimal Treatment Regimen of Using Chemo-Immunotherapy for Solid Tumors

Abstract Chemotherapeutic agents not only cause apoptosis and necrosis of cancer cells but also induce cellular senescence (TIS), resulting in a persistent cell population that re-enters the cell cycle with stemness characteristics via the senescence-associated secretory phenotype. These senescent cancer cells, therefore, seed disease relapses if they are not effectively eliminated. TIS cancer cells lack HLA expression and cannot be targeted by T cells. Herein, we show that chemotherapy-induced senescent B16F10 cells express the NK ligands Rae1e and Ulbp1, and are effectively killed by mouse NK cells in vitro. Docetaxel treatment also induced senescent B16F10 cells in vivo. We have previously shown that the TGF-β trap/IL-15 fusion protein, aka HCW9218, effectively activates NK cells in vivo. Therefore, we treated B16F10 tumor bearing mice with docetaxel and then a single dose of HCW9218 in combination with the ADCC antibody TA99. This treatment regimen exhibited potent antitumor activity compared to docetaxel alone. End point analysis showed that HCW9218-treated tumors have lower mitotic activity, reduced expression of senescence markers, and increased frequencies of infiltrating CD8 T cells. We further showed in this model that HCW9218 treatment followed by anti-PD-L1 therapy was more effective than anti-PD-L1 therapy followed by HCW9218. These results suggest that the optimal sequence of cancer treatment is: (1) standard-of-care chemotherapy to control tumor burden and induce cellular senescence, (2) immunotherapy for removal of senescent cells and enhanced tumor infiltration of CD8+ T cells and (3) checkpoint blockade to maintain infiltrated T cell effector function to enhance therapeutic responses and treatment durability.

The IRE1 endoplasmic reticulum stress sensor activates natural killer cell immunity and promotes natural killer cell memory by regulating c-Myc and mitochondrial respiration

Abstract Natural killer (NK) cells are critical mediators of host immunity to pathogens. Though historically being considered as a part of the innate immune system, NK cells exhibit adaptive features and can ‘remember’ stimuli like inflammation or viral infection and hence are capable of responding more robustly upon re-encountering the stimulation. Successful design of an NK cell-based therapy would ideally rely on harnessing NK cell memory, the key drivers and regulators of which remain poorly understood. Here, we demonstrate that the endoplasmic reticulum stress sensor IRE1α and its substrate transcription factor XBP1 drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mTOR pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. NK cells with c-Myc haploinsufficency phenocopied IRE1α deficiency, while c-Myc overexpression largely rescued the proliferation defect in IRE1α−/− NK cells. Like c-Myc, IRE1α also promotes oxidative phosphorylation in NK cells. Combined, our study identifies an IRE1α-XBP1-cMyc axis in NK cell immunity; and based on this work, we are currently investigating the role of IRE1 in NK cell memory using novel transgenic mouse models and small molecule drug treatment in primary human cells. We are also evaluating the therapeutic benefit of manipulating IRE1 function in CAR-NK cells to treat cancer. Our ongoing study should advance the current knowledge of NK cell memory and also open new opportunities in cancer therapy for patients with otherwise extremely poor prognoses.

IL33 activates CD8+T and NK cells through MyD88 pathway to suppress the lung cancer cell growth in mice.

In this study, we observed the effects of IL-33 on tumor immune response in lung cancer-bearing mice using wild type and MyD88-/- mice respectively. Wild C57BL/6 (C57BL/6WT), MyD88 knockout C57BL/6 mice (C57BL/6 MyD88-/-) and Lewis cells were used in this study. Cell proliferation, cytokine release and cytotoxicity were detected. IL-33 could significantly up-regulate specific cellular immunity, inhibit tumor growth and improve survival time in wild type mice group, and it had dose dependent effect. However, IL-33 had no effect on cell immunity and tumor growth in MyD88-/- mice group. Compared with MyD88-/- mice, IL-33 could significantly increase the ratio of CD8+T cells to neutrophils in wild type mice, while the percentage of tumor infiltrating CD11b+ cells, Mo-MDSC, F4/80+ macrophages and mDC cells decreased significantly in wild type mice group. IL-33 could upregulate the expression of CD107a and IFN-γ in CD8+T cells and NK cells of wild type mice, while IL-33 could not upregulate them in MyD88-/- mice. IL-33 could upregulate the expression of CD40, CD80, CD86 and CD205 in DC cells in wild type mice, induce T cells to differentiate into Th1 cells and enhance tumor cell immunity. IL-33 could promote differentiation and maturation of DC cells through MyD88 pathway, up-regulate the tumor immunity of CD8+T cells and NK cells, and inhibit the proliferation of lung cancer cells.