Cytokine-inducible SH2-containing Protein Research Articles

Abstract C055: Withaferin A offers novel targeted immunotherapy against high-grade serous ovarian carcinoma

Abstract Background: Tumor-induced immunosuppression and cell proliferation are the hallmarks of progression of malignant tumors including high-grade serous ovarian carcinoma (HGSOC), a fatal malignancy of women. Natural killer (NK) cell plays critical roles in killing tumor cells in the tumor vicinity as it does not require antigen presentation. Our earlier studies showed the frequency of tumor infiltrating NK cells is significantly low as compared with that of tumor stroma suggesting that tumor-induced factors may be involved in inhibition of NK cell infiltration, and the tumor continue to metastasize. Cytokines including IL-10/IL-15 are used to stimulate NK cells and tumor continuously secretes IL-10 or IL-15 and it is possible that excessive exposure to these cytokines induces cytokine inducible SH2-containing protein (CISH), a marker of NK cell exhaustion. We have shown earlier that root powder of Ashwagandha (ASH), an anti-stress natural herb improved NK cell population and reduced tumor progression. The goal of this study was to determine whether Withaferin A (WFA), an active ingredient of ASH, targets the induction of CISH or cyclin D1 (CCND1), a stimulator cell growth and prevented tumor progression. Materials & methods: Study-1: An exploratory study was conducted with clinical specimens which included normal ovaries (n=5) and ovarian high grade serous carcinoma (HGSC) at both early and late stages (n=5 each) and expression levels of CISH and CCND1 were examined. Study-2: A prospective study using the laying hen model of OVCA supplemented with or without 2% dietary ASH root powder for 90 days (n=7 each;normal, early and late-stage). Normal and tumor ovarian tissues were collected at the end of the study and used for analysis. Both the clinical and hen specimens were used for immunohistochemistry, western blotting and gene expression assays to determine expression levels of CISH and CCND1. In vitro studies were also conducted with WFA on the OVCAR3, an HGSC cell line, where multiple doses were tested, and expression of markers previously mentioned were examined. Data were collected and analyzed by ANOVA and unpaired t-test and considered significant when P<0.05. Results: Frequency of CISH-expressing cells in both clinical and hen specimens in normal, early and late-stages were localized. Compared to normal, increased expression of both CISH and CCND1 was seen in early-stages and increased further through late stages. Similarly, strong expression of both CISH and CCND1 was seen in proteomic and genomic studies. ASH supplementation is hens compared to untreated showed a significant decrease (P<0.001) in CISH expression as well as in CCND1 gene expression studies. In vitro studies WFA treatment significantly decreased expression of both markers (P<0.05). Conclusion: The results of this study suggest that both CISH and CCND1 are involved in progression of OVCA and dietary supplementation with ASH or treatment with WFA may be an effective bi-targeted immunotherapy against HGSOC. Support: Ovarian Cancer Spore, Roswell Park Citation Format: Jasmin C. Acosta, Animesh Barua. Withaferin A offers novel targeted immunotherapy against high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C055.

ADSC secretome constrains NK cell activity by attenuating IL-2-mediated JAK-STAT and AKT signaling pathway via upregulation of CIS and DUSP4

BackgroundMesenchymal stem cells (MSCs) have immunomodulatory properties and therapeutic effects on autoimmune diseases through their secreted factors, referred to as the secretome. However, the specific key factors of the MSC secretome and their mechanisms of action in immune cells have not been fully determined. Most in vitro experiments are being performed using immune cells, but experiments using natural killer (NK) cells have been neglected, and a few studies using NK cells have shown discrepancies in results. NK cells are crucial elements of the immune system, and adjustment of their activity is essential for controlling various pathological conditions. The aim of this study was to elucidate the role of the adipose tissue-derived stem cell (ADSC) secretome on NK cell activity.MethodsTo obtain the ADSC secretome, we cultured ADSCs in medium and concentrated the culture medium using tangential flow filtration (TFF) capsules. We assessed NK cell viability and proliferation using CCK-8 and CFSE assays, respectively. We analyzed the effects of the ADSC secretome on NK cell activity and pathway-related proteins using a combination of flow cytometry, ELISA, cytotoxicity assay, CD107a assay, western blotting, and quantitative real-time PCR. To identify the composition of the ADSC secretome, we performed LC–MS/MS profiling and bioinformatics analysis. To elucidate the molecular mechanisms involved, we used mRNA sequencing to profile the transcriptional expression of human blood NK cells.ResultsThe ADSC secretome was found to restrict IL-2-mediated effector function of NK cells while maintaining proliferative potency. This effect was achieved through the upregulation of the inhibitory receptor CD96, as well as downregulation of activating receptors and IL-2 receptor subunits IL-2Rα and IL-2Rγ. These changes were associated with attenuated JAK-STAT and AKT pathways in NK cells, which were achieved through the upregulation of cytokine-inducible SH2-containing protein (CIS, encoded by Cish) and dual specificity protein phosphatase 4 (DUSP4). Furthermore, proteomic analysis revealed twelve novel candidates associated with the immunomodulatory effects of MSCs.ConclusionsOur findings reveal a detailed cellular outcome and regulatory mechanism of NK cell activity by the ADSC secretome and suggest a therapeutic tool for treating NK-mediated inflammatory and autoimmune diseases using the MSC secretome.

CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma

BackgroundGlioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs.MethodsThe GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further detected in vivo antitumor effects using xenograft brain tumor mice.ResultsWe successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice.ConclusionThe findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.

Therapeutic potential of icariin in rats with letrozole and high-fat diet-induced polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is characterized by reproductive, endocrine, and metabolic disorders. Icariin has been shown to regulate endocrine and metabolic imbalances. This study aimed to determine the therapeutic effect and pharmacological mechanism of icariin in PCOS rats. Rats were fed a high-fat diet and gavaged with letrozole to induce PCOS. Thirty-six female rats were randomly divided into four groups: control, model, low-dose, and high-dose icariin. After 30 days of treatment, we evaluated the therapeutic effects on weight and diet, sex hormone levels, ovarian morphology, estrous cycle, inflammatory factors, and indicators of glucolipid metabolism. Combined with the ovarian transcriptome, we verified the key markers of apoptosis and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway by RT-qPCR for mRNA level, western blot, and immunohistochemistry for protein expression. Icariin significantly improved ovarian function and reproductive endocrine disorders by regulating sex hormones, restoring the estrous cycle, and reducing ovarian morphological damage in PCOS rats. Icariin-treated rats had lower weight gain and reduced triglycerides, fasting insulin, HOMA-IR, TNF-α, and interleukin-6 with higher high-density lipoprotein cholesterol levels than PCOS rats. TUNEL staining showed icariin improved apoptosis in the ovaries. This was supported by an increase in Bcl2 and a decrease in Bad and Bax. Icariin decreased the ratios of p-JAK2/JAK2, p-STAT1/STAT1, p-STAT3/STAT3, and p-STAT5a/STAT5a, decreased IL-6, gp130 expression, and increased cytokine-inducible SH2-containing protein (CISH) and suppressor of cytokine signaling 1 (SOCS1) expression. The pharmacological mechanism may be related to the reduction in ovarian apoptosis and inhibition of the IL-6/gp130/JAK2/STATs pathway.

CISH impairs lysosomal function in activated T cells resulting in mitochondrial DNA release and inflammaging.

Chronic systemic inflammation is one of the hallmarks of the aging immune system. Here we show that activated T cells from older adults contribute to inflammaging by releasing mitochondrial DNA (mtDNA) into their environment due to an increased expression of the cytokine-inducible SH2-containing protein (CISH). CISH targets ATP6V1A, an essential component of the proton pump V-ATPase, for proteasomal degradation, thereby impairing lysosomal function. Impaired lysosomal activity caused intracellular accumulation of multivesicular bodies and amphisomes and the export of their cargos, including mtDNA. CISH silencing in T cells from older adults restored lysosomal activity and prevented amphisomal release. In antigen-specific responses in vivo, CISH-deficient CD4+ T cells released less mtDNA and induced fewer inflammatory cytokines. Attenuating CISH expression may present a promising strategy to reduce inflammation in an immune response of older individuals.

Intestinal epithelial cell-specific deletion of cytokine-inducible SH2- containing protein alleviates experimental colitis in ageing mice.

