Src Homology Region Research Articles

Endothelial deletion of PKCδ prevents VEGF inhibition and restores blood flow reperfusion in diabetic ischemic limb.

Aims:Peripheral artery disease is a complication of diabetes leading to critical hindlimb ischemia. Diabetes-induced inhibition of VEGF actions is associated with the activation of protein kinase Cδ (PKCδ). We aim to specifically investigate the role of PKCδ in endothelial cell (EC) function and VEGF signaling.Methods:Nondiabetic and diabetic mice, with (ec-Prkcd−/−) or without (ec-Prkcdf/f) endothelial deletion of PKCδ, underwent femoral artery ligation. Blood flow reperfusion was assessed up to 4 weeks post-surgery. Capillary density, EC apoptosis and VEGF signaling were evaluated in the ischemic muscle. Src homology region 2 domain-containing phosphatase-1 (SHP-1) phosphatase activity was assessed in vitro using primary ECs.Results:Ischemic muscle of diabetic ec-Prkcdf/f mice exhibited reduced blood flow reperfusion and capillary density while apoptosis increased as compared to nondiabetic ec-Prkcdf/f mice. In contrast, blood flow reperfusion and capillary density were significantly improved in diabetic ec-Prkcd−/− mice. VEGF signaling pathway was restored in diabetic ec-Prkcd−/− mice. The deletion of PKCδ in ECs prevented diabetes-induced VEGF unresponsiveness through a reduction of SHP-1 phosphatase activity.Conclusions:Our data provide new highlights in mechanisms by which PKCδ activation in EC contributed to poor collateral vessel formation, thus, offering novel therapeutic targets to improve angiogenesis in the diabetic limb.

Phosphatidylinositol-3 kinase mediates the sweet suppressive effect of leptin in mouse taste cells.

Leptin is known to selectively suppress neural and taste cell responses to sweet compounds. The sweet suppressive effect of leptin is mediated by the leptin receptor Ob-Rb, and the ATP-gated K+ (KATP ) channel expressed in some sweet-sensitive, taste receptor family 1 member 3 (T1R3)-positive taste cells. However, the intracellular transduction pathway connecting Ob-Rb to KATP channel remains unknown. Here we report that phosphoinositide 3-kinase (PI3K) mediates leptin's suppression of sweet responses in T1R3-positive taste cells. In in situ taste cell recording, systemically administrated leptin suppressed taste cell responses to sucrose in T1R3-positive taste cells. Such leptin's suppression of sucrose responses was impaired by co-administration of PI3K inhibitors (wortmannin or LY294002). In contrast, co-administration of signal transducer and activator of transcription 3 inhibitor (Stattic) or Src homology region 2 domain-containing phosphatase-2 inhibitor (SHP099) had no effect on leptin's suppression of sucrose responses, although signal transducer and activator of transcription 3 and Src homology region 2 domain-containing phosphatase-2 were expressed in T1R3-positive taste cells. In peeled tongue epithelium, phosphatidylinositol (3,4,5)-trisphosphate production and phosphorylation of AKT by leptin were immunohistochemically detected in some T1R3-positive taste cells but not in glutamate decarboxylase 67-positive taste cells. Leptin-induced phosphatidylinositol (3,4,5)-trisphosphate production was suppressed by LY294002. Thus, leptin suppresses sweet responses of T1R3-positive taste cells by activation of Ob-Rb-PI3K-KATP channel pathway.

Polyphyllin D Shows Anticancer Effect through a Selective Inhibition of Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP2).

Natural products have continued to offer tremendous opportunities for drug development, as they have long been used in traditional medicinal systems. SHP2 has served as an anticancer target. To identify novel SHP2 inhibitors with potential anticancer activity, we screened a library containing 658 natural products. Polyphyllin D was found to selectively inhibit SHP2 over SHP1, whereas two other identified compounds (echinocystic acid and oleanolic acid) demonstrated dual SHP1 and SHP2 inhibition. In a cell-based assay, polyphyllin D exhibited cytotoxicity in Jurkat cells, an acute lymphoma leukemia cell line, whereas the other two compounds were ineffective. Polyphyllin D also decreased the level of phosphorylated extracellular signal-regulated kinase (p-ERK), a proliferation marker in Jurkat cells. Furthermore, knockdown of protein tyrosine phosphatase (PTP)N6 (SHP1) or PTPN11 (SHP2) decreased p-ERK levels. However, concurrent knockdown of PTPN6 and PTPN11 in Jurkat cells recovered p-ERK levels. These results demonstrated that polyphyllin D has potential anticancer activity, which can be attributed to its selective inhibition of SHP2 over SHP1.

