Effect of Human Bone Marrow Mesenchymal Stem Cells with Ectopic High OCT4 Expression on T Lymphocyte Function / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of human bone marrow mesenchymal stem cells (MSCs) with ectopic high OCT4 expression on T-cell proliferation, activation and secretion in vitro.@*METHODS@#Peripheral blood mononuclear cells were isolated from healthy children. Anti-CD3 and anti-CD28 monoclonal antibodies were used to activate T lymphocytes, which were stimulated by interleukin (IL)-2 for one week in vitro. Then MSCs with ectopic high OCT4 expression (MSC-OCT4) were co-cultured with activated T lymphocytes. After one week of co-culture, the supernatant was collected and the levels of Th1/Th2 cytokines [IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ] were determined by flow cytometry. The lymphocytes after one week of co-culture were collected and counted by Countstar software. After the proportions of activated/inactivated T cell subsets were determined by flow cytometry, the absolute lymphocyte counts were calculated and expressed as mean ± standard deviation.@*RESULTS@#Compared with control T cell alone culture group, the proliferation of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells were significantly inhibited in MSC group and MSC-OCT4 group. Compared with MSC, MSC-OCT4 could inhibit CD3+CD8+ T cell proliferation better (P =0.049), and mainly inhibited early T cell activation. Compared with control T cell alone culture group, the levels of IL-2 and INF-γ were significantly down-regulated both in MSC group and MSC-OCT4 group.After co-culture with T cells for one week, the level of IL-6 significantly increased in MSC group and MSC-OCT4 group compared with that before co-culture. Compared with control MSC group, MSC-OCT4 group had higher viable cell numbers after 1 week of co-culture (P =0.019), and could resist the inhibition of proliferation by higher concentration of mitomycin C.@*CONCLUSION@#Both MSC and MSC-OCT4 can inhibit the proliferation and activation of IL-2-stimulated T cells in vitro. After overexpression of OCT4, MSC has better proliferation ability in vitro and can inhibit the proliferation of CD3+CD8+ T cells more effectively, which may have a better and more lasting immunosuppressive ability to regulate the balance of Th1/Th2.

Formononetin enhances the antitumor effect of H22 hepatoma transplanted mice / 细胞与分子免疫学杂志

Objective To explore the effect of formononetin on immunity of mice with transplanted H22 hepatocarcinoma. Methods Male C57BL/6 mice were subcutaneously inoculated with H22 cells (4×105) to establish a tumor-bearing mouse model. The mice were treated with formononetin [10 mg/(kg.d)] or [50 mg/(kg.d)] for 28 days, and then the tumor inhibition rate was calculated. Carrilizumab was used as a positive control drug. The expressions of CD8, granzyme B and forkbox transcription factor 3 (FOXP3) in HCC tissues were analyzed by immunohistochemical staining. The mRNA and protein expression of programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) in HCC tissues were detected by real-time PCR or Western blot analysis, respectively. The serum levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were detected by ELISA. Results Formononetin increased the tumor inhibition rate and the positive rate of CD8 and granzyme B staining in tumor-bearing mice. There was no significant difference in the positive rate of FOXP3 staining in tumor tissues of mice in each group. Formononetin decreased the levels of IL-10 and TGF-β in serum of tumor-bearing mice, and decreased the relative expression of mRNA and protein of PD-1 and PD-L1 in tumor tissue of tumor-bearing mice. Conclusion Formononetin can activate CD8+ T cells and reduce the release of immunosuppressive factors in regulatory T cells by blocking PD-1/PD-L1 pathway and play an antitumor role.

Astragalus polysaccharide inhibits IDO1 expression in colon tumor microenvironment to increase intratumoral CD8~+ T cell infiltration / 中国中药杂志

This study aims to investigate the regulatory effects of Astragalus polysaccharide(APS) and APS combined with 5-fluorouracil(5-FU) on indoleamine-2,3-dioxygenase(IDO1) in the colon tumor microenvironment. Sixty Balb/c mice were randomized into a blank group, a model group, an APS group, an APS + 5-FU group, an APS + low-dose 5-FU group, and a 5-FU group. A tumor model was established by subcutaneous transplantation with CT-26 mouse colon cancer cells in other groups except the blank group. After successful modeling, each group was treated with corresponding drugs for 7 days. The general condition, body weight, and tumor volume of the mice were observed and measured daily during the treatment period. The mice were sacrificed at the end of treatment, and the tumor suppression rate and spleen index of the mice were calculated. Western blot and fluorescence quantitative PCR were employed to determine the protein and mRNA levels, respectively, of IDO1 in the tumor tissue of mice. High performance liquid chromatography was employed to measure the levels of tryptophan(Trp) and kynurenine(Kyn) in the tumor tissue of mice. Hematoxylin-eosin(HE) staining was performed to observe the histological changes of the tumor tissue, and immunohistochemistry to detect the changes of CD4 and CD8 expression in the tumor tissue. Compared with that in the model group, the tumor volume of mice in each treatment group significantly reduced. The body weights of mice in APS + 5-FU group and 5-FU group significantly reduced from day 4 to day 7 of treatment. In addition, the APS + 5-FU group and 5-FU group showed significantly decreased spleen index. The protein and mRNA levels of IDO1 were significantly down-regulated in the APS, APS + 5-FU, and APS + low-dose 5-FU groups. The drug interventions significantly increased the Trp content and decreased the Kyn content. The APS + 5-FU group showed significantly reduced infiltration of CD4~+ T lymphocytes and increased infiltration of CD8~+ T lymphocytes. APS inhibited the expression of IDO1 in the colon tumor microenvironment to increase CD8~+ T lymphocyte infiltration, and the combination of APS with 5-FU demonstrated better effect.