The incidence of inflammatory bowel disease (IBD) in the elderly has increased in recent years. However, the mechanisms underlying the ageing-related IBD susceptibility remain elusive. Cytokine-inducible SH2-containing protein (CISH) involved in regulating metabolism, the expansion of intestinal tuft cells and type-2 innate lymphoid cells, and ageing-related airway inflammation. Here, we investigated the role of CISH in ageing-related colitis susceptibility. CISH and phosphorylated signal transducer and activator of transcription-3 (p-STAT3) levels were evaluated in the colons of ageing mice and older ulcerative colitis (UC) patients. Mice with intestinal epithelial cell-specific knockout of Cish (CishΔIEC) and Cish-floxed mice were administered dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce colitis. Colonic tissues were analysed in quantitative real-time polymerase chain reaction, immunoblotting, immunohistochemical, and histological staining experiments. Differentially expressed genes from colonic epithelia were analysed by RNA-sequencing. Ageing increased the severity of DSS-induced colitis and the expression of colonic epithelial CISH in mice. CishΔIEC prevented against the DSS or TNBS-induced colitis in middle-aged mice but not, in the young mice. RNA-sequencing analysis revealed that CishΔIEC significantly suppressed DSS-induced oxidative stress and proinflammatory responses. During ageing CCD841 cell model, knockdown of CISH decreased ageing-induced oxidative stress and proinflammatory responses, whereas these effects were compromised by knocking down or inhibiting STAT3. The increase in CISH expression was higher in the colonic mucosa of older patients with UC than that in healthy controls. CISH might be a proinflammatory regulator in ageing; therefore, targeted therapy against CISH may provide a novel strategy for treating ageing-related IBD.

Expression of CISH, an Inhibitor of NK Cell Function, Increases in Association with Ovarian Cancer Development and Progression

Epithelial ovarian cancer (OVCA), a fatal malignancy of women, disseminates locally. Although NK cells mount immune responses against OVCA, tumors inhibit NK cells, and the mechanism is not well understood. Cytokines stimulate NK cells; however, chronic stimulation exhausts them and induces expression of cytokine-inducible SH2-containing protein (CISH). Tumors produce anti-inflammatory cytokine interleukin (IL)-10 which may induce NK cell exhaustion. The goal of this study was to examine if CISH expression in NK cells increases during OVCA development and to determine the mechanism(s) of OVCA-induced CISH expression in NK cells. Normal ovaries (n = 7) were used for CISH, IL-10 and GRP78 expression. In tumor ovaries, CISH was examined in early and late stages (n = 14 each, all subtypes) while IL-10 and GRP78 expression were examined in early and late stage HGSC (n = 5 each). Compared to normal, the population of CISH-expressing NK cells increased and the intensity of IL-10 and GRP78 expression was significantly higher in OVCA (p < 0.05). CISH expression was positively correlated with IL-10 expression (r = 0.52, r = 0.65, p < 0.05 at early and late stages, respectively) while IL-10 expression was positively correlated with GRP78 expression (r = 0.43, r = 0.52, p < 0.05, respectively). These results suggest that OVCA development and progression are associated with increased CISH expression by NK cells which is correlated with tumor-induced persistent cellular stress.

Disruption of CISH promotes the antitumor activity of human T cells and decreases PD-1 expression levels

Tumor cells and the immunosuppressive tumor microenvironment suppress the antitumor activity of Tcells through immune checkpoints, including the PD-L1/PD-1 axis. Cytokine-inducible SH2-containing protein (CISH), a member of the suppressor of cytokine signaling (SOCS) family, inhibits JAK-STAT and Tcell receptor (TCR) signaling in T and natural killer (NK) cells. However, its role in the regulation of immune checkpoints in Tcells remains unclear. In this study, we ablated CISH in Tcells with CRISPR-Cas9 and found that the sensitivity of Tcells to TCR and cytokine stimulation was increased. In addition, chimeric antigen receptor Tcells with CISH deficiency exhibited longer survival and higher cytokine secretion and antitumor activity. Notably, PD-1 expression was decreased in activated CISH-deficient Tcells invitro and invivo. The level of FBXO38, a ubiquitination-regulating protein that reduces PD-1 expression, was elevated in activated Tcells after CISH ablation. Hence, this study reveals a mechanism by which CISH promotes PD-1 expression by suppressing the expression of FBXO38 and proposes a new strategy for augmenting the therapeutic effect of CAR-T cells by inhibiting CISH.

CIS controls the functional polarization of GM-CSF-derived macrophages.