A natural product phillygenin suppresses osteosarcoma growth and metastasis by regulating the SHP-1/JAK2/STAT3 signaling.

Osteosarcoma represents one of the most devastating cancersdue to its high metastatic potency and fatality. Osteosarcoma is insensitive to traditional chemotherapy. Identification of a small molecule that blocks osteosarcoma progression has been a challenge in drug development. Phillygenin,a plant-derived tetrahydrofurofuran lignin, has shown to suppress cancer cell growth and inflammatory response. However, how phillygenin plays functional roles in osteosarcoma has remained unveiled. In this study, we showed that phillygenin inhibited osteosarcoma cell growth and motility in vitro. Further mechanistic studies indicated that phillygenin blocked STAT3 signaling pathway. Phillygenin led to significant downregulation of Janus kinase 2 and upregulation of Src homology region 2 domain-containing phosphatase 1. Gene products of STAT3 regulating cell survival and invasion were also inhibited by phillygenin. Therefore, our studies provided the first evidence that phillygenin repressed osteosarcoma progression by interfering STAT3 signaling pathway. Phillygenin is a potential candidate in osteosarcoma therapy.

PIR-B Regulates CD4+ IL17a+ T-Cell Survival and Restricts T-Cell-Dependent Intestinal Inflammatory Responses.

Background & AimsCD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype.MethodsWe used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non–inflammatory bowel disease patients and sorted human memory CD4+ T cells.ResultsWe identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B– Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1–dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population.ConclusionsOur findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses.

Astragalus Polysaccharide Regulates Proliferation, Migration and Invasion of Lung Cancer Cells via Janus Kinase/Signal Transducer and Activator of Transcription Signaling Pathway

To evaluate the regulatory effects of Astragalus polysaccharide on the proliferation, migration and invasion of lung cancer cells via the Janus kinase/signal transducer and activator of transcription signaling pathway. The inhibitory effects of treatment with Astragalus polysaccharide at different concentrations (0, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/ml) for different time (24, 48 and 72 h) on the proliferation of adenocarcinomic human alveolar basal epithelial cells in the logarithmic growth phase were detected by methyl thiazolyl tetrazolium assay and its inhibitory effects on the migration and invasion of adenocarcinomic human alveolar basal epithelial cells were determined by wound healing assay and Transwell migration and invasion assays respectively. In contrast with control group, the proliferation of adenocarcinomic human alveolar basal epithelial cells treated with different concentrations of Astragalus polysaccharide for 24, 48 and 72 h was remarkably inhibited in a dose and time dependent manner under the same drug concentration, longer the action time, higher the proliferation inhibition rate of adenocarcinomic human alveolar basal epithelial cells; under the same action time, higher the drug concentration, higher the proliferation inhibition rate of adenocarcinomic human alveolar basal epithelial cells. The number of migrating cells dropped significantly in Astragalus polysaccharide groups (p<0.05), which was more obvious with the increase of Astragalus polysaccharide concentration (p<0.05). Astragalus polysaccharide can dramatically inhibit the proliferation of lung cancer adenocarcinomic human alveolar basal epithelial cells in a concentration and time-dependent manner and also effectively suppress their migration and invasion. The mechanism may be related to its ability to up-regulate the protein expressions of Src homology region 2 containing protein tyrosine phosphatase 2 and suppressor of cytokine signaling 3 and inhibit the over-activation of the Janus kinase/signal transducer and activator of transcription signaling pathway in lung cancer adenocarcinomic human alveolar basal epithelial cells.

The BTLA and PD-1 signaling pathways independently regulate the proliferation and cytotoxicity of human peripheral blood γδ T cells.

