Weijuan Gong

Ex vivo expansion of natural killer cells with high cytotoxicity by K562 cells modified to co-express major histocompatibility complex class I chain-related protein A, 4-1BB ligand, and interleukin-15.

A large number of natural killer (NK) cells with high function are expected to generate especially in tumor adoptive immunotherapy. Here K562 cells were genetically modified to co-express major histocompatibility complex class I chain-related protein A (MICA), 4-1BB ligand, and IL-15, called K562-MICA-4-1BBL-IL-15. The modified K562 cells not only promoted activation, proliferation, and survival of NK cells, but also enhanced NK cell cytotoxicity. In long-term culture tests, K562-MICA-4-1BBL-IL-15 cells stimulated NK cell to expand mean 550 folds in 24-day culture and to cover from 14.8% of total peripheral blood monoclonal lymphocytes on day 1 to 86.7% on day 24. Prevalent NK cells after expansion enhanced the ability of killing targets and producing interferon gamma (IFN-γ), and kept high expression of activating receptors. The results indicated that K562-MICA-4-1BBL-IL-15 cells would be developed for expansion of NK cells ex vivo and may have important implications for clinical immunotherapy.

Induction of TGF-β and IL-10 production in dendritic cells using astilbin to inhibit dextran sulfate sodium-induced colitis

Astilbin, a major bioactive compound from Rhizoma smilacis glabrae, has been reported to possess anti-inflammatory properties. Our study first evaluated astilbin on dextran sulfate sodium (DSS)-induced acute colitis in mice. By intraperitoneal injection of astilbin, the severity of colitis was attenuated, and the serum levels of IL-10 and TGF-β were increased. Using flow cytometry, a higher number of IL-10(+) dendritic cells (DCs) and TGF-β(+) DCs and a lower number of CD86(+) DCs, IL-12 p40(+) DCs, and IL-1β(+) DCs were detected in the spleen of mice with colitis after astilbin treatment. The administration of astilbin also resulted in the upregulation of CD103(+) expression in colonic DCs. In a coculture system, murine bone marrow-derived DCs pretreated with astilbin resulted in an enhanced production of CD4(+)CD25(+)Foxp3(+) T cells. The results of this study show that astilbin could be a candidate drug for inflammatory bowel disease by mediating the regulatory functions of DCs.

Human fused NKG2D-IL-15 protein controls xenografted human gastric cancer through the recruitment and activation of NK cells.

Interleukin (IL)-15 plays an important role in natural killer (NK) and CD8+ T-cell proliferation and function and is more effective than IL-2 for tumor immunotherapy. The trans-presentation of IL-15 by neighboring cells is more effective for NK cell activation than its soluble IL-15. In this study, the fusion protein dsNKG2D–IL-15, which consisted of two identical extracellular domains of human NKG2D coupled to human IL-15 via a linker, was engineered in Escherichia coli. DsNKG2D–IL-15 could efficiently bind to major histocompatibility complex class I chain-related protein A (MICA) of human tumor cells with the two NKG2D domains and trans-present IL-15 to NK or CD8+ T cells. We transplanted human gastric cancer (SGC-7901) cells into nude mice and mouse melanoma cells with ectopic expression of MICA (B16BL6–MICA) into C57BL/6 mice. Then, we studied the anti-tumor effects mediated by dsNKG2D–IL-15 in the two xenografted tumor models. Human dsNKG2D–IL-15 exhibited higher efficiency than IL-15 in suppressing gastric cancer growth. Exogenous human dsNKG2D–IL-15 was centrally distributed in the mouse tumor tissues based on in vivo live imaging. The frequencies of human CD56+ cells infiltrated into the tumor tissues following the injection of peripheral blood mononuclear cells into nude mice bearing human gastric cancer were significantly increased by human dsNKG2D–IL-15 treatment. Human dsNKG2D–IL-15 also delayed the growth of transplanted melanoma (B16BL6–MICA) by activating and recruiting mouse NK and CD8+ T cells. The anti-melanoma effect of human dsNKG2D–IL-15 in C57BL/6 mice was mostly decreased by the in vivo depletion of mouse NK cells. These data highlight the potential use of human dsNKG2D–IL-15 for tumor therapy.Cellular & Molecular Immunology advance online publication, 14 September 2015; doi:10.1038/cmi.2015.81

Establishment and Characterization of a Cell Based Artificial Antigen-Presenting Cell for Expansion and Activation of CD8 + T Cells Ex Vivo

