Natural Killer Cell Signaling Research Articles

Combinatorial cellular therapy in pediatric solid tumors with natural killer (NK) and genetically engineered myeloid cells (GEMys).

2561 Background: Early human studies with Natural Killer (NK) cells have shown promise, harnessing their ability to infiltrate into tumors and be tumoricidal. However, these effects can be limited by adenosine, reactive oxygen species, and TGF-β in the highly immune suppressive tumor microenvironment (TME). TGF-β and downstream SMAD3 signaling in Natural Killer (NK) cells have been shown to cause decreased cytokine production, downregulation of activating cell surface receptors, and attenuated cytotoxic function against solid tumors. Exposure to TGF-β during cell culture generates expanded NK cells with increased resistance to TGF-β signaling, dubbed TGF-β imprinted NK cells. In vitrostudies have shown that these cells have increased cytokine production and cytotoxic function in the presence of TGF-β. However, their in vivo trafficking, modulation of the TME, and therapeutic efficacy remain understudied. Our lab has previously shown that when given to tumor-bearing mice, myeloid cells modified to produce IL-12 (IL-12-Genetically Engineered Myeloid cells, aka IL-12 GEMys) are effective at homing to tumor and metastatic sites and, via IL-12 secretion and TME modulation, increase NK cell presence and activation in these sites. They hold great promise as combination therapy, enhancing the effectiveness of other cellular therapies such as NK cell therapy. A current clinical trial exploring TGF-β Imprinted, Ex Vivo Expanded, Universal Donor NK Cell Infusions in relapsed sarcomas is underway (NCT05634369). Methods: In vitro experiments were conducted with the xCELLigence real-time cell analysis system. The human osteosarcoma cell line 143B and human rhabdomyosarcoma cell line RH-30 were evaluated for cell death after co-culture with healthy donor NK cells or TGF-β imprinted NK cells (E:T ratio of 5:1) in isolation and in combination with IL-12 GEMys (E:T ratio of 2:1). Results: TGF-β imprinted NK cells are significantly more effective at tumor cell killing of both 143B and RH30 cell lines in-vitro compared to regular expanded NK cells (WT NK cells), with >90% decrease in cell index compared to 50-60%, respectively, after 48 hours of co-culture. IL-12 GEMys also significantly enhance the cytotoxic effects of WT NK cells, increasing cell killing to 70-80% compared to co-culture with untransduced GEMy controls. Conclusions: These initial in-vitro experiments provide insight into the promising effectiveness of NK-myeloid cell combination therapies in the treatment of pediatric sarcomas. Ongoing in vitroexperiments include cytokine quantification by ELISA and flow cytometric analysis of NK cell activation and proliferation markers. Our preliminary results provide rationale to continue studying the in vivo efficacy of TGF-β imprinted NK cell monotherapy and in combination with IL-12 GEMys in the treatment of tumor-bearing NSG mice.

Abstract 3615: Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells

Abstract Background: Neuroblastoma (NB) is a common malignant extracranial solid tumor in children, responsible for 11% of cancer deaths in pediatric patients under 15 (Smith et al, Cancer, 2014). High-risk neuroblastoma is a metastatic disease in children aged 18 months or older with L2, M, or MS disease with amplification of the MYCN oncogene (Chuang Pediatr Blood Cancer 2021). Recent analyses by COG show that HR Neuroblastoma still has a 5-year event free survival of 51% despite an extensive treatment regimen (Steven et al, ASCO, 2022). To address improved tumor targeting and overcome antigen escape, chimeric antigen receptor (CAR) technology to Natural Killer (NK) cell therapy has been adapted (Chu/Cairo, et al, Frontiers in Oncology, 2019). TGFβ is a potent mechanism of the NK cell anti-tumor resistance and is present at elevated levels in the NB tumor microenvironment (TME) (Foltz, Cancers, 2018). Studies demonstrated that blockade of TGF-β signaling in NK cells caused the accumulation of NK cells that produce IFN-γ and neutralization of TGF-β prevented NKG2D downregulation and also restored NK cell anti-tumor reactivity (Nayyar/Chu/Cairo, et al Frontierls in Oncology, 2018). ROR1, a transmembrane protein is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al Cancer Immunol Res, 2015). Objective: To determine in vitro cytotoxicity against neuroblastoma cells by ex vivo expanded TGFβ imprinting (TGFβi) NK cells. Methods: TGFβi NK cells are generated by culturing expanded NK cells with irradiated K562-41BBL-mbIL21 in 50 IU/mL IL-2 plus 10 ng/mL TGFβ every 2-3 days for two weeks. NK and TGFβi NK cells specific in-vitro cytotoxicity were compared via luciferase reporter assay against ROR1+ NB cell lines as we previously described (Chu/Cairo, et al, JITC, 2022). Cytokine/chemokine levels secreted by NK and TGFβi NK cells were compared using ELISA assays (Chu/Cairo, et al, JITC, 2022). Results: We have successfully expanded TGFβi NK cells. These TGFβi NK cells showed significantly enhanced in vitro cytotoxicity against NB cells compared to NK cells under TGFβ environment (20+2.4% vs 0%; p<0.001) at E:T=3:1 and consistently, TGFβ-imprinting NK secreted significantly high level of TNF-alpha (393.7pg/ml+16.77pg/ml) against NB cells (p<0.001) compared to NK cells (79.7pg/ml+14.7pg/ml) under TGFβ environment. Conclusion: Our data indicate TGFβi NK cells may be a promising novel therapy to overcome the immunosuppression of TGFβ in the tumor microenvironment for pediatric NB tumors. We will further engineer these TGFβi NK cells to express anti-ROR1 CAR to enhance the targeting specificity against NB tumors and investigate the anti-tumor effects of anti-ROR1 CAR TGFβi NK cells against NB tumors in-vitro and in-vivo (Funded by U54 CA232561). Citation Format: Jessica A. Ayala-Cuesta, Yaya Chu, Keira Foley, Meijuan Tian, Kayleigh Klose, Alyssa S. Mendelowitz, Timothy P. Cripe, Dean A. Lee, Mitchell S. Cairo. Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3615.