BackgroundB‐ and T‐lymphocyte attenuator (BTLA) and programmed cell death‐1 (PD‐1) inhibit γδ T cell homeostasis and activation. This study aimed to determine whether BTLA and PD‐1 signaling pathways were convergent or independent in human peripheral blood γδ T cells. Herein we demonstrate that the signalings of BTLA and PD‐1 regulated proliferation and cytotoxicity of human γδ T cells, respectively.MethodsHuman peripheral blood γδ T cells were cultured with inactivated Jurkat cells in the presence of interleukin‐2 and zoledronate (Zol) for 14 days. Flow cytometry was performed to evaluate the phenotypes and functions of γδ T cells.ResultsThe proliferation of the γδ T cells was increased when PBMCs were cocultured with inactivated herpes virus entry mediator (HVEM)low Jurkat cells. The cytotoxicity of the expanded γδ T cells was not affected by coculture with inactivated HVEMlow Jurkat cells and was further increased in the presence of anti‐PD‐L1 mAb. These results suggest that the inactivation of the BTLA signaling pathway during expansion could help produce more γδ T cells without compromising γδ T cell function. The inhibition of BTLA or PD‐1 signaling repressed phosphorylation of the src homology region 2‐containing protein tyrosine phosphatase 2 and increased the phosphorylation of protein kinase B in γδ T cells. However, there were no synergistic or additive effects by a combination of BTLA and PD‐1 blockade.ConclusionThese results suggest that BTLA signaling is crucial in regulating γδ T cell proliferation and function and that the BTLA and PD‐1 signaling pathways act independently on the proliferation and cytotoxicity of human peripheral γδ T cells.

Therapeutic effect of the injectable thermosensitive hydrogel loaded with SHP099 on intervertebral disc degeneration

AimsIntervertebral disc (IVD) degeneration (IDD), a common musculoskeletal disease with limited self-healing ability, is challenging to treat. The development of innovative therapies to reverse IDD depends on the elucidation of its regulatory mechanisms. Therefore, the role of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) in the pathogenesis of IDD and the therapeutic effect of its small-molecule inhibitor, SHP099, were investigated. Materials and methodsThe expression of SHP2 by nucleus pulposus (NP) cells in IVD was investigated in vitro and in vivo, and its molecular mechanism in IDD was explored using transfection technology. Injectable N-isopropylacrylamide-based thermosensitive hydrogels were synthesized for SHP099 delivery. Key findingsSHP2 was highly expressed in degenerated IVDs, where its overexpression in NP cells inhibited the expression of Sry-related HMG box-9 (Sox9), leading to the decreased expression of key proteins (collagen II and aggrecan) and consequently to IDD. SHP099 reversed the degeneration of NP cells in vitro. Moreover, its administration in rats via the injectable thermosensitive hydrogel had a therapeutic effect on IDD. SignificanceOur results suggest that SHP2 is a key factor in IDD progression, and SHP099 inhibits both its expression and NP cell degeneration. Therefore, SHP099 delivery via injectable thermosensitive hydrogels is a potential treatment strategy for IDD.

NH2 -terminal fragment of ZF21 protein suppresses tumor invasion via inhibiting the interaction of ZF21 with FAK.

Cellular migration, coupled with the degradation of the extracellular matrix (ECM), is a key step in tumor invasion and represents a promising therapeutic target in malignant tumors. Focal adhesions (FAs) and invadopodia, which are distinct actin‐based cellular structures, play key roles in cellular migration and ECM degradation, respectively. The molecular machinery coordinating the dynamics between FAs and invadopodia is not fully understood, although several lines of evidence suggest that the disassembly of FAs is an important step in triggering the formation of invadopodia. In a previous study, we identified the ZF21 protein as a regulator of both FA turnover and invadopodia‐dependent ECM degradation. ZF21 interacts with multiple factors for FA turnover, including focal adhesion kinase (FAK), microtubules, m‐Calpain, and Src homology region 2‐containing protein tyrosine phosphatase 2 (SHP‐2). In particular, the dephosphorylation of FAK by ZF21 is a key event in tumor invasion. However, the precise role of ZF21 binding to FAK remains unclear. We established a method to disrupt the interaction between ZF21 and FAK using the FAK‐binding NH2‐terminal region of ZF21. Tumor cells expressing the ZF21‐derived polypeptide had significantly decreased FA turnover, migration, invadopodia‐dependent ECM degradation, and Matrigel invasion. Furthermore, the expression of the polypeptide inhibited an early step of experimental lung metastasis in mice. These findings indicate that the interaction of ZF21 with FAK is necessary for FA turnover as well as ECM degradation at the invadopodia. Thus, ZF21 is a potential regulator that coordinates the equilibrium between FA turnover and invadopodia activity by interacting with FAK.