The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the capacity to differentiate monocytes into macrophages (MØs) with opposing functions, namely, proinflammatory M1-like MØs and immunosuppressive M2-like MØs. Despite the importance of these opposing biological outcomes, the intrinsic mechanism that regulates the functional polarization of MØs under GM-CSF signaling remains elusive. Here, we showed that GM-CSF-induced MØ polarization resulted in the expression of cytokine-inducible SH2-containing protein (CIS) and that CIS deficiency skewed the differentiation of monocytes toward immunosuppressive M2-like MØs. CIS deficiency resulted in hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss- and gain-of-function approaches highlighted IRF8 as a critical regulator of the M1-like polarization program. In vivo, CIS deficiency induced the differentiation of M2-like macrophages, which promoted strong Th2 immune responses characterized by the development of severe experimental asthma. Collectively, our results reveal a CIS-modulated mechanism that clarifies the opposing actions of GM-CSF in MØ differentiation and uncovers the role of GM-CSF in controlling allergic inflammation.

Alpha7 Nicotinic Acetylcholine Receptor Antagonists Prevent Meningitic Escherichia coli-Induced Blood-Brain Barrier Disruptions by Targeting the CISH/JAK2/STAT5b Axis.

Despite the availability of antibiotics over the last several decades, excessive antibiotic treatments for bacterial sepsis and meningitis (BSM) in children may result in several adverse outcomes. Hematogenous pathogens may directly induce permeability increases in human brain microvascular endothelial cells (HBMECs) and blood–brain barrier (BBB) dysfunctions. Our preliminary studies demonstrated that the alpha7 nicotinic acetylcholine receptor (α7nAChR) played an important role in the pathogenesis of BSM, accompanied by increasing cytokine-inducible SH2-containing protein (CISH) at the transcriptome level, but it has remained unclear how α7nAChR-CISH works mechanistically. The study aims to explore the underlying mechanism of α7nAChR and CISH during E. coli-induced BSM in vitro (HBMECs) and in vivo (α7nAChR-KO mouse). We found that in the stage of E. coli K1-induced BBB disruptions, α7nAChR functioned as the key regulator that affects the integrity of HBMECs by activating the JAK2–STAT5 signaling pathway, while CISH inhibited JAK2–STAT5 activation and exhibited protective effects against E. coli infection. Notably, we first validated that the expression of CISH could be regulated by α7nAChR in HBMECs. In addition, we determined the protective effects of MLA (methyllycaconitine citrate) and MEM (memantine hydrochloride) (functioning as α7nAChR antagonists) on infected HBMECs and suggested that the α7nAChR–CISH axis could explain the protective effects of the two small-molecule compounds on E. coli-induced HBMECs injuries and BBB disruptions. In conclusion, we dissected the α7nAChR/CISH/JAK2/STAT5 axis as critical for the pathogenesis of E. coli-induced brain microvascular leakage and BBB disruptions and provided novel evidence for the development of α7nAChR antagonists in the prevention of pediatric E. coli BSM.

Hepatic cytokine-inducible SH2-containing protein (CISH) regulates gluconeogenesis via cAMP-responsive element binding protein (CREB).

Impairment of gluconeogenesis is a key factor responsible for hyperglycemia in patients with type 2 diabetes. As an important member of the suppressors of cytokine signaling (SOCS) protein family, many physiological functions of cytokine-inducible SH2-containing protein (CISH) have been described; however, the role of hepatic CISH in gluconeogenesis is poorly understood. In the present study, we observed that hepatic CISH expression was reduced in fasted wild-type (WT) mice. Overexpression of CISH decreased glucose production in mouse primary hepatocytes, while silencing of CISH had the opposite effects. In addition, adenovirus-mediated hepatic CISH overexpression resulted in improved glucose tolerance and decreased gluconeogenesis in WT and leptin receptor-deficient diabetic (db/db) mice. In contrast, adenovirus-mediated hepatic CISH knockdown impaired glucose tolerance and increased gluconeogenesis in WT mice. We also generated liver-specific CISH knockout (LV-CISH KO) mice and discovered that these mice had a similar phenotype in glucose tolerance and gluconeogenesis as mice injected with adenoviruses that knockdown CISH expression. Mechanistically, we found that CISH overexpression decreased and CISH knockdown increased the mRNA and protein levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase 1 (PEPCK), two key enzymes involved in gluconeogenesis, in vitro, and in vivo. Moreover, we discovered that the phosphorylation of cAMP-responsive element binding protein 1 (CREB), a transcription factor of G6pase and Pepck, was required for regulating gluconeogenesis by CISH. Taken together, this study identifies hepatic CISH as an important regulator of gluconeogenesis. Our results also provide important insights into the metabolic functions of the SOCS protein family and the potential targets for the treatment of diabetes.