Abstract TP284: Transcriptome Profiling of Peripheral Blood Mononuclear Cells in Moyamoya Disease

Objectives: Moyamoya Disease (MMD) is a progressive occlusive cerebrovascular disease with unknown etiology and pathogenesis. Studies have reported dysregulation in extracellular matrix-related molecules and increased circulating inflammatory cytokines/chemokines in MMD patients, suggesting a role of immune responses in the development of MMD. In this study, we utilized single-cell RNA sequencing (scRNA-seq) to investigate the circulating transcriptome immune cell signatures in MMD. Methods: Whole blood samples were collected from bilateral MMD (n=3), non-MMD patients (n=2, aneurysm, or cavernous malformation) and healthy controls (HC) (n=2). Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved. Single Cell 3’ Gene Expression libraries were generated and sequenced. Data were demultiplexed and mapped using the CellRanger. Differentially expressed genes (DEGs) were identified and top biological functions and pathways in each cell type were analyzed. Results: ScRNA-seq data showed differences in several key cell types between MMD and HC, such as CD8+ NKT, Naïve CD8+ T, CD4+ NKT, Naïve B cells and non-classical monocytes. Further analyses of DEGs for CD8+ NKT, Naïve CD8+ T and CD4+ NKT highlighted in MMD revealed a predicted activation in cell death and predicted inhibition in cell viability, cell movement, proliferation, and migration. In addition, eIF2 (eukaryotic initiation factor-2) activation was detected in all three cell types, most prominently in CD8+ NKT cells. Pathway analysis revealed an inhibition in natural killer cell signaling and autophagy pathways. Conclusion: In this study, we identified circulating immune cell signatures in MMD and highlighted the role of CD8+ NKT cells, Naïve CD8+ T cells and CD4+ NKT cells. These cell types manifest distinct pathological processes involved in cellular stress, including inhibition of natural killer cell signaling and disruption in autophagy, which can lead to impaired cytotoxic functions. Active investigations address validation of key cell-type and genes with additional patient cohort. This investigation elucidates the intricate immune responses associated with MMD, providing integral insights for discerning the disease's pathophysiology.

