CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity.

The incidence of autoimmune diseases has significantly increased over the past 20 years. Excessive host immunoreactions and disordered immunoregulation are at the core of the pathogenesis of autoimmune diseases. The traditional anti-tumor chemotherapy drug CPT-11 is associated with leukopenia. Considering that CPT-11 induces leukopenia, we believe that it is a promising drug for the control of autoimmune diseases. Here, we show that CPT-11 suppresses T cell proliferation and pro-inflammatory cytokine production in healthy C57BL/6 mice and in complete Freund's adjuvant-challenged mice. We found that CPT-11 effectively inhibited T cell proliferation and Th1 and Th17 cell differentiation by inhibiting glycolysis in T cells. We also assessed CPT-11 efficacy in treating autoimmune diseases in models of experimental autoimmune encephalomyelitis and psoriasis. Finally, we proved that treatment of autoimmune diseases with CPT-11 did not suppress long-term immune surveillance for cancer. Taken together, these results show that CPT-11 is a promising immunosuppressive drug for autoimmune disease treatment.

Regulatory T cell therapy suppresses inflammation of oral mucosa.

Oral inflammatory diseases, including oral lichen planus (OLP) and recurrent aphthous ulcer (RAU), seriously affect the patient's quality of life. Due to the lack of ideal disease models, it is difficult to determine whether novel immunotherapy strategies are effective in treating oral inflammatory diseases. Here, we show that the deficiency of Foxp3 or IL-2 caused oral mucosa inflammation in mice, proving that Treg cells are important in maintaining the immune homeostasis in the oral mucosa. Then we determined that adoptive transfer of CD4+CD25-CD45Rbhigh T cells could induce oral inflammation in Rag1 -/- mice, and co-transfer of Treg cells together with CD4+CD25-CD45Rbhigh T cells could suppress the development of oral inflammation in this mouse model. Our study showed that adoptive transfer of CD4+CD25-CD45Rbhigh T cells into Rag1 -/- mice could be a novel disease model of oral inflammation. Our data provides direct evidence that Treg cell therapy is effective in suppressing oral mucosa inflammation in mice. Therefore, Treg cell therapy may be a promising novel strategy to treat oral inflammatory diseases.

Role of Cytokines in Thymic Regulatory T Cell Generation: Overview and Updates.

CD4+CD25+Foxp3+ Regulatory (Treg) T cells are mainly generated within the thymus. However, the mechanism of thymic Treg cell (tTreg cell) generation remains to be fully revealed. Although the functions of TCR/CD28 co-stimulation have been widely accepted, the functions of cytokines in the generation of tTreg cells remain highly controversial. In this review, we summarize the existing studies on cytokine regulation of tTreg cell generation. By integrating the key findings of cytokines in tTreg cell generation, we have concluded that four members of γc family cytokines (IL-2, IL-4, IL-7 and IL-15), transforming growth factor ß (TGF-ß), and three members of TNF superfamily cytokines (GITRL, OX40L and TNF-α) play vitally important roles in regulating tTreg cell generation. We also point out all disputed points and highlight critical scientific questions that need to be addressed in the future.

Study on the preparation and function of regulatory T cells from human peripheral blood.

BACKGROUND: Regulatory T cells (Tregs) are an important cell subgroup of CD4+ T cells. Treg cells are critically involved in inducing immune tolerance, maintaining immune environment homeostasis, and preventing the occurrence of autoimmune diseases. Under normal conditions, the number of Tregs in the body is very small. This research was designed to establish an effective method to expand human peripheral blood Tregs in vitro and to analyze phenotype, purity, and function of Treg cells post-expansion. METHODS: Peripheral blood was obtained from healthy donors. CD4+CD25+CD127dim/- Treg cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic-activated cell sorting (MACS), and an optimized culture system was used for amplification. The in vitro amplification ability of Treg cells was evaluated to determine the expression and purity of Treg cell-specific surface markers in different culture cycles. The suppressive function of Treg was determined by in vitro lymphocyte proliferation assay. RESULTS: Treg cells could be successfully isolated by magnetic activated cell sorting (MACS). After 21 days of in vitro culture, the mean expansion fold was 2,009±452.2 in ≤60 years, and there was a significant difference between the younger group and the older than 60 years group (1,238±330.0). Flow cytometry analysis revealed that the percentages of CD4+CD25+ cells and FOXP3+ cells were (93.25±3.05)% and (94.19±4.21)% on day 14, and (92.86±4.36)% and (91.55±5.62)% on day 21, respectively. In addition, the proportions of CD8+ T, CD19+ B, CD3-CD56+ natural killer cell (NK), and CD3+ CD56+ natural killer T cell (NKT) were extremely low. Lymphocyte proliferation assay demonstrated that Tregs could inhibit the proliferation of CD8+ T cells more effectively than that of CD4+ T cells. Furthermore, the suppressive capacity of Tregs was correlated with Treg-to-PBMCs ratios. CONCLUSIONS: We successfully established a technical protocol for manufacturing a large quantity of Tregs with high efficiency in vitro. The expanded Tregs have a steady FOXP3 expression and exhibited a potent immune suppression, which might have great significance in adoptive Treg therapy for treating graft-versus-host disease and autoimmune diseases.