Concurrent transposon engineering and CRISPR/Cas9 genome editing of primary CLL-1 chimeric antigen receptor–natural killer cells

BackgroundNatural killer (NK) cell genome editing promises to enhance the innate and alloreactive anti-tumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development. Aims and MethodsWe set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein–Barr virus–transformed lymphoblastoid feeder cell-mediated activation and expansion. ResultsThis approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1–positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype. ConclusionsThis report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols.

CIS and TGF-β regulatory pathways influence immunity to bacterial infection.

Immunotherapy has revolutionized cancer therapy by reactivating tumour-resident cytotoxic lymphocytes. More recently, immunotherapy has emerged to restore immunity against infectious agents, including bacterial infections. Immunotherapy primarily targets inhibitory pathways in T cells, however interest in other effector populations, such as natural killer (NK) cells, is growing. We have previously discovered that NK cell metabolism, proliferation and activation can be neutralized through the immunosuppressive transforming growth factor (TGF)-β pathway by inducing plasticity of NK cells and differentiation into innate lymphoid cell (ILC)1-like subsets. NK cells are also regulated through cytokine-inducible SH2-containing protein (CIS), which is induced by interleukin (IL)-15 and is a potent intracellular checkpoint suppressing NK cell survival and function. Targeting these two distinct pathways to restore NK cell function has shown promise in cancer models, but their application in bacterial infection remains unknown. Here, we investigate whether enhancement of NK cell function can improve anti-bacterial immunity, using Salmonella Typhimurium as a model. We identified conversion of NK cells to ILC1-like for the first time in the context of bacterial infection, where TGF-β signalling contributed to this plasticity. Future study should focus on identifying further drivers of ILC1 plasticity and its functional implication in bacterial infection model. We further describe that CIS-deficient mice displayed enhanced pro-inflammatory function and dramatically enhanced anti-bacterial immunity. Inhibition of CIS may present as a viable therapeutic option to enhance immunity towards bacterial infection.

Abstract 5512: CBLB, CISH and CD70 multiplexed gene knockout with CRISPR/Cas9 enhances cytotoxicity of CD70-CAR NK cells and provides greater resistance to TGF-β for cancer immunotherapy

Abstract Natural killer (NK) cells provide an attractive platform for development of effective cancer immunotherapies. NK cells are known for their ability to kill tumor cells and do not elicit graft-versus-host disease, making them a potential source of ‘off-the-shelf’ allogeneic cell therapy. NK cells are also amenable to CRISPR genomic engineering to enhance the antitumor activity of NK cells by increasing their cytotoxicity, overcoming suppression within the tumor microenvironment, or promoting their persistence and homing to tumor sites. Cytokine inducible SH2-containing protein (CISH) is a NK cell checkpoint for IL-15 mediated NK survival, proliferation, cytotoxicity, and anti-tumor immunity. The E3 ubiquitin ligase CBLB is also a negative regulator of NK cell function and has been shown to mediate TGF-β sensitivity by downregulating inhibitory SMAD7 in primary T cells. We hypothesized that knockout of both CISH and CBLB would not only improve NK cell effector function and but also render NK cells resistant to TGF-β mediated suppression. In this study, we utilized CRISPR-Cas9 ribonucleoproteins (RNPs) to disrupt CISH and CBLB genes in isolated peripheral blood NK cells from healthy donors. Given that CD70 expression is present on activated NK cells, to target CD70 on renal cell carcinoma (RCC) with CAR NK cells, CD70 was knocked out in NK cells to avoid fratricide. Western blotting, FACS and TIDE/Amplicon NGS Sequencing data confirmed all three genes were successfully disrupted. Then we expanded these edited NK cells by using IL-2 and stimulation using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation. Furthermore, we were able to transduce CRISPR/Cas9 edited NK cells to express a CD70-CAR construct and membrane bound IL-15. CAR expression was assessed by flow cytometry. In vitro cytotoxicity was measured using the IncuCyte S3 live cell analysis system. CD70/CISH/CBLB triple knockout CD70-CAR NK cells could be produced efficiently and exhibited similar persistence as CD70/CISH or CD70/CBLB double knockout CD70-CAR NK cells in culture. Cytotoxicity assays demonstrated that CD70/CISH/CBLB triple knockout CD70-CAR NK cells had greater tumor growth control after multiple rechallenges. In the presence of exogenous TGF-β, CD70/CISH/CBLB triple knockout CD70-CAR NK cells showed greater resistance to TGF-β inhibition of cytotoxicity. In summary, we show CD70/CISH/CBLB triple knockout CD70-CAR NK cells demonstrate enhanced anti-tumor activity against relevant solid tumor cell lines and provide greater resistance to tumor microenvironment inhibition. These data support the further exploration of CD70/CISH/CBLB triple gene knockout CD70 CAR NK cells for clinical application. Citation Format: Chao Guo, Yanying Fan, Alexander Aronov, Qi Zhang, Sombeet Sahu, Mary-Lee Dequéant, Changan Guo, Sushant Karnik, Glenn D. Leary, Chandirasegaran Massilamany, Ivan H. Chan, James B. Trager. CBLB, CISH and CD70 multiplexed gene knockout with CRISPR/Cas9 enhances cytotoxicity of CD70-CAR NK cells and provides greater resistance to TGF-β for cancer immunotherapy [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 5512.