G-CSFR Is a Checkpoint of Natural Killer Cells Against Tumor

G-CSFR is a cell surface receptor that can activate cell proliferation and differentiation after binding to granulocyte colony stimulating factor(G-CSF). However, in natural killer (NK) cells, the biological role of G-CSFR has not been fully elucidated. Here we show that granulocyte colony-stimulating factor receptor, that is encoded by the CSF3R gene, is a novel and potent checkpoint in both human and mouse NK cells fighting against tumors. Previous studies have shown that NK cells under resting state with low expression of G-CSFR, but highly expressed in peripheral blood and bone marrow NK cells after G-CSF mobilization. We therefore used G-CSFR blocking agent in vitro to evaluate the effect of G-CSFR on human NK cells proliferation and cytotoxicity capacity. The results showed that G-CSFR agonist significantly down-regulated the proliferation and cytotoxicity of NK cells in vitro and this is independent of G-CSF stimulation. Since NK92 and YT cell lines highly express G-CSFR, we then knocked down G-CSFR in NK92 and YT cells. The results showed that the low expression of G-CSFR enhanced the expression of CD107a in vitro, indicating G-CSFR may be an inhibitory signaling in NK cells. To the further evaluate the specific role of G-CSFR in NK cells and we therefore constructed NK-specific CSF3R deficiency mice (CSF3R △NK/NK; CSF3Rfl/flNCR1cre, thecre group) and the control group ( CSF3Rfl/fl ( CSF3RWT/WT mice, the flox group). We then evaluated the phenotype, differentiation and functional state of NK cells between the two group. NK-specific CSF3R deficiency increased mature NK subsets in mice, and these CSF3R-deficient NK cells exhibited enhanced activation, cytotoxicity and proliferation, which is in accordance with the role in human NK cells. After constructing four tumor-bearing models, namely MC38 (colon tumor), LLC (lung tumor), MLL-AF8(acute myeloid leukemia), B16-F10(melanoma), we found that CSF3R specific depletion in NK cells reduced the tumor burden, prolonged the survival and reduced tumor metastasis in CSF3R△NK/NK mice (Figure 1A-D), indicating CSF3R depletion in NK cells promotes increased ability against tumors. To further evaluate the molecular mechanism of G-CSFR regulation of NK cell function, single cell RNA sequencing on bone marrow cells from both CSF3R△NK/NK mice and CSF3RWT/WT mice and NK cells were extracted for further subpopulation analysis. UMAP analysis revealed the clustering of bone marrow NK cells from both genotypes into ten subsets and on the basis of the key gene expression of NK cell effector molecules, transcriptional factors, maturation markers, and proliferation marker, we identified NK cell maturation program as mBM5+mBM8, mBM2+mBM3+ mBM6 and mBM0, by which immature NK cells should go through to gain optimal effector function, according to this model, CSF3R△NK/NK mice contained more NK cells belonging to the terminally mature mBM0 than CSF3RWT/WT mice. “Cell Killing” and “leukocyte mediated cytotoxicity” pathway was enriched in CSF3R△NK/NK mice, indicating enhanced activation state of NK cells. In conclusion, our results uncover a potent checkpoint in NK cells protecting against tumors, suggesting a promising approach of targeting G-CSFR for NK cell-based immunotherapies.

Identification of Immune-Related Genes as Biomarkers for Uremia.

Uremia, which is characterized by immunodeficiency, is associated with the deterioration of kidney function. Immune-related genes (IRGs) are crucial for uremia progression. The co-expression network was constructed to identify key modular genes associated with uremia. IRGs were intersected with differentially expressed genes (DEGs) between uremia and control groups and key modular genes to obtain differentially expressed IRGs (DEIRGs). DEIRGs were subjected to functional enrichment analysis. The protein-protein interaction (PPI) network was constructed. The candidate genes were identified using the cytoHubba tool. The biomarkers were identified using various machine learning algorithms. The diagnostic value of the biomarkers was evaluated using receiver operating characteristic (ROC) analysis. The immune infiltration analysis was implemented. The biological pathways of biomarkers were identified using gene set enrichment analysis and ingenuity pathway analysis. The mRNA expression of biomarkers was validated using blood samples of patients with uremia and healthy subjects with quantitative real-time polymerase chain reaction (qRT-PCR). In total, four biomarkers (PDCD1, NGF, PDGFRB, and ZAP70) were identified by machine learning methods. ROC analysis demonstrated that the area under the curve values of individual biomarkers were > 0.9, indicating good diagnostic power. The nomogram model of biomarkers exhibited good predictive power. The proportions of six immune cells significantly varied between the uremia and control groups. ZAP70 expression was positively correlated with the proportions of resting natural killer (NK) cells, naïve B cells, and regulatory T cells. Functional enrichment analysis revealed that the biomarkers were mainly associated with translational function and neuroactive ligand-receptor interaction. ZAP70 regulated NK cell signaling. The PDCD1 and NGF expression levels determined using qRT-PCR were consistent with those determined using bioinformatics analysis. PDCD1, NGF, PDGFRB, and ZAP70 were identified as biomarkers for uremia, providing a theoretical foundation for uremia diagnosis.

Multiplex interrogation of the NK cell signalome reveals global downregulation of CD16 signaling during lentivirus infection through an IL-18/ADAM17-dependent mechanism.

