Objective:To evaluate the role of IL-12 and CpG as adjuvants to promote the differentiation of antigen-specific CD4~+T cells in vivo.Methods:Naive OVA-TCR-Tg CD4~+T cells were isolated from spleens and lymph nodes of DO11.10 mice.The cells were labeled with CFSE in vitro and then were adoptively transferred into normal mice.One day after transfer,mice were immunized i.v.either with OVA,OVA+IL-12 or OVA+CpG.3 days post transfer,single cell suspensions were prepared from spleens,lymph nodes and lungs.The proliferation and IFN-? expression of transferred CD4~+T cells in different tissues were assessed by FACS.Results:No proliferation of antigen-specific CD4~+T cells was observed in the absence of immunization.After immunization with OVA for 3 days,cells were proliferated.The frequency of IFN-? producing cells in spleens,lymph nodes and lungs was at a range of 0.93%～2.17%.IL-12 promoted IFN-?~+ cells(4.53%～26.79%);Moreover,CpG enhanced the frequency of IFN-?-producing cells(3.84%) by the cells only from lymph nodes.In consistent with the results reported previously,cells divided 3-5 times revealed higher frequency of IFN-?-producing cells than the rest of them.Conclusion:IL-12 and CpG as adjuvants can promote the differentiation and expression of IFN-? by antigen-specific CD4~+T cells,and thus mediate cellular immune responses.

Objective:Understanding of subset、memory cell generation、effector function and organ distribution of Th1 cells in vitro and in vivo.Methods:Different stages of polarized Th1 cells are generated from antigen-specific CD4 +T cells.The expression of cell surface markers and intracellular cytokines(IL-2、IFN-? and IL-4) is determined at a single cell level by FACS. Equal numbers of CFSF-labeled naive CD4 +T cells and different stages of Th1 cells are adoptively transferred into mouse.The production of IFN-? and distribution of Th1 memory cells in lymphoid and non-lymphoid organs are measured. Results:Th1 cell populations can be subdivided into several subsets based upon the production of IL-2 and IFN-?. The percentage of IFN-?-producing cells is increased and IL-2-producing cells is decreased following repeatedly stimulation under Th1 culture condition. Moreover,the expression of CD62L on Th1 cells is reduced after activation and differentiation of CD4 +T cells. 2 weeks post-transfer,the frequency of naive CD4 +T cells is comparable from lymph nodes、spleens and lungs,whereas memory Th1 cells are preferentially detected in spleens and lungs and rapidly produce IFN-? following re-exposure to the same antigen.Conclusion:Th1 cell population is composed of distinct subsets of cells. In comparson with naive CD4 +T cells,memory Th1 cells reveal the changes of surface molecule expression、biologic function and distribution in tissues.

Objective:To investigate the correlation between CD4+T cell division and surface molecule expression or cytokine production after stimulation with antigen.Methods:Isolation of CD4+T cells from spleens and lymph nodes of OVA-T cell-receptor transgenic mouse (TCR-Tg ). After stimulation with OVA peptide antigen in the presence of antigen-presenting cells, CD4+T cell division, surface molecule expression and intracellular cytokine production are determined in flow cytometry.Results:CD4+T cells are divided 1-5 times after stimulation for 3 days with antigen. The cell division is associated with both up- and down-regulation of surface molecules such as CD25, CD44, CD62L and CD69 and of cytokines such as IFN-?, IL-4 and IL-10.IL-12 promotes cell division and enhances IFN-? but inhibits IL-4 and IL-10 production.Conclusion:After stimulation with antigen, CD4+T cells are divided in association with the quantitative and qualitative changes of surface molecules and cytokine production.

