Allosteric inhibition reveals SHP2-mediated tumor immunosuppression in colon cancer by single-cell transcriptomics

Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING-TBK1-IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68+ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy.

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC

Tyrosine phosphatase SHP2 inhibitors in tumor-targeted therapies

Src homology containing protein tyrosine phosphatase 2 (SHP2) represents a noteworthy target for various diseases, serving as a well-known oncogenic phosphatase in cancers. As a result of the low cell permeability and poor bioavailability, the traditional inhibitors targeting the protein tyrosine phosphate catalytic sites are generally suffered from unsatisfactory applied efficacy. Recently, a particularly large number of allosteric inhibitors with striking inhibitory potency on SHP2 have been identified. In particular, few clinical trials conducted have made significant progress on solid tumors by using SHP2 allosteric inhibitors. This review summarizes the development and structure-activity relationship studies of the small-molecule SHP2 inhibitors for tumor therapies, with the purpose of assisting the future development of SHP2 inhibitors with improved selectivity, higher oral bioavailability and better physicochemical properties.

The Role of B and T Lymphocyte Attenuator in Immune Regulation and Immune Related Diseases / 中国生物化学与分子生物学报

B / T lymphuocyte attenuator (BTLA) is one of the important checkpoint molecules in immune regulation. BTLA belongs to the CD28 superfamily, and its protein structure is similar to programmed cell death-1 (PD-1) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4). BTLA is mainly expressed in B and T cells, and has also been found in some innate immune cells such as dendritic cells and monocytes. To date, the herpesvirus entry mediator (HVEM) is the only ligand for BTLA found in human cells. HVEM belongs to the Tumor necrosis factor receptor (TNFR) superfamily, and its interaction with BTLA directly connects CD28 and TNFR families. The binding of BTLA with HVEM often mediates immunosuppressive effects and plays an important role in maintaining immune tolerance. However, recent studies have found that the BTLA / HVEM pathway not only provides negative regulatory signals, but also promotes the survival of T cells. This bidirectional signaling system renders BTLA-mediated immune regulation more sophisticated and complex. Growing evidence has shown that BTLA is involved in T cells, B cells and dendritic cell-mediated immune regulation, and therefore plays an important role in a variety of immune related diseases, such as inflammation, tumor, infectious diseases and autoimmune diseases. Based on the latest research progress at home and abroad, this paper reviews the role of BTLA in immune regulation and immune related diseases.

Role of CD160/BTLA-HVEM-LIGHT/LT-α signaling pathway related to herpetic stromal keratitis / 中华实验眼科杂志

Ocular infection of herpes simplex virus-1 (HSV1) can result in herpetic stromal keratitis (HSK),which impairs vision and is a common cause of human blindness.Studies indicated that HSK lesions are mainly orchestrated by CD4+ T cells.Herpesvirus entry mediator (HVEM),a tumor necrosis factor receptor superfamily member,facilitates virus entry through interactions with viral glycoprotein D (gD).HVEM,a widely expressed tumor necrosis factor (TNF) receptor superfamily member with diverse roles in immune signaling.Intriguingly,HVEM has five receptors:two costimulatory molecules (LIGHT and LT-α),two coinhibitory molecules (BTLA and CD160),and the HSV-gD.HVEM is referred to as a molecular switch because of its capacity to deliver costimulatory signals when bound to LIGHT/LT-α and to produce inhibitory signals when bound to BTLA/CD160.In this paper,the researching progress of the five receptors functions of HVEM and CD160/BTLA-HVEM-LIGHT/LT-α signaling pathway in the HSK were reviewed.We have to provide an insight into the pathogenesis of HSK and clinical ideas for the effective treatment of HSK.Through effective clinical intervention,the inflammatory immune response is reduced,thereby achieving therapeutic effects on recurrence of autoimmune diseases and chronic immune diseases.

Expression of B and T lymphocyte attenuation factor on regulatory T cell of HIV-infected patients with different diseases progression / 中国免疫学杂志

Objective:To detect the expression of BTLA on Treg cells of HIV-infected patients and investigate the role of BTLA in HIV infection.Methods: Forty-four HIV-1-infected patients (twenty-four early HIV infection,fourteen chronic HIV-infected patients with CD4+ T counts> 200 cells/μl,AIDS patients with CD4+T counts<200 cells/μl) and nine healthy people served as normal controls were selected to detect the expression of BTLA on Treg cells by flow cytometry.The correlations between BTLA expression on Treg cells and disease progression or immune activation were studied.Results: There was a higher percentage of BTLA on Treg cells in chronic HIV patients and AIDS patients than that in early HIV infected patients(P<0.05,P<0.01),and the expression of BTLA on Treg cells in AIDS patients was higher than that in normal controls(P<0.05).The expression of BTLA on Treg cells was negatively correlated with CD4+T lymphocyte counts and positively correlated with viral load (P<0.001,P<0.01).The percentage of BTLA on Treg cells was positively correlated with CD4+CD38+T lymphocytes and CD4+HLA-DR+T lymphocytes(P<0.001,P<0.001).Conclusion: Increased BTLA expression on HIV-infected Treg cells is associated with disease progression,suggesting that it may accelerate disease progression by enhancing Treg cells inhibitory function and may provide intervention information for HIV infection in the future.

