Mingchun Ji

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.

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

Neisseria Gonorrhoeae NspA Induces Specific Bactericidal and Opsonic Antibodies in Mice

ABSTRACT Neisseria gonorrhoeae surface protein A (NspA) is a highly conserved gonococcal antigen. To explore the potential of NspA in vaccine development against gonorrhea, BALB/c mice were immunized with pcNspA containing the NspA gene from N. gonorrhoeae strain WHO-A via intramuscular (i.m.) injection, intranasal (i.n.) immunization, or intravaginal (i.vag.) immunization. Following the last DNA immunization, mice were boosted with recombinant NspA (rNspA). Enzyme-linked immunosorbent assays (ELISAs) indicated that all immunized mice generated measurable NspA-specific IgG and IgA in serum and secretory IgA (sIgA) in vaginal wash fluids. The antisera had bactericidal and opsonic activities. These data demonstrated that NspA induced antibodies with antigonococcal activity.

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.

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.

Immobilized MHC class I chain-related protein A synergizes with IL-15 and soluble 4-1BB ligand to expand NK cells with high cytotoxicity ex vivo

Major histocompatibility complex (MHC) class I chain-related protein A (MICA), which is a ligand for human NKG2D, is expressed by a variety of epithelial tumor cells and promotes the activation of natural killer (NK), CD8+ and γδ-T cells. Although ectopic expression of MICA on tumor cells elicits anti-tumor responses, soluble MICA downregulates the activities of lymphocytes. In this study, we showed that recombinant, immobilized MICA (iMICA) molecules coated on plastic wells weakly promote peripheral NK cell activation, secretion of interferon (IFN)-γ and degranulation without inducing apoptosis. In addition, iMICA synergized with IL-15 and soluble 4-1BB ligand (s4-1BBL) to expand NK cells 25- to 42-fold in a 13-day culture, whereas NK cells stimulated only with IL-15 and s4-1BBL expanded 10- to 16-fold. In contrast to NK cells expanded by IL-15 and s4-1BBL stimulation, NK cells expanded long term in the presence of iMICA exhibited increased cytotoxicity against leukemia cells. These results suggest that large numbers of NK cells with high cytotoxicity can be generated by stimulation with IL-15 and s4-1BBL in the presence of iMICA and that these cells can be used for adoptive cancer immunotherapy.

Construction of a plasmid for co-expression of mouse membrane-bound form of IL-15 and RAE-1ε and its biological activity.

Interleukin 15 (IL-15) is a pivotal cytokine for the proliferation and activation of a specific group of immune cells such as natural killer (NK), IFN-producing killer dendritic cells (IKDC) and CD8 T cells. RAE-1ε, the ligand for the activating NKG2D receptor, which also play an important role in the proliferation and activation of NK cells and IKDCs. In this study, a membrane-bound form of IL-15 (termed mb15) encoding sequence and RAE-1ε gene were obtained by SOE-PCR or PCR amplification. The amplified mb15 and RAE-1ε gene were then digested and inserted into the multiple cloning site1 (MCS1) and MCS2 of pVITRO2-mcs vector, respectively. A recombinant eukaryotic expression vector for co-expression of mb15 and RAE-1ε was successfully constructed. After it was transfected to BaF3 cells, the expression of IL-15 and RAE-1ε in recombinant BaF3/mb15/RAE-1ε cells were verified by RT-PCR, western blot and FCM analysis. Furthermore, BaF3/mb15/RAE-1ε cells had the ability of promoting NK cells proliferation and IFN-γ secretion. In conclusion, BaF3/mb15/RAE-1ε cells were successfully constructed, which is very useful for further studies, especially for the expansion and activation of certain subsets of immune cells such as NK cells and IKDCs.