Artificial antigen-presenting cells are expected to stimulate the expansion and acquisition of optimal therapeutic features of T cells before infusion. Here CD32 that binds to a crystallizable fragment of IgG monoclonal antibody was genetically expressed on human K562 leukemia cells to provide a ligand for T-cell receptor. CD86 and 4-1BBL, which are ligands of co-stimulating receptors of CD28 and 4-1BB, respectively, were also expressed on K562 cells. Then we accomplished the artificial antigen-presenting cells by coupling K32/CD86/4-1BBL cell with OKT3 monoclonal antibody against CD3, named K32/CD86/4-1BBL/OKT3 cells. These artificial modified cells had the abilities of inducing CD8+ T cell activation, promoting CD8+ T cell proliferation, division, and long-term growth, inhibiting CD8+ T cell apoptosis, and enhancing CD8+ T cell secretion of IFN-γ and perforin. Furthermore, antigen-specific cytotoxic T lymphocytes could be retained in the culture stimulated with K32/CD86/4-1BBL/OKT3 cells at least within 28 days. This approach was robust, simple, reproducible and economical for expansion and activation of CD8+ T cells and may have important therapeutic implications for adoptive immunotherapy.

CD4+ NKG2D+ T cells induce NKG2D down-regulation in natural killer cells in CD86-RAE-1ε transgenic mice

The binding of NKG2D to its ligands strengthens the cross‐talk between natural killer (NK) cells and dendritic cells, particularly at early stages, before the initiation of the adaptive immune response. We found that retinoic acid early transcript‐1ε (RAE‐1ε), one of the ligands of NKG2D, was persistently expressed on antigen‐presenting cells in a transgenic mouse model (pCD86‐RAE‐1ε). By contrast, NKG2D expression on NK cells, NKG2D‐dependent cytotoxicity and tumour rejection, and dextran sodium sulphate‐induced colitis were all down‐regulated in this mouse model. The down‐regulation of NKG2D on NK cells was reversed by stimulation with poly (I:C). The ectopic expression of RAE‐1ε on dendritic cells maintained NKG2D expression levels and stimulated the activity of NK cells ex vivo, but the higher frequency of CD4+ NKG2D+ T cells in transgenic mice led to the down‐regulation of NKG2D on NK cells in vivo. Hence, high levels of RAE‐1ε expression on antigen‐presenting cells would be expected to induce the down‐regulation of NK cell activation by a regulatory T‐cell subset.

Expression of human cytomegalovirus pp150 gene in transgenic Vicia faba L. and immunogenicity of pp150 protein in mice.

The pp150 gene of human cytomegalovirus (HCMV) was transferred into Vicia faba plants by Agrobacterium tumefaciens-mediated transformation. Three of five hygromycin resistant V. faba plants were identified as positive by PCR and dot-blot hybridization. The ELISA results indicated that pp150 protein from three plants of transformed V. faba leaves and seeds made up 0.005-0.015% of the total soluble protein. The results of detection by immunoblot and inhibition of immunofluorescent assay (IFA) showed that pp150 soluble protein had immunity activity. HCMV pp150-specific antibody (IgG, IgA) and IFN-gamma producing T cells were detected in 100% of the mice immunized with pp150 transgenic V. faba seeds by ELISA and intracellular staining and flow cytometry analysis, respectively. The transgenic V. faba plants will provide new material for the development of edible vaccination against HCMV infection.

NKG2D ligand RAE1ε induces generation and enhances the inhibitor function of myeloid‐derived suppressor cells in mice

Expression of surface NKG2D ligands on tumour cells, which activates nature killer (NK) cells and CD8+ T cells, is crucial in antitumour immunity. Some types of tumours have evolved mechanisms to suppress NKG2D‐mediated immune cell activation, such as tumour‐derived soluble NKG2D ligands or sustained NKG2D ligands produced by tumours down‐regulate the expression of NKG2D on NK cells and CD8+ T cells. Here, we report that surface NKG2D ligand RAE1ε on tumour cells induces CD11b+Gr‐1+ myeloid‐derived suppressor cell (MDSC) via NKG2D in vitro and in vivo. MDSCs induced by RAE1ε display a robust induction of IL‐10 and arginase, and these MDSCs show greater suppressive activity by inhibiting antigen‐non‐specific CD8+ T‐cell proliferation. Consistently, upon adoptive transfer, MDSCs induced by RAE1ε significantly promote CT26 tumour growth in IL‐10‐ and arginase‐dependent manners. RAE1ε moves cytokine balance towards Th2 but not Th1 in vivo. Furthermore, RAE1ε enhances inhibitory function of CT26‐derived MDSCs and promotes IL‐4 rather than IFN‐γ production from CT26‐derived MDSCs through NKG2D in vitro. Our study has demonstrated a novel mechanism for NKG2D ligand+ tumour cells escaping from immunosurveillance by facilitating the proliferation and the inhibitory function of MDSCs.