Objective: IFN-y is produced by both activated T and NK cells in response to mitogen or antigen and has a broad range of immunoregulatory activity. IL-12 has been described as a strong inducer of IFN-?production and promotes the differentiation of naive CD4+T cells toward the Th1 phenotype, priming them for IFN-?production, and consequent induction of cell-mediated immunity. Aim is to know endogenous production of IL,12 from PBMC inducing production of IFN-?in vitro, which is involving in mechanism of T cells to be activated. Methods: Induced IFT-? secretion from human PBMC by stimulated with anti-CD3 , PHA, anti-CD3 plus anti-CD28 and antigen(MLC) Also inhibited IFN-?production by neutralizing antibodies to IL-12 and IL-12R?1 significantly. Results: IFN-?secretion from human activated PBMC is endogenous IL-12dependent, and activated T cells induce the production of IL-12 from APC by a mechanism involving the interaction between CD40L on T cells and CD40 on APC. Conclusion: These results suggest that endogenous IL-12 plays an important role in the normal host defense against infection by a variety of intracellular pathogens and also plays a central role in the genesis of some forms of immunopathology including autoimmune diseases and transplantation rejections.

Objective: To investigate the reverse effect and mechanism of IL-12 on chemotherapeutic medicine suppressing the immune function of NK cells. Methods: Purified NK cells were stimulated with PMAplus Ionomycin in the presence or absent of Cisplatin (DDP) and IL-12. The levels of IFN-γ and TNF-α in culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA); The content of IFN-γ and TNF-α, TRAIL (TNF-related apoptosis inducing ligand) and transcription factors including T-bet and p-STAT-4 in NK cells were analyzed by Flow cytometry. The cytotoxicity of purified NK cells (pretreated with/without chemotherapeutics and IL-12 for 48 h) to Jurkat cells was measured by Flow cytometry. Results: Chemotherapeutics significantly inhibited the production of IFN-γ, TNF-α and the expression of TRAILin NK cells, which were significantly reversed by IL-12 (P<0.05 or P<0.01). Further study revealed that chemotherapeutics down-regulated while IL-12 reversed the expression of p-STAT4 to restore cytokine production. In addition, DDP also inhibited but IL-12 recovered the cytotoxicity of NK cells against tumor cells by inducing the expression of TRAIL (P<0.05 or P<0.01). Conclusion: Chemotherapeutics inhibited the cytotoxicity of NK cells and its secretion of cytokines (IFNγ and TNF-α), which were reversed by IL-12 via up-regulating TRAIL and p-STAT-4; this might provide experimental evidence for the clinical application of IL-12 for rebuild the immune function of tumor patients receiving chemotherapy.

Objective To investigate the efficacy and safety of transarterial chemoembolization (TACE) combined with camrelizumab and apatinib in the treatment of advanced hepatocellular carcinoma (HCC). Methods From July 2019 to June 2021, 19 patients with advanced HCC who met the inclusion and exclusion criteria in the First Affiliated Hospital of Soochow University were enrolled in this study. All patients received TACE combined with camrelizumab and apatinib. Tumor response was assessed according to Modified Response Evaluation Criteria in Solid Tumors (mRECIST), and adverse events were assessed according to Common Terminology Criteria for Adverse Events (v5.0). The Kaplan-Meier method was used to analyze progression-free survival and overall survival and calculate 95% confidence interval (CI). Results The median follow-up time was 14.0 months for the 19 patients. As of the last follow-up based on mRECIST, 7 patients (7/19, 36.8%) achieved complete response, 9 (9/19, 47.4%) achieved partial response, and 2 (2/19, 10.5%) achieved stable disease. During follow-up, overall objective response rate and overall disease control rate reached 84.2% and 94.7%, respectively; the median duration of response reached 8.0 (3.4-13.0) months, and median progression-free survival reached 9.5 (95% CI : 4.7-14.3) months; the 6-month survival rate reached 100%, and the 12-month survival rate reached 78.9%. Among the 19 patients, 7 (36.8%) experienced serious adverse events. The most common adverse events of all grades included post-embolization syndrome after TACE (17/19, 89.5%), liver injury (14/19, 73.7%), hematologic toxicity (8/19, 42.1%), and proteinuria (8/19, 42.1%). Conclusion TACE combined with camrelizumab and apatinib has marked efficacy and controllable adverse events in the treatment of advanced HCC, which provides a potential new option for the first-line treatment of advanced HCC.