Perlecan, a modular instructive proteoglycan with diverse functional properties.

This study reviewed some new aspects of the modular proteoglycan perlecan, a colossal proteoglycan with a 467 kDa core protein and five distinct functional domains. Perlecan is a heparan sulphate proteoglycan that transiently displays native CS sulphation motifs 4-C-3 and 7-D-4 during tissue morphogenesis these are expressed by progenitor cell populations during tissue development. Perlecan is susceptible to fragmentation by proteases during tissue development and in pathological tissues particularly in domains IV and V. The fragmentation pattern of domain IV has been suggested as a means of grading prostate cancer. Domain V of perlecan is of interest due to its interactive properties with integrin α5β1 that promotes pericyte migration enhancing PDGF-BB-induced phosphorylation of PDGFRβ, Src homology region 2 domain-containing phosphatase-2, and focal adhesion kinase supporting the repair of the blood brain barrier following ischaemic stroke. Fragments of domain V can also interact with α2β1 integrin disrupting tube formation by endothelial cells. LG1-LG2, LG3 fragments can antagonise VEGFR2, and α2β1 integrin interactions preventing angiogenesis by endothelial cells. These domain V fragments are of interest as potential anti-tumour agents. Perlecan attached to the luminal surfaces of endothelial cells in blood vessels acts as a flow sensor that signals back to endothelial and smooth muscle cells to regulate vascular tone and blood pressure. Perlecan also acts as a flow sensor in the lacuno-canalicular space regulating osteocytes and bone homeostasis. Along with its biomechanical regulatory properties in cartilaginous tissues this further extends the functional repertoire of this amazingly diverse functional proteoglycan.

Abstract 2173: CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma

Abstract Early clinical trials have demonstrated the therapeutic potential of chimeric antigen receptor (CAR) T cells against glioblastoma (GBM), but also highlighted the obstacles that need to be addressed to improve their anti-tumor activity. GBM is highly immunosuppressive and thus necessitates endowing adoptively transferred CAR T cells with resistance mechanisms to achieve sustained treatment responses. This is, in part, due to the effect of immune-inhibitory receptors, including PD1, CTLA4, LAG3 and BTLA, that antagonize the tumor-directed T cells. A common mechanism of T cell inhibition involves the recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of multiple co-inhibitory receptors. SHP2, in turn, de-phosphorylates key signaling proteins involved in T cell activation. We hypothesized that disrupting SHP2 expression will offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Using CRISPR/Cas9 gene editing, we disrupted the SHP2-encoding gene locus, PTPN11, in primary human T cells by generating and testing a library of single-guide RNA (sgRNA). We selected the top candidate that reproducibly reduced the SHP2 protein expression to &amp;lt;10%. A HER2-targeting second-generation CAR (HER2-CAR) was then expressed in SHP2 knock-out (SHP2KO) T cells via gamma retroviral vector transduction. SHP2KO CAR T cell manufacturing following a single round of stimulation was optimized to minimize ex vivo manipulation of the cells. The phenotypic profiles of SHP2KO and wild-type HER2-CAR T cells were comparable at baseline. Loss of SHP2 did not alter the CAR T cell viability, expansion or proliferative capacity in comparison to the wild-type CAR T cells. Mass cytometry (CyTOF)-based analysis of SHP2KO CAR T cells after encountering GBM cells demonstrated the preservation of the central memory (CCR7+CD45RA-) T cell subset. The majority of PD1+ SHP2KO CAR T cells co-expressed functional activation markers such as ICOS and CD107a. The PD1+ T cell subset was comprised of mostly T-betHi progenitor population and only a smaller percentage of EomesHi terminally exhausted population. Functionally, HER2-specific SHP2KO CAR T cells from different donors (n=4) exhibited improved anti-tumor activity that was sustained in long-term cytotoxicity assays. In an experimental orthotopic LN229-GBM mouse model, SHP2KO HER2-CAR T cells better controlled the growth of established xenografts and improved survival in comparison to wild-type HER2-CAR T cells. In conclusion, our results show that disrupting SHP2 expression using a CRISPR/Cas9 gene editing strategy could improve the CAR T cell function against GBM and warrants further clinical testing. Citation Format: Khaled Sanber, Zeid Nawas, Vita Salsman, Ahmed Gad, Pretty Rose Matthew, Daniel Landi, Ciaran Lee, Amel Sengal, Rikhia Chakraborty, Sujith Joseph, Ahmed Nabil, Meenakshi Hegde. CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma [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 2173.