Abstract 2520: Ovarian tumors escape anti-tumor function of NK cells by inducing CISH expression and its inhibition prevents tumor progression

Abstract Background: Ovarian cancer (OVCA), a lethal malignancy of women, mainly disseminates locally in the peritoneal cavity and the local immune system plays important roles in inhibiting its progression. Although NK cells, a member of innate immunity, recognize and exert anti-tumor immune responses, tumors escape NK cell immunity and progress to late stages by inducing factors like inhibitory receptors and cytokines. IL-15, a cytokine, stimulates NK cell function and persistent exposure to IL-15 induces cytokine inducible SH2-containing protein (CISH), an inhibitory receptor and a marker of NK cell exhaustion. It is unknown if ovarian tumors use this phenomenon in their favor as the tumor secretes enhanced levels of IL-15. Furthermore, dietary supplementation with root powder of Ashwagandha (ASH), a natural herb and an anti-stress compound has shown to decrease the rates of OVCA metastasis and enhance the frequency of intratumoral NK cells in a preclinical model. However, it is unknown if ASH supplementation improves NK cell frequency by inhibiting CISH. The goal of this study was to examine if OVCA progresses by inducing CISH-expression in NK cells and if dietary ASH supplementation reduces NK cell exhaustion by decreasing CISH expression. Materials and methods: Two studies were conducted. Study 1: Exploratory study with clinical specimens including normal ovaries (n=5) and ovarian high grade serous carcinoma at early and late stages (n=5 each). Study 2: Prospective study using the laying hen model of OVCA supplemented with or without 2% dietary ASH root powder for 90 days (n=7 for each group; normal, early and late stage). Hen normal and tumor ovarian tissues were collected at the end of the study. Both the clinical and hen specimens were used for immunohistochemistry, Western blotting and gene expression assays to determine the frequency of CISH-expressing NK-cells and intensity of GRP78 expression (a marker of cellular stress). Data were collected and analyzed by ANOVA and unpaired t-test and considered significant when P&amp;lt;0.05. Results: CISH-expressing NK cells were localized in normal and tumor ovaries in women and hens. Compared with normal ovaries, frequency of CISH-expressing NK cells was significantly higher in tumors at early and late stages in patients and hens. Similarly, strong expression of CISH gene and protein (29kDa) was detected in tumor tissues. Enhanced expression of CISH positively correlated with expression of GRP78. Compared with untreated, ASH supplementation reduced tumor progression in 5 out of 7 hens and significantly decreased the frequency of CISH-expressing NK cells and GRP78 (P&amp;lt;0.001). Conclusion: The results of this study suggest that expression of CISH by NK cells and cellular stress were associated with OVCA progression and dietary ASH supplementation reduced NK cell exhaustion by decreasing CISH and GRP78. Support: Swim Across America and NIH CA187309 Citation Format: Jasmin C. Acosta, Amy K. Stasik, Janice M. Bahr, Animesh Barua. Ovarian tumors escape anti-tumor function of NK cells by inducing CISH expression and its inhibition prevents tumor progression [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 2520.

Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity

BackgroundThe success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK)...

AMFR drives allergic asthma development by promoting alveolar macrophage–derived GM-CSF production

Alveolar macrophages (AMs) are specialized tissue-resident macrophages that orchestrate the immune response in allergic inflammation and asthma. However, what signals direct AMs to cross talk with other immune cells remains unclear. Here, we report that autocrine motility factor receptor (AMFR), an endoplasmic reticulum-resident E3 ubiquitin ligase, is upregulated in AMs of asthma and is critical for this condition. AMFR deficiency significantly decreased allergy-induced T helper 2 (Th2) and eosinophilic inflammation, with less granulocyte-macrophage colony-stimulating factor (GM-CSF) production in AMs. Mechanistically, following thymic stromal lymphopoietin (TSLP) stimulation, AMFR associated directly with cytokine-inducible SH2-containing protein (CIS), induced the ubiquitination of Lys48-linked polyubiquitination of CIS, and consequently blocked the inhibitory effect of CIS on signal transducer and activator of transcription 5 (STAT5) phosphorylation and the downstream pathway activation in AMs. In conclusion, our results demonstrate that AMFR serves a crucial role in promoting inflammation in asthma through regulating AM function, and may emerge as a new potential drug target for asthma therapy.

A 12-bp indel in the 3’UTR of porcine CISH gene associated with Landrace piglet diarrhea score

Variations in Cytokine inducible SH2-containing protein (CISH) gene influence human susceptibility to common infectious diseases, but little is known about CISH in swine. The objectives of this study were to 1) determine porcine CISH (pCISH) mRNA expression level in different tissues of piglets, 2) predict putative functional genetic variations within pCISH, 3) investigate the association between a identified variation in the 3’UTR and piglets phenotype traits in Min (n = 226) and Landrace (n = 186) population, and explore the function of this variation. Results of quantitative PCR showed pCISH mRNA expressed in all the collected tissues with higher level in lung and ileum than colon (p < 0.05). In-silico analysis indicated none of the functional ns-SNPs existed in pCISH coding region. Results from the characterizing of 3’UTR presented a novel 12-bp insertion/deletion (indel) mutation. Statistical analysis demonstrated that this 12-bp indel associated with piglets diarrhea score in the Landrace population, and animals with AA genotype (12-bp insertion) presented lower diarrhea score when compared with BB (p < 0.05) or AB (p < 0.01) carriers. The in vitro study indicated that the luciferase activity of reconstruct plasmid psiCHECK-2-CISH-AA or psiCHECK-2-CISH-BB was significantly lower than the negative control (p < 0.05), and luciferase activity of psiCHECK-2-CISH-AA was higher than that of the psiCHECK-2-CISH-BB (p < 0.05). Although results herein suggested the 12-bp indel might affect Landrace piglet susceptibility to diarrhea, further association studies in more populations are needed before this preliminary finding could be used for pig breeding.

Allulose Attenuated Age-Associated Sarcopenia via Regulating IGF-1 and Myostatin in Aged Mice.

Allulose is shown to increase the muscle weight in diet-induced obese mice. However, there are no studies on the effects of allulose in age-associated sarcopenia. This study aims to elucidate the mechanisms of action for allulose in age associated by analyzing the transcriptional patterns in aged mice. The 48-week-old mice are fed with AIN-93diet containing allulose for 12 weeks. Allulose supplementation increases the muscle mass and grip strength in aged mice. Allulose increases the insulin-like growth factor 1 (IGF-1) and its downstream factor expressions which 40 are related protein synthesis, while inhibits the myostatin expression related protein degradation. In mRNA-seq analysis, allulose supplementation significantly decreases in Adiponectin, Adipsin, cell death inducing DFFA like effector (CIDEC), Haptoglobin, Neuroglobin, and stearoyl-CoA desaturase-1 (SCD1) and increases in cytokine-inducible SH2-containing protein (CISH) and ceramide synthase 1 (CerS1) that are regulate protein turn over in gastrocnemius. Also, allulose alleviates autophagy in muscle with regulated mammalian target of rapamycin (mTOR) signaling pathway and increases the anti-oxidant enzyme activity. These findings suggest that allulose improves the age-associated sarcopenia with enhancing antioxidant properties by altering mRNA and protein expression.