Despite their importance, natural killer (NK) cell responses are frequently dysfunctional during human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) infections, even irrespective of antiretroviral therapies, with poorly understood underlying mechanisms. NK cell surface receptor modulation in lentivirus infection has been extensively studied, but a deeper interrogation of complex cell signaling is mostly absent, largely due to the absence of any comprehensive NK cell signaling assay. To fill this knowledge gap, we developed a novel multiplex signaling analysis to broadly assess NK cell signaling. Using this assay, we elucidated that NK cells exhibit global signaling reduction from CD16 both in people living with HIV-1 (PLWH) and SIV-infected rhesus macaques. Intriguingly, antiretroviral treatment did not fully restore diminished CD16 signaling in NK cells from PLWH. As a putative mechanism, we demonstrated that NK cells increased surface ADAM17 expression via elevated plasma IL-18 levels during HIV-1 infection, which in turn reduced surface CD16 downregulation. We also illustrated that CD16 expression and signaling can be restored by ADAM17 perturbation. In summary, our multiplex NK cell signaling analysis delineated unique NK cell signaling perturbations specific to lentiviral infections, resulting in their dysfunction. Our analysis also provides mechanisms that will inform the restoration of dysregulated NK cell functions, offering potential insights for the development of new NK cell-based immunotherapeutics for HIV-1 disease.

Influenza A Infection Stimulates RIG-I and Enhances Effector Function of Primary Human NK Cells.

Immune surveillance by natural killer (NK) cells and their recruitment to sites of inflammation renders them susceptible to viral infection, potentially modulating their effector function. Here, we analyzed innate RNA receptor signaling in NK cells downstream of direct Influenza A virus (IAV) infection and its impact on NK cell effector function. Infection of NK cells with IAV resulted in the activation of TBK1, NF-ϰB and subsequent type-I IFN secretion. CRISPR-generated knockouts in primary human NK cells revealed that this effect depended on the antiviral cytosolic RNA receptor RIG-I. Transfection of NK cells with synthetic 3p-dsRNA, a strong RIG-I agonist that mimics viral RNA, resulted in a similar phenotype and rendered NK cells resistant to subsequent IAV infection. Strikingly, both IAV infection and 3p-dsRNA transfection enhanced degranulation and cytokine production by NK cells when exposed to target cells. Thus, RIG-I activation in NK cells both supports their cell intrinsic viral defense and enhances their cytotoxic effector function against target cells.

Single-Cell RNA Sequencing Analysis in Murine Biliary Atresia Model Reveals Subpopulation of Biliary Epithelial Cells with Proinflammatory and Fibrogenic Traits

INTRODUCTION: Understanding the pathogenesis of liver fibrosis is critical to impacting outcomes of infants with biliary atresia (BA). In BA, Prominin-1 (Prom1)-expressing hepatic progenitor cell (HPC) populations give rise to biliary epithelial cells (BECs) with ductular reactions (DR). Loss of Prom1 function significantly decreases DRs and fibrosis. Hypothesis: Prom1-expressing HPCs give rise to BECs manifesting activated fibrogenic pathways in BA. METHODS: Experimental BA was induced in neonatal Balb/c mice with intraperitoneal injection of rhesus rotavirus (RRV) vs saline on postnatal day (p) 3. Livers were collected at p3, 10, and 17. EPCAM+/CD31–/CD45–/TER119– live BECs were isolated by fluorescence-activated cell sorting for single-cell RNA sequencing. RESULTS: A total of 44,877 cells were sequenced (saline: 29,195 cells, n = 11; RRV: 15,682 cells, n = 6), graph-based clustering identified 19 clusters of transcriptionally distinct cell types/states. Four distinct BEC subtypes were identified: stem/progenitor, immature, hepatocyte-like, and mature (Figure). All demonstrated activation of inflammation and fibrogenic pathways with RRV treatment. UMAP clustering revealed a unique population of Prom1-expressing immature BECs in RRV p17 samples. Ingenuity pathway analysis (IPA) of this population showed upregulation of several pathways specific to innate immune response, ie natural killer cell and Toll-like receptor signaling. IPA also showed evidence of NFκB signaling with upregulation of proinflammatory cytokines, IL-1β, IL-6, and TNF, and profibrogenic cytokines, Tgfβ1 and Ccl2, in this population.FigureCONCLUSION: A subset of Prom1-expressing immature BECs upregulate proinflammatory and profibrogenic cytokines that likely contribute to fibrosis in BA. These cells and these signaling pathways represent potential therapeutic targets.

Abstract 015: Hypertensive Stretch On The Regulation Of The Immunological Synapse And Secretomes Of Male And Female Renal Endothelial Cells