Effect of IL-15 addition on asbestos-induced suppression of human cytotoxic T lymphocyte induction

BACKGROUND@#Asbestos fibers possess tumorigenicity and are thought to cause mesothelioma. We have previously reported that exposure to asbestos fibers causes a reduction in antitumor immunity. Asbestos exposure in the mixed lymphocyte reaction (MLR) showed suppressed induction of cytotoxic T lymphocytes (CTLs), accompanied by a decrease in proliferation of CD8@*METHODS@#For MLR, human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated allogenic PBMCs upon exposure to chrysotile B asbestos at 5 μg/ml for 7 days. After 2 days of culture, IL-15 was added at 1 ng/ml. After 7 days of MLR, PBMCs were collected and analyzed for phenotypic and functional markers of CD8@*RESULTS@#IL-15 addition partially reversed the decrease in CD3@*CONCLUSION@#These findings indicate that CTLs induced upon exposure to asbestos possess dysfunctional machinery that can be partly compensated by IL-15 supplementation, and that IL-15 is more effective in the recovery of proliferation and granzyme B levels from asbestos-induced suppression of CTL induction compared with IL-2.

N-glycosylation modification of heat shock protein gp96 affects its immunological function / 生物工程学报

N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.

The Expression of Programmed Death-1 in Circulating CD4+ and CD8+ T Cells during Hepatitis B Virus Infection Progression and Its Correlation with Clinical Baseline Characteristics

BACKGROUND/AIMS: Programmed death-1 (PD-1) expression was investigated in CD4+ and CD8+ T cells from hepatitis B virus (HBV)-infected patients at the chronic hepatitis B (CHB) infection, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) stages. METHODS: PD-1 expression in circulating CD4+ and CD8+ T cells was detected by flow cytometry. The correlations between PD-1 expression and HBV viral load, alanine aminotransaminase (ALT) levels and aspartate aminotransferase (AST) levels were analyzed using GraphPad Prism 5.0. RESULTS: PD-1 expression in CD4+ and CD8+ T cells was significantly increased in both the CHB group and advanced-stage group (LC plus HCC). In the CHB group, PD-1 expression in both CD4+ and CD8+ T cells was positively correlated with the HBV viral load, ALT, and AST levels. However, in the LC plus HCC group, significant correlations between PD-1 expression and the clinical parameters were nearly absent. CONCLUSIONS: PD-1 expression in peripheral CD4+ and CD8+ T cells is dynamic, changes with HBV infection progression, and is related to HBV viral load and liver function, especially in CHB. PD-1 expression could be utilized as a potential clinical indicator to determine the extent of virus replication and liver injury.

The Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein, a toll-like receptor 4 agonist, enhances dendritic cell-based cancer vaccine potency

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.

HIV-Specific Cellular Immune Responses Are Stimulated by Structured Treatment Interruption in Chronically HIV-1 Infected Koreans

We evaluated the enhancing effect of structured treatment interruptions (STIs) on HIV-specific immunity in chronically HIV-1 infected Korean patients. A prospective case-control study was done with a total of 10 subjects for a period of 26 weeks. Six subjects were on STIs and four subjects were on continuous HAART for comparison. The STI subjects underwent four periods of STIs. For those on STIs, HAART was stopped at week 0 for two weeks, and resumed thereafter for six weeks. Viral load and CD4+/CD8+ T cells were measured by HIV RNA RT-PCR and flow cytometry, and HIV-specific immunity was measured by an ELISPOT assay. HIV-specific cytotoxic T cell immunity was more pronounced in the STI subjects than in the continuous HAART subjects after 26 weeks (p = 0.011). The difference in cytotoxic T cell response in the STI group was more prominent than in the continuous HAART group (p = 0.011). Viral load after 26 weeks was higher in the STI subjects than in the continuous HAART subjects (p = 0.008). An HIV-specific cellular immune response can be stimulated by STIs in chronically HIV-infected Koreans. A larger study is warranted in order to further characterize viral and immunological parameters of treatment with STIs in cases of chronic HIV infection.

Distribution of CD38 molecules on CD3+ and CD8+ T- lymphocyte in adulthood HIV-1-uninfected Thais.