Chitosan nanoparticle-based delivery of fused NKG2D–IL-21 gene suppresses colon cancer growth in mice

Nanoparticles can be loaded with exogenous DNA for the potential expression of cytokines with immune-stimulatory function. NKG2D identifies major histocompatibility complex class I chain-related protein in human and retinoic acid early induced transcript-1 in mouse, which acts as tumor-associated antigens. Biologic agents based on interleukin 21 (IL-21) have displayed antitumor activities through lymphocyte activation. The NKG2D–IL-21 fusion protein theoretically identifies tumor cells through NKG2D moiety and activates T cells through IL-21 moiety. In this study, double-gene fragments that encode the extracellular domains of NKG2D and IL-21 genes were connected and then inserted into the pcDNA3.1(−) plasmid. PcDNA3.1–dsNKG2D–IL-21 plasmid nanoparticles based on chitosan were generated. Tumor cells pretransfected with dsNKG2D–IL-21 gene nanoparticles can activate natural killer (NK) and CD8+ T cells in vitro. Serum IL-21 levels were enhanced in mice intramuscularly injected with the gene nanoparticles. DsNKG2D–IL-21 gene nanoparticles accumulated in tumor tissues after being intravenously injected for ~4–24 h. Treatment of dsNKG2D–IL-21 gene nanoparticles also retarded tumor growth and elongated the life span of tumor-bearing mice by activating NK and T cells in vivo. Thus, the dsNKG2D–IL-21 gene nanoparticles exerted efficient antitumor activities and would be potentially used for tumor therapy.

Treatment with a fusion protein of the extracellular domains of NKG2D to IL-15 retards colon cancer growth in mice.

Tumor-targeted cytokines are a new class of pharmaceutical anticancer agents often considered superior to the corresponding unconjugated cytokines for therapeutic purposes. We generated a new fusion protein, dsNKG2D-IL-15, in which double NKG2D extracellular domains were fused to IL-15, in Escherichia coli. This fusion protein promoted the activation, proliferation, and cytotoxicity of NK cells, and bound to NKG2D ligand-positive tumor cells. These tumor cells were also more susceptible to NK-cell attack when decorated with dsNKG2D-IL-15. The administration of mouse dsNKG2D-IL-15 protein in vivo significantly retarded the growth of transplanted colon cancers and prolonged the survival of tumor-bearing mice. Treatment with dsNKG2D-IL-15 increased the frequencies of NK and CD8+ T cells in spleen and tumor tissues. The antitumor effect mediated by dsNKG2D-IL-15 was significantly decreased with in vivo depletion of NK cells or CD8+ T cells. Recombinant dsNKG2D-IL-15 thus inhibited NKG2D ligand-positive tumor growth effectively by activating lymphocytes. This new biological fusion protein could potentially be used to elicit immunity in tumor-targeting treatments.

Application and expression of Toxoplasma gondii surface antigen 2 (SAG2) and rhoptry protein 2 (ROP2) from recombinant Escherichia coli strain

The gene encoding surface antigen 2 (SAG2) or rhoptry protein 2 (ROP2) of Toxoplasma gondii was cloned into the plasmid pGEX-4T-1 and subsequently expressed in Escherichia coli as a glutathione-s-transferase (GST) fusion protein. The characteristics of purified GST-SAG2 or GST-ROP2 were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. The specific IgG of a panel of serum samples provided by the National Institute for the Control of Pharmaceutical and Biological Products were tested with commercial ELISA and the lateral flow immunoassay (LFIA) based on GST-SAG2, GST-ROP2 or GST-SAG2+ROP2. A total of 1096 sera and saliva samples from pregnant women were tested by GST-SAG2+ROP2-LFIA. In total, 20 T. gondii IgM positive sera (1.82%), 81 T. gondii IgG positive sera (7.4%) and 23 T. gondii IgA positive saliva (2.1%) were finally confirmed. The SAG2+ROP2 specific IgG and IFN-γ producing CD8+ T cells were induced in mice immunised with GST-SAG2+ROP2. The results indicate that GST-SAG2+ROP2 protein can be used as an antigen for diagnosing T. gondii infection and provide a strategy for development of subunit vaccines for protection against T. gondii infection.