Abstract 5340: Novel small-molecule antagonists of the PD1/PD-L1 axis

Abstract Immuno-therapy has become a leading strategy to fight cancer. Over the past few years, immuno-therapies using checkpoint inhibitor monoclonal antibodies (mAbs) against programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) have demonstrated improved survival compared with chemotherapy. We describe the identification and characterization of an innovative series of synthetic compounds (named PyrDLones, patented) endowed with nanomolar activity against PD-L1. PyrDLones properties were characterized using several biophysical techniques including microscale thermophoresis (MST) and fluorescence resonance energy transfer (FRET) measurements. In vitro, selected small molecules demonstrate a high affinity for human PD-L1, potently disrupt the PD-L1:PD-1 interaction (&amp;lt;2nM), and inhibit Src homology region 2 domain-containing phosphatase (SHP2) recruitment to PD-1 (&amp;lt;4nM). As a result, upon binding to and inhibiting PD-L1, these molecules reactivate proliferation of CTL-L2 cells expressing PD-1 (EC50=44nM). More than 150 molecules have been synthesized and a dozen of highly potent “PD-L1 silencing compounds” have been identified, based on in vitro measurements. Structure-activity relationships have been defined and an in silico drug-target model supporting the mechanism of action has been built. Experiments are on-going to delineate further the molecular mechanism of action of the PyrDLones (drug-induced PD-L1 dimer formation), the cellular consequences of the PD-L1 silencing (reactivation of cytotoxic T cells in the tumor microenvironment) and to characterize their activity in vivo. In summary, we have discovered a novel series of potent, small molecule PD-L1 antagonists, amenable to the design of orally active drugs for immuno-therapy of cancers. Citation Format: Romain Magnez, Natascha Leleu-Chavain, Morgane Tardy, Hassiba El Bouazzati, Fréderique Klupsch, Christian Bailly, Régis Millet, Bruno Quesnel, Xavier Thuru. Novel small-molecule antagonists of the PD1/PD-L1 axis [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 5340.

SHP-1 ameliorates nonalcoholic steatohepatitis by inhibiting proinflammatory cytokine production.

Inflammation is the main contributor for the pathogenesis of nonalcoholic steatohepatitis (NASH). Src homology region 2 domain-containing phosphatase 1 (SHP-1, also known as PTPN6) is regarded as a negative regulator of inflammation, but its role in NASH remains unknown. Here, hepatocyte-specific Ptpn6 knockout mice (Ptpn6HKO ) and adenovirus vector-mediated ectopic expression of SHP-1 (AdSHP1) were used to evaluate the role of SHP-1 in a methionine- and choline-deficient diet-induced NASH model. Compared with the control littermates, Ptpn6HKO mice show exacerbated hepatic steatosis, inflammation, and fibrosis. Additionally, administration of AdSHP1 significantly ameliorates steatohepatitis and inhibits the expression of proinflammatory cytokines, including transforming growth factor-β, interleukin-6, and tumor necrosis factor-α. Our data indicate that SHP-1 could be a potential therapeutic target for NASH.