PKCι regulates the expression of PDL1 through multiple pathways to modulate immune suppression of pancreatic cancer cells.

To investigate the impact of oncogenic protein kinase C isoform ι (PKCι) on the microenvironment and the immunogenic properties of pancreatic tumors, we prohibit PKCι activity in various pancreatic ductal adenocarcinoma (PDAC) cell lines and co-culture them with human natural killer NK92 cells. The results demonstrate that PKCι suppression enhances the susceptibility of PDAC to NK cytotoxicity and promotes the degranulation and cytolytic activity of co-cultured NK92 cells. Mechanistic studies pinpoint that downstream of KRAS, both YAP1 and STAT3 are recruited by oncogenic PKCι to elevate the expression of PDL1, contributing to constitute an immune suppressive microenvironment in PDAC. Co-culture with NK92 further induces PDL1 upregulation via STAT3 to stimulate immune escape of PDAC cells. Subsequently, inhibition of PKCι in PDAC alleviates the immune suppression and enhances the cytotoxicity of NK92 towards PDAC through restraining PDL1 overexpression. Combined with PD1/PDL1 blocker, PKCι inhibitor remarkably elevates the cytotoxicity of NK92 against PDAC cells in vitro, establishing PKCι inhibitor as a promising candidate for boosting the immunotherapy of PDAC.

[Study on the anti-NTHi infection of Hap recombinant protein in vivo].

AIM: To observe the immune effect of Hap recombinant protein on murine model of bronchopneumonia infected with NTHi, and explore the mechanism about the anti-NTHi infection. METHODS: The C57BL/6 mice intranasally immunized with purified Hap recombinant protein and CT-B were challenged by NTHi encased in agar beads. The immunifaction of anti-infection was observed through encocyte counting of BALF, bacteria detection of lung and the pathologyical change of lung tissue. RESULTS: In the challenge with NTHi experiment, the inflammatory exudation of the infected murine and pathological change of lung tissue was relieved by combined immunization of Hap recombinant protein and CT-B, and quantity of NTHi in lung of the infected murine was reduced obviously. CONCLUSION: The Hap recombinant protein also had good ability of anti-NTHi infection in the murine model of NTHi bronchopneumonia. This study could offer the oretical and experimental basis for development of new vaccine against NTHi.

[Construction and expression characteristics in vitro of human papillomavirus type 16 E7 suicidal DNA vaccine].

OBJECTIVE: To construct the suicidal DNA vaccine of human papillomavirus type 16 E7 gene (HPV16), and explore the DNA vaccine expression characteristics in vitro and capacity of inducing the transfected cells into apoptosis. METHODS: HPV16 E7 gene cloned by PCR from pET32/E7 was inserted into the plasmid pSCA1 to construct the recombinant plasmid pSCA/E7, followed by identification with PCR, BamH I and Sma I digestion and sequencing. pSCA/E7 was then used to transfect BHK-21 cell line. The transient expression of HPV16 E7 gene was confirmed by immuno-fluorescent staining, and the apoptosis induced by pSCA/E7 was checked with TDT-mediated dUTP nick end-labeling (TUNEL). RESULTS: The cloned E7 gene fragment was about 400 bp in length. PCR, restriction endonuclease digestion and sequence analysis revealed that the HPV16 E7 gene was cloned into the eukaryotic expression plasmid pSCA1 successfully. Immunofluorescent staining confirmed that the E7 gene could express in BHK-21 cell line. The BHK-21 cells transfected with pSCA/E7 could be induced into apoptosis which was confirmed by TUNEL. CONCLUSION: The results show that HPV16 E7 suicidal DNA vaccine can express in BHK-21 cell line, and induce the pSCA/E7 transfected cells into apoptosis. These findings may provide the foundation for exploring the therapeutic vaccine against HPV16-associated cervical cancer.

[The effects of activities of Ca(2+)-ATPase and Na(+)-K(+)-ATPase in plasma membranes of hepatocytes on the formation of calcium bilirubinate gallstone].

OBJECTIVE: To investigate the effects of activities of Ca(2+)-ATPase and Na(+)-K(+)-ATPase in plasma membranes of hepatocytes on the formation of calcium bilirubinate gallstone. METHODS: The rabbit models for studying calcium bilirubinate gallstone were used. One hundred and three rabbits were randomly divided into the control (sham operation) group (Con, n = 28), the simple biliary obstruction group (BO, n = 36), and the biliary obstruction and infection group (BOI, n = 39). The activities of Ca(2+)-ATPase and Na(+)-K(+)-ATPase in plasma membranes of hepatocytes and the intracellular calcium content were measured on the 3rd, 7th, 14th and 20th days after operation. RESULTS: The activities of Ca(2+)-ATPase and Na(+)-K(+)-ATPase decreased remarkably in all phases of BOI and BO groups as compared with those of Con group (P < 0.01). The over-loaded intracellular calcium was found in both BOI and BO groups. The above-mentioned changes were more significant in BOI group than in BO group (P < 0.05). CONCLUSION: The progressive decrease of the activities of Ca(2+)-ATPase and Na(+)-K(+)-ATPase is in close relationship with the continuous increase of intracellular calcium content during the formation of calcium bilirubinate gallstone in rabbit models. Infection can aggravate those changes and further the formation of stone.