Immune cells and inflammation contribute to the development of hypertension and renal injury. While endothelial cells are recognized as professional antigen presenting cells, they can express both Major Histocompatibility Complex (MHC) I and II and possess antigen-presenting functions. Endothelial cell activation and inflammation are critical in the development of hypertension. However, the mechanisms by which inflammatory hypertensive stimuli regulate renal endothelial immunological synapse proteins remains unknown. We hypothesize that inflammatory hypertensive mechanical stretch promotes the regulation of MHC I and II and costimulatory molecules on mouse renal endothelial cells (MRECs) in a sex-dependent manner. MRECs derived from both sexes underwent normotensive (5%; 1 Hz), hypertensive (10%, 1 Hz), and supra-hypertensive (15% 1 Hz) uniaxial cyclical stretch for 48 hours. We then assessed CD80, CD86, MHC II, MHC I, CD70, and CD40 by immunoblot analysis. A proteome multiplex assay was used to analyze male and female MREC secretomes during 5%, 10%, and 15% stretch. Hypertensive 10% stretch significantly increased only CD40 expression in males, while it upregulated CD80 and CD70 expression in females (p<0.05). Under 15% stretch, males significantly upregulated MHC I, CD70, and CD40 compared to I-Ab, CD80, CD86, and CD70 in females MRECs (p<0.05). We investigated sex differences and found that females significantly increased CD70 in both 10% and 15% stretch compared to males MRECs (p<0.05). We compared sex differences from our secretome proteomics in male and female MRECs at 10% and 15% stretch. We found that pathways associated with wound healing, Th17 activation, IL-6, and NF-κB signaling were upregulated in male MRECs undergoing 10% stretch. Interestingly, Th17 activation, natural killer cell signaling, and IL-23 signaling were downregulated in female MRECs undergoing 10% stretch. Under 15% stretch, IL-13 signaling, HMBG1 signaling, B cell signaling, and IL-13 signaling were downregulated in MRECs from both sexes. Our data provides novel insights into how inflammatory hypertensive stretch regulates immunological synapse proteins and secretomes in renal endothelial cells by stretch and sex-dependent manner.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling.

FACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated in both epithelial and natural killer (NK) cells. The histone acetyltransferase TIP60 contributes to the acetylation of SUPT16H middle domain (MD) at lysine 674 (K674). Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H in both epithelial and NK cells. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (HDAC1, EZH2), and further regulates their activation status and/or promoter association as well as affects the relevant histone marks (H3ac, H3K9me3 and H3K27me3). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, depletion or inhibition of SUPT16H is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that depletion or inhibition of SUPT16H also causes the remarkable activation of IFN signaling in NK cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in coordinating with BRD4 and regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of the FACT inhibitor CBL0137 to treat viral infections.

The HIV Latency Reversal Agent HODHBt Enhances NK Cell Effector and Memory-Like Functions by Increasing Interleukin-15-Mediated STAT Activation.

ABSTRACTElimination of human immunodeficiency virus (HIV) reservoirs is a critical endpoint to eradicate HIV. One therapeutic intervention against latent HIV is “shock and kill.” This strategy is based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) with the consequent killing of the reactivated cell by either the cytopathic effect of HIV or the immune system. We have previously found that the small molecule 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) acts as an LRA by increasing signal transducer and activator of transcription (STAT) factor activation mediated by interleukin-15 (IL-15) in cells isolated from aviremic participants. The IL-15 superagonist N-803 is currently under clinical investigation to eliminate latent reservoirs. IL-15 and N-803 share similar mechanisms of action by promoting the activation of STATs and have shown some promise in preclinical models directed toward HIV eradication. In this work, we evaluated the ability of HODHBt to enhance IL-15 signaling in natural killer (NK) cells and the biological consequences associated with increased STAT activation in NK cell effector and memory-like functions. We showed that HODHBt increased IL-15-mediated STAT phosphorylation in NK cells, resulting in increases in the secretion of CXCL-10 and interferon gamma (IFN-γ) and the expression of cytotoxic proteins, including granzyme B, granzyme A, perforin, granulysin, FASL, and TRAIL. This increased cytotoxic profile results in increased cytotoxicity against HIV-infected cells and different tumor cell lines. HODHBt also improved the generation of cytokine-induced memory-like NK cells. Overall, our data demonstrate that enhancing the magnitude of IL-15 signaling with HODHBt favors NK cell cytotoxicity and memory-like generation, and thus, targeting this pathway could be further explored for HIV cure interventions.IMPORTANCE Several clinical trials targeting the HIV latent reservoir with LRAs have been completed. In spite of a lack of clinical benefit, they have been crucial to elucidate hurdles that “shock and kill” strategies have to overcome to promote an effective reduction of the latent reservoir to lead to a cure. These hurdles include low reactivation potential mediated by LRAs, the negative influence of some LRAs on the activity of natural killer and effector CD8 T cells, an increased resistance to apoptosis of latently infected cells, and an exhausted immune system due to chronic inflammation. To that end, finding therapeutic strategies that can overcome some of these challenges could improve the outcome of shock and kill strategies aimed at HIV eradication. Here, we show that the LRA HODHBt also improves IL-15-mediated NK cell effector and memory-like functions. As such, pharmacological enhancement of IL-15-mediated STAT activation can open new therapeutic avenues toward an HIV cure.