Regulation of autoimmune arthritis by the SHP-1 tyrosine phosphatase

BackgroundThe Src homology region 2 domain-containing phosphatase-1 (SHP-1) is known to exert negative regulatory effects on immune cell signaling. Mice with mutations in the Shp1 gene develop inflammatory skin disease and autoimmunity, but no arthritis.We sought to explore the role of SHP-1 in arthritis using an autoimmune mouse model of rheumatoid arthritis. We generated Shp1 transgenic (Shp1-Tg) mice to study the impact of SHP-1 overexpression on arthritis susceptibility and adaptive immune responses.MethodsSHP-1 gene and protein expression as well as tyrosine phosphatase activity were evaluated in spleen cells of transgenic and wild type (WT) mice. WT and Shp1-Tg (homozygous or heterozygous for the transgene) mice were immunized with human cartilage proteoglycan (PG) in adjuvant, and arthritis symptoms were monitored. Protein tyrosine phosphorylation level, net cytokine secretion, and serum anti-human PG antibody titers were measured in immune cells from WT and Shp1-Tg mice. WT mice were treated with regorafenib orally to activate SHP-1 either before PG-induced arthritis (PGIA) symptoms developed (preventive treatment) or starting at an early stage of disease (therapeutic treatment). Data were statistically analyzed and graphs created using GraphPad Prism 8.0.2 software.ResultsSHP-1 expression and tyrosine phosphatase activity were elevated in both transgenic lines compared to WT mice. While all WT mice developed arthritis after immunization, none of the homozygous Shp1-Tg mice developed the disease. Heterozygous transgenic mice, which showed intermediate PGIA incidence, were selected for further investigation. We observed differences in interleukin-4 and interleukin-10 production in vitro, but serum anti-PG antibody levels were not different between the genotypes. We also found decreased tyrosine phosphorylation of several proteins of the JAK/STAT pathway in T cells from PG-immunized Shp1-Tg mice. Regorafenib administration to WT mice prevented the development of severe PGIA or reduced disease severity when started after disease onset.ConclusionsResistance to arthritis in the presence of SHP-1 overexpression likely results from the impairment of tyrosine phosphorylation (deactivation) of key immune cell signaling proteins in the JAK/STAT pathway, due to the overwhelming tyrosine phosphatase activity of the enzyme in Shp1-Tg mice. Our study is the first to investigate the role of SHP-1 in autoimmune arthritis using animals overexpressing this phosphatase. Pharmacological activation of SHP-1 might be considered as a new approach to the treatment of autoimmune arthritis.

Hypoxia Impairs NK Cell Cytotoxicity through SHP-1-Mediated Attenuation of STAT3 and ERK Signaling Pathways.

Natural killer (NK) cells are innate immune effectors with potent antitumor activity. However, tumor cells can create an immunosuppressive microenvironment to escape immune surveillance. Although accumulating evidence indicates that microenvironmental hypoxia plays an important role in favoring tumor development and immune evasion, it remains unclear by what means hypoxia directly impairs NK cell antitumor activity. In this study, we confirmed that hypoxic NK cells showed significantly lower cytotoxicity against tumor cells. Consistent with this finding, we found that the reduction in NK cell cytotoxicity resulting from hypoxia correlated to the lower expression of granzyme B, IFN-γ, and degranulation marker CD107a, as well as activating receptors including NKp30, NKp46, and NKG2D expressed on the surface of NK cells. More importantly, we further demonstrated that a reduction in the phosphorylation levels of ERK and STAT3 secondary to hypoxia was strongly associated with the attenuated NK cell cytotoxicity. Focusing on the mechanism responsible for reduced phosphorylation levels of ERK and STAT3, we reveal that the activation of protein tyrosine phosphatase SHP-1 (Src homology region 2 domain-containing phosphatase-1) following hypoxia might play an essential role in this process. By knocking down SHP-1 or blocking its activity using a specific inhibitor TPI-1, we were able to partially restore NK cell cytotoxicity under hypoxia. Taken together, we demonstrate that hypoxia could impair NK cell cytotoxicity by decreasing the phosphorylation levels of ERK and STAT3 in a SHP-1-dependent manner. Therefore, targeting SHP-1 could provide an approach to enhance NK cell-based tumor immunotherapy.

Modulation of inhibitory signals in CAR T cells leads to improved activity against glioblastoma.