Promising Preliminary Activity of Optimized Affinity, CD38 CAR NK Cells Generated Using a Non-Viral Engineering Approach in Gene Edited Cord Blood Derived NK Cells for the Treatment of Multiple Myeloma

CD38 is a multifunctional cell surface protein that is frequently overexpressed on malignant plasma cells as well as on immune suppressive cells within the tumor microenvironment and constitutes a validated immunotherapeutic target in the treatment of multiple myeloma (MM). At ONK Therapeutics we are developing a gene edited, cord blood-derived NK (CBNK) cell product targeting CD38 for treatment of patients with relapsed and/or refractory MM.The product will be generated using a workflow shown in Figure 1A. This involves starting with cord blood that is processed for NK expansion using a clinically validated, Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) feeder layer. The NK cells would undergo genetic engineering that involves gene editing followed by a non-viral chimeric antigen receptor (CAR) introduction process mediated by the TcBuster (TcB) DNA transposon system (Biotechne). This is followed by a second round of expansion on the EBV-LCL feeder layer resulting in a characterized NK cell product that can then be cryopreserved.In order to develop protocols for optimizing the best transfection efficiencies using the Maxcyte ATx instrument, GFP mRNA (TriLink) was used for transfecting CBNK cells using different electroporation programs. High transfection efficiency was obtained using all programs (Figure 1B.), with the best from program NK4.Since the product employs an optimized affinity second generation anti CD38 CAR (Stikvoort et al., Hemasphere 2021) which could also target CD38 expressed on neighbouring activated NK cells, it is imperative to knock out (KO) the cell surface expression of CD38 on the CAR-NK cells. To achieve this we carried out CRISPR Cas9 based KO studies of CD38 (Figure 1C. left top), using guide RNAs targeting CD38 (Synthego) in the form of a ribonucleoprotein (RNP) complex with Cas9. CBNK cells were transfected using the Maxcyte ATx instrument and CD38 cell surface expression monitored. As shown in Figure 1C. (left top), complete CD38 KO was achieved 11 days post transfection.ONK Therapeutics is actively involved in targeting and downregulating the negative regulator of cytokine signalling, cytokine inducible SH2-containing protein (CIS), which is encoded by the CISH gene, as part of their CBNK products. It has been demonstrated that in addition to facilitating greater cytokine signalling, CISH KO also confers greater metabolic capacity to NK cells resulting in their increased persistence (Daher et al., Blood 2021). Therefore, ONK Therapeutics have evaluated CISH KO in CBNK cells (Figure 1C, top right) using the same scheme that was used for the CD38 KO. Guide RNAs in the form of a RNP complex with Cas9 (Synthego) were transfected into CBNK cells and intracellular CIS protein levels monitored over time. Almost complete KO was attained by 9 days post transfection. In order to dial in CISH KO as part of the product, we further carried out a simultaneous KO of CD38 and CISH, in addition to individual KO of CD38 or CISH (Fig 1C, bottom). Simultaneous multiplexing of the CD38 and CISH KOs resulted in efficient double KO (DKO) . The extent of knock down leading to KO in the DKO setting was very similar to that of individual gene KOs.We then introduced the anti CD38 CAR as part of a transposon that could be transposed by TcB transposase in CBNK cells. After DKO of CD38 and CISH in CBNK cells, the transposon DNA and mRNA for transposase were electroporated. CAR expression was detected 4-5 days post transposition (Figure 1D) with more than 50% of cells expressing the anti CD38 CAR. These CAR expressing CBNK cells were then tested for functionality in a co-culture kill assay against the CD38 positive MM cell line, RPMI8226, which was engineered to express firefly luciferase. In a 4 hour killing assay, robust killing of the RPMI8226 cells was achieved by the CAR-CBNK cells with an EC 50 ten times lower (more potent) than that of mock electroporation control CBNK cells.To our knowledge this is the first successful expression of an anti CD38 CAR in cord-derived NK cells, and with a double CD38/CISH KO, using non-viral CAR insertion approaches. Current work is focusing on designing and developing a manufacturing-ready workflow for this potential product and further examining the effects of CAR NK cell activity in a DKO setting where both KOs contribute to improved metabolism and potentially NK cell persistence, as well as exploring the added benefit of a DR5 TRAIL variant to enhance cytotoxicity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.