Phospho-proteomics reveals that RSK signaling is required for proliferation of natural killer cells stimulated with IL-2 or IL-15

Natural killer (NK) cells are cytotoxic lymphocytes that play a critical role in the innate immune system. Although cytokine signaling is crucial for the development, expansion, and cytotoxicity of NK cells, the signaling pathways stimulated by cytokines are not well understood. Here, we sought to compare the early signaling dynamics induced by the cytokines interleukin (IL)-2 and IL-15 using liquid chromatography-mass spectrometry (LC-MS)-based phospho-proteomics. Following stimulation of the immortalized NK cell line NK-92 with IL-2 or IL-15 for 5, 10, 15, or 30 min, we identified 8,692 phospho-peptides from 3,023 proteins. Comparing the kinetic profiles of 3,619 fully quantified phospho-peptides, we found that IL-2 and IL-15 induced highly similar signaling in NK-92 cells. Among the IL-2/IL-15-regulated phospho-peptides were both well-known signaling events like the JAK/STAT pathway and novel signaling events with potential functional significance including LCP1 pSer5, STMN1 pSer25, CHEK1 pSer286, STIM1 pSer608, and VDAC1 pSer104. Using bioinformatic approaches, we sought to identify kinases regulated by IL-2/IL-15 stimulation and found that the p90 ribosomal S6 kinase (p90RSK) family was activated by both cytokines. Using pharmacological inhibitors, we then discovered that RSK signaling is required for IL-2 and IL-15-induced proliferation in NK-92 cells. Taken together, our analysis represents the first phospho-proteomic characterization of cytokine signaling in NK cells and increases our understanding of how cytokine signaling regulates NK cell function.

Abstract 5003: Gene expression to copy number alterations analysis in colorectal cancers: Organ specificity of transcriptomic impact on copy number alterations

Abstract Background: Colorectal cancers (CRCs) is the third most commonly diagnosed cancer and the second most lethal cancer worldwide. CRCs carry high degrees of genomic instability (GI), which enables cancer evolution and makes prognosis poorer. Thus, GI is an exploitable target for therapy purposes. GI can be caused by gene mutation, alteration in gene expression, epigenetic modification, and other infectious or environmental factors. Several mutated genes involved in Chromosome instability and/or microsatellite instability have been identified. Yet it has not been technically feasible to modify functions of mutated genes (e.g., APC, TP53). From the drug development’ standpoint, inhibition of over-expressed target gene is preferred. However, specific genes involved in GI via expression alterations in CRCs have not been comprehensively identified. The gap in knowledge hinders development of CRC therapies targeting GI in CRC. In a previous study, we developed a data mining strategy (Gene Expression to Copy Number Alterations; “GE-CNA”) and comprehensively identified 1,578 genes that associate with CNA in lung adenocarcinoma, among which 39 were survival-critical (i.e., expression levels correlate with significant differences in patients’ survival). Rationale: Causal genes for GI via expressions have not been comprehensively identified in CRCs. Thus, the mechanistic understanding of GI generation in CRC remains incomplete. Few attempts have been made to understand the organ-specificity of genomic instability in cancer. Main Results: We applied the GE-CNA approach to 592 TCGA CRC datasets, and identified 513 genes whose expression levels associate with CNA. Among these, 27 were survival-critical. Comparison with previous results from lung adenocarcinoma indicated striking differences between lung adenocarcinoma and CRC: (a) overall CNA numbers are higher in lung adenocarcinoma than CRC in all stages, (b) 262 CRC-CNA facilitator genes did not show significant concentration in a specific pathway, and (c) 251 CRC-CNA suppressor genes are concentrated in the following pathways: Interferon Signaling, Antigen Presentation Pathway, Heme Biosynthesis II, Natural Killer Cell Signaling, Retinoic acid Mediated Apoptosis Signaling, JAK/Stat Signaling, Glucocorticoid Receptor Signaling, Heme Biosynthesis from Uroporphyrinogen-III I, and Glutathione Redox Reactions II. Implications: The 27 survival-critical genomic instability genes are potential targets to suppress CRC genomic instability and therefore are targets for CRC drug development. Causal genes for genomic instability via expressions differ among organs. Hence, the “targeting genomic instability and/or aneuploidy” approach will need to be tailored for the specific target organ. Citation Format: Chinthalapally V. Rao, Chao Xu, Yuting Zhang, Adam S. Asch, Hiroshi Y. Yamada. Gene expression to copy number alterations analysis in colorectal cancers: Organ specificity of transcriptomic impact on copy number alterations [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 5003.

Endometrial Epithelial ARID1A Is Required for Uterine Immune Homeostasis during Early Pregnancy.