3031 Background: Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the immunosuppressive tumor microenvironment. Adoptively transferred CAR T cells remain susceptible to inhibition via the engagement of co-inhibitory receptors on their surface such as PD1, BTLA, CTLA4 and LAG3. The subsequent recruitment of Src homology region 2 containing protein tyrosine phosphatase 2 (SHP2) by these receptors to the immune synapse may represent a common mechanism of T cell inhibition, as SHP2 can de-phosphorylate key signaling molecules that mediate T cell activation (including CD28 and CD3ζ). We hypothesized that SHP2 deletion will simultaneously offset the effects of multiple co-inhibitory receptors, thereby improving the anti-tumor activity of CAR T cells. Methods: Electroporation of sgRNA/Cas9 ribonucleoprotein complexes into human T cells was used to knockout (KO) SHP2. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 endodomain) targeting HER2. The phenotype of wild-type (WT) and SHP2KO CAR T cells was evaluated with mass cytometry and flow cytometry. Their anti-tumor function was tested in vitro using the xCELLigence assay (an impedance-based cytotoxicity assay), and in vivo, in an orthotopic xenograft mouse model of GBM. Results: Efficient and reproducible depletion of the SHP2 protein in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect CAR T cell expansion, proliferation or baseline phenotype. However, following co-culture with HER2+ LN229-GBM cells, the CD8+ central memory (CCR7+ CD45RA-) and effector memory (CCR7- CD45RA-) subsets were enriched to a greater extent in the SHP2KO CAR T cells. The pattern of cytokine co-expression varied between donors in a single-cell analysis comparing SHP2KO to WT CAR T cells after encountering LN229 cells. Functionally, SHP2KO CAR T cells derived from the majority of healthy donor and patient peripheral blood eliminated LN229 cells more rapidly in vitro. In an orthotopic mouse model of GBM, SHP2KO CAR T cells showed better early control of established LN229 xenografts and improved survival in comparison to WT CAR T cells. Conclusions: SHP2 deletion in CD28ζ.CAR T cells improves their anti-tumor activity.

Extracellular adenosine enhances pulmonary artery vasa vasorum endothelial cell barrier function via Gi/ELMO1/Rac1/PKA-dependent signaling mechanisms.

The vasa vasorum (VV), the microvascular network around large vessels, has been recognized as an important contributor to the pathological vascular remodeling in cardiovascular diseases. In bovine and rat models of hypoxic pulmonary hypertension (PH), we have previously shown that chronic hypoxia profoundly increased pulmonary artery (PA) VV permeability, associated with infiltration of inflammatory and progenitor cells in the arterial wall, perivascular inflammation, and structural vascular remodeling. Extracellular adenosine was shown to exhibit a barrier-protective effect on VV endothelial cells (VVEC) via cAMP-independent mechanisms, which involved adenosine A1 receptor-mediated activation of Gi-phosphoinositide 3-kinase-Akt pathway and actin cytoskeleton remodeling. Using VVEC isolated from the adventitia of calf PA, in this study we investigated in more detail the mechanisms linking Gi activation to downstream barrier protection pathways. Using a small-interference RNA (siRNA) technique and transendothelial electrical resistance assay, we found that the adaptor protein, engulfment and cell motility 1 (ELMO1), the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2, and atypical Gi- and Rac1-mediated protein kinase A activation are implicated in VVEC barrier enhancement. In contrast, the actin-interacting GTP-binding protein, girdin, and the p21-activated kinase 1 downstream target, LIM kinase, are not involved in this response. In addition, adenosine-dependent cytoskeletal rearrangement involves activation of cofilin and inactivation of ezrin-radixin-moesin regulatory cytoskeletal proteins, consistent with a barrier-protective mechanism. Collectively, our data indicate that targeting adenosine receptors and downstream barrier-protective pathways in VVEC may have a potential translational significance in developing pharmacological approach for the VV barrier protection in PH.

