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@#Objective: To investigate the function of anti-PD-1 (scFv)/IL-15/IL-15Rα-sushi fusion protein (PD-S15) to specifically bind to PD-1 in vitro and to explore its effect on NK/T cell proliferation. Methods: The human anti-PD-1 (scFv) gene sequence and human IL-15/IL-15Rα-sushi fusion gene sequence were synthesized chemically. The recombinant expression plasmid pUC57-PD-S15 was constructed by enzyme digestion and ligation of the two target genes, and then transiently transfected into HEK293T cells by lipofectamineTM 2000. The supernatants of cell culture medium were acquired, and the expression of PD-S15 fusion protein in cell culture supernatants was detected by Wb assay. PBMCs and TILs were cultured in mediums with different proportion of PD-S15/X-VIVOTM15, respectively. Then, the capacity of PD-S15 fusion protein to bind to PD-1 in vitro and its effect on the proliferation of PBMCs and the proportion of CD3+CD8+, CD3+CD4+ and CD3-CD56+ subsets were detected by flow cytometry. The effect of PD-S15 fusion protein on the proliferation of TILs was detected by cytometry. Results: The successful construction of pUC57-PD-S15 eukaryotic expression plasmid was confirmed by double enzyme digestion and sequencing, and then successfully transfected into HEK293T cells. The relative molecular weight of the target protein was approximately 55 000, and was in line with expectations. PD-S15 fusion protein could specifically combine with PD-1 in vitro (P<0.05) and stimulate NK/T cell proliferation (P<0.05). Compared with classical TILs culture method, the efficiency of activation and amplification of T cells in vitro by PD-S15 culturemethodwasbetter (P<0.01). Conclusion: PD-S15 fusion protein can specifically target PD-1 and rapidly expand NK/T cells in vitro, which lays a foundation for the selective expansion of CD8+PD-1+ antigen-specific T lymphocytes from tumor tissues and even peripheral blood.
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Objective To investigate the antigen-specific T cell functionality in type 2 diabetes mellitus patients. Methods Peripheral blood from 38 type 2 diabetes mellitus patients and 47 health controls (control group) have been collected. The proportions of CD4+and CD8+T cell as well as the ratio of CD4+/CD8+were monitored by flow cytometry. Meanwhile, antigen- nonspecific and specific Th1 responses were compared between two groups through detecting interferon (IFN)-γ, interleukin 2 (IL-2), and tumor necrosis factor (TNF)-α producing cells upon propylene glycol monomethyl ether acetate (PMA)/ionomycine and epstein-barr virus ( EBV) peptides stimulation, respectively followed by an intracellular cytokine staining. Results Compared to control group, the proportion of CD4+T cell and the ratio of CD4+/CD8+were significantly increased in type 2 diabetes mellitus group (P＜0.05) whereas CD8+T cells exhibited no significant difference between two groups. Antigen-nonspecific Th1 responses in type 2 diabetes mellitus patients were significantly decreased, demonstrated by lower percentages of IFN-γ, IL-2, and TNF-α producing CD4+T cells when compared to control group , while CD8+T cells in type 2 diabetes mellitus patients exhibited similar cytokine production patterns. However, when stimulated by EBV specific peptides, the percentages of IFN-γ, IL-2, and TNF-α producing CD8+T cells were significantly higher in type 2 diabetes mellitus patients than those in control group (P＜0.05). HbA1Cwas positively correlated with the percentage of EBV-specific TNF-α producing CD8+T cells (P＜0.05). Conclusion In type 2 diabetes mellitus, the secretion capacity of CD4+and CD8+T cell was significantly decreased and the antigen-specific responses represent the presence of an abnormal activated status, which indicates that chronic hyperglycemia may damage T cells function and aggravate chronic inflammation.
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BACKGROUND: Collagen-induced arthritis (CIA) in mice is animal model of autoimmune disease known as rheumatic arthritis in human. We investigated CII-specific CD4+ T cell receptor usage in CIA mice. METHODS: In CIA model, draining lymph node (dLN) CD4+ T cells and splenocytes at 3rd, 5th, 8th week, we investigated CII-specific T cell proliferation, production of IL-17, IFN-gamma, TNF-alpha, IL-4 and IL-10. And we also performed anti-CII IgG Ab measurements in serum level, TCRVbeta usage and T cell clonality with RT-PCR-SSCP analysis. Also, we performed proliferative response against CII when CII-specific T cell subset is deleted. RESULTS: CIA mice showed more increase in the serum level of anti-CII IgG than normal mice after induction of arthritis. And the level of anti-CII IgG2a in CIA mice was increased after 3rd week after primary immunization, while anti-CII IgG1 was decreased. Draining LN CD4+T cells have proliferated against CII stimulation at 3rd week after 1st immunization. CD4+T cells derived from dLN of CIA mice produced proinflammatory cytokine IFN-gamma, IL-17 etc. Draining LN CD4 T cells of CIA presented higher proportion of CD4+Vbeta +subsets compared to those of normal mice at 3rd week after 1st immunization, and they were increased in proportion by CII stimulation. Draining LN CD4+ T cells without TCRVbeta +/Vbeta 8.1/8.2+/Vbeta 10b+cells were not responsive against CII stimulation. But, CII-reactive response of TCRVbeta 3-/Vbeta 8.1/8.2-/Vbeta 10b- T cells was recovered when Vbeta 3+ T cells were added in culture. CONCLUSION: Our results indicate that CD4+Vbeta 3+ T cells cells are selectively expanded in dLN of CIA mice, and their recovery upon CII re-stimulation in vitro, as well as the production Th1-type cytokines, may play pivotal role in CIA pathogenesis.