A growing body of work suggests epigenetic dysregulation contributes to endometriosis pathophysiology and female infertility. The chromatin remodeling complex subunit AT-rich interaction domain 1A (ARID1A) must be properly expressed to maintain normal uterine function. Endometrial epithelial ARID1A is indispensable for pregnancy establishment in mice through regulation of endometrial gland function; however, ARID1A expression is decreased in infertile women with endometriosis. We hypothesized that ARID1A performs critical operations in the endometrial epithelium necessary for fertility besides maintaining gland function. To identify alterations in uterine gene expression resulting from loss of epithelial ARID1A, we performed RNA-sequencing analysis on pre-implantation uteri from LtfiCre/+Arid1af/f and control mice. Differential expression analysis identified 4181 differentially expressed genes enriched for immune-related ingenuity canonical pathways including agranulocyte adhesion and diapedesis and natural killer cell signaling. RT-qPCR confirmed an increase in pro-inflammatory cytokine and macrophage-related gene expression but a decrease in natural killer cell signaling. Immunostaining confirmed a uterus-specific increase in macrophage infiltration. Flow cytometry delineated an increase in inflammatory macrophages and a decrease in uterine dendritic cells in LtfiCre/+Arid1af/f uteri. These findings demonstrate a role for endometrial epithelial ARID1A in suppressing inflammation and maintaining uterine immune homeostasis, which are required for successful pregnancy and gynecological health.

CD3ζ adaptor structure determines functional differences between human and mouse CD16 Fc-gamma receptor signaling in natural killer cells

Abstract Natural killer (NK) cells are innate lymphocytes capable of distinguishing between healthy and pathogenic cells using a myriad of receptors expressed on their cell surface. The CD16 receptor detects the Fc portion of IgG on antibody-coated cells resulting in NK cell activation. The importance of this receptor on human NK cell biology has long been appreciated; however, how CD16 functions in mouse NK cells remains poorly understood. Here, we report drastic differences between human and mouse CD16 functions in NK cells. We demonstrate that one of the adaptor molecules that CD16 associates with and signals through, CD3ζ, plays a critical role in these functional differences. Using a systematic approach, we demonstrate that key residues in the transmembrane domain of the mouse CD3ζ molecule prevent efficient complex formation with mouse CD16, thereby dampening receptor function. Mutating these residues in mouse CD3ζ to those encoded by human CD3ζ resulted in rescue of CD16 receptor function. Supported by grants from the Cancer Research Institute (CRI), Burroughs Wellcome Fund (BWF), the Parker Institute for Cancer Immunotherapy (PICI) and NIH grants AI068129 and AI146581.

Impaired Interleukin-18 Signaling in Natural Killer Cells From Patients With Systemic Juvenile Idiopathic Arthritis.

ObjectiveSystemic juvenile idiopathic arthritis (sJIA) is characterized by fever, arthritis, rash, hepatosplenomegaly, and macrophage activation syndrome; however, its pathogenesis is still unclear. Elevated serum interleukin (IL)‐18 concentrations and decreased natural killer (NK) cell activity are characteristic of active disease; thus, we examined IL‐18 signaling in NK cells from sJIA.MethodsWe analyzed mitogen‐activated protein kinase (MAPK) p38 and nuclear factor κ light chain enhancer of activated B cells (NFκB) p65 phosphorylation in NK cells after in vitro recombinant IL‐18 (rIL‐18) stimulation in 31 patients with sJIA. Associations between clinical features, serum IL‐18, and phosphorylation intensity were analyzed. Furthermore, we investigated the effects of high IL‐18 concentrations on phosphorylation in NK cells.ResultsPatients were divided according to their disease activity: systemic features (n = 8), chronic arthritis (n = 7), remission on medication (n = 10), and remission off medication (n = 6). MAPK p38 and NFκB p65 phosphorylation intensity were the highest in healthy controls, followed by remission off medication, remission on medication (vs. control; MAPK p38, P < 0.01; NFκB p65, P < 0.05), chronic arthritis (P < 0.001, P < 0.001), and systemic features (P < 0.001, P < 0.001). The systemic features group showed a complete defect in phosphorylation. Serum IL‐18 was the highest in the systemic features group followed by chronic arthritis, remission on medication (P < 0.01), remission off medication (P < 0.01), and healthy controls (P < 0.01). Phosphorylation intensity was negatively correlated with serum IL‐18 (MAPK p38, r 2 = 0.42; NFκB p65, r 2 = 0.54). Furthermore, healthy control NK cells were cultured with rIL‐18; impaired phosphorylation was reproduced in vitro.ConclusionImpaired IL‐18 signaling in NK cells correlated with disease activity in sJIA. High serum IL‐18 exposure induces impaired MAPK and NFκB phosphorylation in NK cells.

C3aR Signaling Inhibits NK-cell Infiltration into the Tumor Microenvironment in Mouse Models.

Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells, suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells, but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration, we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells, we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1, which led to a high-affinity LFA-1 conformation, decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME, through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1, represent a new strategy to improve the efficiency of immunotherapy for cancer treatment.

Chronic IL-15 Stimulation and Impaired mTOR Signaling and Metabolism in Natural Killer Cells During Acute Myeloid Leukemia

Natural Killer (NK) cells are potent anti-leukemic immune effectors. However, they display multiple defects in acute myeloid leukemia (AML) patients leading to reduced anti-tumor potential. Our limited understanding of the mechanisms underlying these defects hampers the development of strategies to restore NK cell potential. Here, we have used a mouse model of AML to gain insight into these mechanisms. We found that leukemia progression resulted in NK cell maturation defects and functional alterations. Next, we assessed NK cell cytokine signaling governing their behavior. We showed that NK cells from leukemic mice exhibit constitutive IL-15/mTOR signaling and type I IFN signaling. However, these cells failed to respond to IL-15 stimulation in vitro as illustrated by reduced activation of the mTOR pathway. Moreover, our data suggest that mTOR-mediated metabolic responses were reduced in NK cells from AML-bearing mice. Noteworthy, the reduction of mTOR-mediated activation of NK cells during AML development partially rescued NK cell metabolic and functional defects. Altogether, our data strongly suggest that NK cells from leukemic mice are metabolically and functionally exhausted as a result of a chronic cytokine activation, at least partially IL-15/mTOR signaling. NK cells from AML patients also displayed reduced IL-2/15Rβ expression and showed cues of reduced metabolic response to IL-15 stimulation in vitro, suggesting that a similar mechanism might occur in AML patients. Our study pinpoints the dysregulation of cytokine stimulation pathways as a new mechanism leading to NK cell defects in AML.

The Role of Arginine Metabolism in Oral Tongue Squamous Cell Carcinoma.

Simple SummaryCancers that are ‘arginine auxotrophic’ rely on extracellular arginine as a crucial substrate for proliferation and growth. Capitalizing on this vulnerability, there are numerous clinical trials evaluating the therapeutic benefits of depleting arginine in multiple types of cancer, including those occurring in the head and neck. However, head and neck cancers are different and are nonauxotrophic for arginine. Here, we explored the intricacies of arginine metabolism in tongue cancer in order to better understand the therapeutic potential of this biological vulnerability. We showed that reprogramming arginase 1 (ARG1) expression in tongue cancer cells inhibits growth compared with controls. Further, RNA-sequencing showed that HIFα, natural killer cell and interferon signaling were concordantly deregulated.Early diagnosis and treatment do not prevent the high morbidity and poor prognosis of oral tongue squamous cell carcinoma (TSCC). Earlier studies have shown that ARG1 signaling is deregulated in TSCC. Here, we investigated the complexity of ARG1 metabolism in this cancer subsite to appreciate the therapeutic potential of this potential biological vulnerability. Various functional studies show that ARG1 overexpression in oral cancer cells inhibits cell proliferation and invasion compared with controls. Further, RNA-sequencing revealed numerous differentially expressed genes (DEGs) and associated networks were dysregulated by ARG1 overexpression, including hypoxia-inducible factor (HIFα) signaling, the natural killer cell signaling pathway and interferon signaling. Our work provides a foundation for understanding the mechanism of action of disrupted arginine metabolism in oral tongue squamous cell carcinoma. This may impact the community for developing further therapeutic approaches.

Colon cancer cells acquire immune regulatory molecules from tumor-infiltrating lymphocytes by trogocytosis

Cancer cells can develop an immunosuppressive tumor microenvironment to control tumor-infiltrating lymphocytes. The underlying mechanisms still remain unclear. Here, we report that mouse and human colon cancer cells acquire lymphocyte membrane proteins including cellular markers such as CD4 and CD45. We observed cell populations harboring both a tumor-specific marker and CD4 in the tumor microenvironment. Sorted cells from these populations were capable of forming organoids, identifying them as cancer cells. Live imaging analysis revealed that lymphocyte membrane proteins were transferred to cancer cells via trogocytosis. As a result of the transfer invivo, cancer cells also acquired immune regulatory surface proteins such as CTLA4 and Tim3, which suppress activation of immune cells [T. L. Walunas etal, Immunity 1, 405-413 (1994) and L. Monney etal., Nature 415, 536-541 (2002)]. RNA sequencing analysis of ex vivo-cocultured splenocytes with trogocytic cancer cells showed reductions in Th1 activation and natural killer cell signaling pathways compared with the nontrogocytic control. Cancer cell trogocytosis was confirmed in the patient-derived xenograft models of colorectal cancer and head and neck cancer. These findings suggest that cancer cells utilize membrane proteins expressed in lymphocytes, which in turn contribute to the development of the immunosuppressive tumor microenvironment.