Regulatory role of BTLA signal in the initiation and early phase of T cell activation / 中国免疫学杂志

Objective:To observe the expression of BTLA on T cells during activation and further analyze its inhibitory effects on T cell activation in different phases.Methods: T cells from PBMC were enriched by negative selection using magnetic beads.Expression of BTLA,CTLA-4 and PD-1 on freshly isolated human T cells and kinetics expression of BTLA,CILA-4 and PD-1 on CD3 mAb stimulated T cells were examined by flow cytometry.T cells were stimulated by anti-CD3 mAb combined with anti-CD28 mAb in the presence of anti-BTLA mAb 8H9,then T cell proliferation was tested by MTT assay in the different culture time.Immature DCs were generated from monocytes cultured in the medium containing GM-CSF and IL-4, and further driven to maturation by anti-CD40 mAb.Expression of HVEM on DCs was measured by flow cytometry.T cells were co-cultured with DCs in the presence of soluble 8H9 or anti-HVEM antibody to block HVEM-BTLA interaction,T cell proliferation was measured by MTT assay.Results:Freshly isolated T cells exhibited high levels of BTLA expression, but not CTLA-4 and PD-1.After T cell activation, BTLA expression decreased on first 2 days, with rapidly increasing to high levels.Unlike BTLA, expression of CTLA-4 and PD-1 was gradually increased during T cell activation.8H9 significantly inhibited the proliferation of T cell stimulated by CD3 mAb and CD28 mAb.8H9 could still exhibit inhibitory effect on T cell proliferation after 24 h or 48 h of preactivation by CD3 mAb plus CD28 mAb stimulation.HVEM was highly expressed on immature DCs, and down-regulated on mature DCs.Blockade of BTLA by soluble 8H9 or anti-HVEM antibody enhanced DC-mediated T cell proliferation within 48 h.Conclusion: BTLA signal enhances the threshold of T cell activation and plays importantly negative regulatory role in the initiation and early phase of T cell activation.

Effect of BTLA-HVEM inhibitory pathway blockade on DCs in vitro and in vivo / 中华微生物学和免疫学杂志

Objective To explore the effect of blocking BTLA-HVEM (herpesvirus entry mediator-B and T lymphocyte attenuator) pathway on dendritic cell function and the related immunological mechanisms. Methods Murine BTLA extracellular domain eukaryotic expression vector psBTLA was constructed by gene recombination and transfected CHO by Lipofection method. Mouse bone marrow cells were induced to differentiate into DCs by GM-CSF plus IL-4. Expression of BTLA and HVEM on DCs was detected after HSPT0-TC-1 peptide complex stimulation by FACS. Expression of BT-1 and secretion of IL-12 were detected after HSP70-TC-1 peptide complex plus psBTLA transfected CHO culture supernatant stimulation on DCs. Pretreated DCs co-cultured with the same genetic background mouse splenocytes and lymphocytes proliferation and cytokine secretion were detected. Effect of psBTLA gene transfer in vivo on BT-1 expression of DCs and tumor growth on tumor-bearing mice was detected. Results Extracellular domain of murine BTLA was successfully constructed, psBTLA stable transfection CHO cells were obtained and expression of BTLA extracellular domain(sBTLA) was detected the in its culture supernatant. BTLA and HVEM expression of DCs were increased after stimulation by the antigen peptide complex. When DCs were treated with antigen peptide complex plus culture supernatant containing sBTLA, B7-1 expression and IL-12 secretion were increased. Co-cultured with splenocytes, lymphocytes proliferation and cytokine secretion, such as IL-2 and IFN-γ,, were also increased. Gene transfection with psBTLA in vivo promoted B7-1 expression on DCs and inhibited cervical cancer cells growth. Conclusion Blockade of BTLA-HVEM inhibitory pathway with sBTLA can further improve DCs function, activation of lymphocytes and promote antitumor immune response.

Aberrant expression of B7-H4 and BTLA in tumormodel / 中国癌症杂志

Purpose:To further investigate the role of the novel negative immuno-regulatory molecules in tumor evasion by testing B7-H4 e xpression on tumor tissues and spleen macrophages induced by tumor antigen of mi ce and BTLA expression on T cells of the tumor bearing mice. Methods:Via RT-PCR, we evaluated the differences in the expres sion of B7-H4 mRNA of the tumor cell lines (3LL and Renca) in vitro,tumor t issues and the expression of BTLA mRNA of the tumor bearing mice T cells;via flo w cytometry, we evaluated the differences in the expression of B7-H4 surface pr otein of the spleen macrophages from tumor bearing mice and the normal spleen ma crophages after stimulation with the tumor antigen. Moreover, via immunohistoche mistry , we also evaluated the differences in the expression of B7-H4 surface p rotein of tumor tissues. Results:Tumor cell lines (3LL and Renca) did not express the B 7-H4 mRNA in vitro.All tumor tissues or spleen macrophages in tumor bearing mice have a high expression of both B7-H4 mRNA and B7-H4 protein. Compared wi th those from normal mice, T cells from tumor bearing mice showed a higher expre ssion of BTLA. Stimulated by tumor antigen in vitro, normal mice spleen macr ophages B7-H4 expression increased significantly; while no apparent difference was found in the groups stimulated by the corresponding normal tissue antigen. Conclusions:The aberrant expression of the costimulatory molecu les, B7-H4 and its receptor BTLA, may have a regulative effect in tumor evasion immunity.