The expression of CD38 on CD8+ T-lymphocyte is a significant predictive value in disease progression of HIV infected individuals and in monitoring a response to therapy. CD38 molecules expressing on CD3+ and CD8+ T-cells were measured quantitatively by flow cytometry in 30 healthy Thai adults. In each experiment, the known amount of fluorochrome in CD38 antibodies bound per cell of QuantiBRITE PE beads was plotted, and set a regression line. With this line, the amount of CD38 molecules bound to CD3 and CD8 target cells was estimated. The aim of this study was to determine the reference value of CD38 molecules on CD8+ T-lymphocyte, which is the baseline in comparison to the CD38 molecule expressing on CD8+ T-lymphocyte in HIV-infected individuals. The present results showed that the amount of CD38 expressions on CD8+ T-lymphocyte in HIV negative Thai adults was about 2 times higher than those from Caucasian's lymphocyte. The reference range of CD38 molecules in the present study would best be used as baseline in prognosis and drug monitoring of HIV-1 infection in Thailand.

Dendritic cell, the immunotherapeutic cell for cancer.

Dendritic cells play an important role in the development of effective cancer vaccines. These cells have the potential to present tumour-specific antigens and thereby induce an immune response. Various studies involving clinical trials have investigated the efficacy of administering antigen-loaded dendritic cells for cancer therapy. In order to design such experiments it is important to consider specific antigens, which initiate either a CD4+ or CD8+ response or both. The present review discusses the unique properties of dendritic cells as an immunotherapeutic cell for cancer.

Bystander-Mediated Regression of Murine Neuroblastoma via Retroviral Transfer of the HSV-TK Gene

Selective introduction of genes conferring chemosensitivity into proliferating tumor cells may be used to treat cancer. We investigated the bystander effect of retrovirusmediated gene transfer of herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblastoma cell line (neuro-2a) in vitro and in vivo, and we examined whether the mechanism of bystander effect in neuroblastoma would also depend on connexin-dependent gap junction and/or immune response. A strong bystander effect was observed in vitro, whereby nontransduced tumor cells in proximity to transduced cells acquired susceptibility to ganciclovir (GCV) killing. Implanted mixtures of wildtype cells and HSV-TK transduced cells showed a potent bystander effect upon administration of GCV in A/J mice. HSV-TK/GCV system in murine neuroblastoma induced systemic immunity. Immunohistochemical staining showed many CD4+ and CD8+ cell infiltration but did not show anti-connexin 43+ cells. In conclusion, a strong bystander effect was observed in vitro and in vivo. The bystander effect in murine neuroblastoma might be dependent on immune response and/or on other mechanism such as protein phosphorylation or transfer of apoptotic vesicle, rather than connexin-dependent gap junction.

Natural killer cells in HIV-1 infection: role of NK cell-mediated non-cytolytic mechanisms in pathogenesis of HIV-1 infection.

Natural killer (NK) cells exhibit both cytolytic and non-cytolytic effector functions against HIV-infected targets. Their precise role in immunopathogenesis of HIV-1 infection is yet to be fully understood. This review addresses the non-cytolytic functions exhibited by NK cells, their potential role in pathogenesis of HIV-1 infection and the effect of HIV-1 viremia on NK cell functions. Activated NK cells are capable of secreting CC-chemokines and suppressing HIV-1 replication in a non-cytolytic fashion. However, HIV-1 viremia suppresses the ability of NK cells to secrete CC-chemokines. Suppression of HIV-1 viremia by highly active antiretroviral therapy (HAART) restores the ability of NK cells to secrete CC-chemokines and suppress endogenous HIV-1 replication by non-cytolytic mechanisms. Better understanding of the mechanisms involved in HIV-1-NK cell interactions would be helpful in delineating novel therapeutic strategics against HIV-1.

Comportamiento de la forma soluble de L-selectina en niños infectados con HIV / Behavior of soluble L-selectin in HIV infected children

La L-selectina es una molécula de adhesión responsable de la adhesión inicial de los leucocitos al endotelio. Esta molécula es liberada desde la superficie celular por clivaje proteolítico después de la activación leucocitaria. Se midieron los niveles séricos de la forma soluble de la L-selectina (sL-selectina) en 51 niños HIV(+) y en 15 controles sanos HIV(-). Estos valores se compararon con parámetros de activación inmune y de progresión de enfermedad. La concentración de sL-selectina se encontró significativamente aumentada en el grupo de pacientes HIV(+). Dichos niveles eran más altos en los pacientes con mayor inmunosupresión. Se encontró una correlación positiva entre los niveles de sL-selectina y el número relativo de LTCD8, y entre sL-selectina y los niveles de la forma soluble de la molécula de adhesión intercelular-1(sICAM-1) y la inmunoglobulina A(IgA). No se obtuvo correlación significativa entre sL-selectina y los valores de la carga viral (CV) plasmática. El aumento en los niveles de sL-selectina sería otro componente entre las múltiples alteraciones inmunológicas producidas por el HIV