FCRL3 promotes IL-10 expression in B cells through the SHP-1 and p38 MAPK signaling pathways.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that is caused by the interaction of genetic and environmental factors. Current studies have shown that Fc-receptor like-3 (FCRL3) is closely related to MS, but the specific role of FCRL3 in MS has not yet been clarified. This study further found that FCRL3 and interleukin 10 (IL-10) expression was downregulated in MS patients, but the expression of these proteins was higher in the remission phase than that in the acute phase. The C allele of rs7528684 was associated with MS, and the CC genotype could lead to the upregulation of FCRL3 expression and the increase in IL-10 secretion. Further in vitro experiments with B cells found that lipopolysaccharide (LPS) promoted FCRL3 expression in a dose- and time-dependent manner, thereby promoting IL-10 secretion. LPS regulated Src homology region 2 domain-containing phosphatase-1 (SHP-1) expression and p38 mitogen-activated protein kinase (MAPK) pathway activation through FCRL3, and FCRL3 upregulated the SHP-1 expression and p38 phosphorylation levels. When SHP-1 small interfering RNA or a p38 pathway inhibitor was added, the effect of FCRL3 on IL-10 secretion was significantly inhibited. In addition, FCRL3 inhibited the secretion of inflammatory factors (tumor necrosis factor-α, IL-1β, IL-6, and IL-8); after inhibiting the expression of IL-10, the abovementioned effects of FCRL3 were blocked. These results suggest that FCRL3 can activate the SHP-1 and p38 MAPK pathways and then promote the secretion of IL-10 in B cells, thus inhibiting the secretion of inflammatory factors. Therefore, FCRL3 may play an immunoprotective role in MS, and it will be an effective target for the diagnosis and treatment of MS.

Fine tuning CD8+ T cell functional avidity by the PD-1/SHP2 axis

Abstract T cells of high functional avidity recognize low antigen levels and have the most effective cytotoxic responses against viruses and tumor cells. Understanding the molecular regulations of T cell functional avidity is essential to aid novel vaccine design as well as adoptive immunotherapy by improving the T cell quality. Programmed cell death protein 1 (PD-1) is known to negatively regulate the signal strength of T cell receptor (TCR). Activated PD-1 recruits Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (Shp2), which dephosphorylates kinases involved in TCR signaling. However, it is unclear whether PD-1/SHP2 can modulate T cell functional avidity. To address this question, we treated splenocytes from OT-1 transgenic mice with anti-PD-1 or SHP099 (SHP2 inhibitor), or a combination of both for 3 days in a selective culture condition of APC-pulsed stimulation with 10−6 M or 10−9 M SINNFEKL, respectively. Our preliminary data showed that blocking SHP2/PD-1 enhanced functional avidity and antigen sensitivity of CD8+ T cell in vitro when stimulated with limited SINNFEKL antigen. Blocking of PD1/SHP2 also enhanced CD8 beta expression, which has been shown correlated with high functional avidity CD8+ T cells. These data suggest that PD1/SHP2 is involved in regulating T cell functional avidity.

Modulation of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma

Background & Aim Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the highly immunosuppressive tumor microenvironment. These adoptively transferred CAR T cells are susceptible to inhibition via the engagement of inhibitory receptors on their surface including PD1, CTLA4, LAG3 and BTLA. The recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of these co-inhibitory receptors may represent a common mechanism of T cell inhibition. Upon its recruitment and activation, SHP2 dephosphorylates key stimulatory signaling molecules, thus limiting T cell activation. We hypothesized that disrupting SHP2 expression will simultaneously offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Methods, Results & Conclusion CRISPR/Cas9 technology was used to knockout (KO) SHP2 from human T cells. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 signaling endodomain) targeting HER2. The phenotype of wild-type and edited CAR T cells was investigated using mass cytometry and confirmed with flow cytometry. Their function was tested in vitro using xcelligence (an electrical impedance-based long-term cytotoxicity assay) and in vivo in an orthotopic xenograft mouse model of GBM. Efficient SHP2 KO in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect T cell expansion, proliferation or phenotypic profile. However, SHP2KO CAR T cells eliminated LN229 cells (a GBM cell line) in vitro more efficiently compared to wild-type CAR T cells. In vivo, SHP2KO CAR T cells showed better control of engrafted LN229 tumor cells and improved the survival of treated mice in comparison to wild-type CAR T cells. In conclusion, these results suggest that SHP2 may limit the function of CAR T cells and that SHP2 deletion may improve their anti-GBM activity.