Costimulatory Molecule B7-H1 on the Immune Escape of Bladder Cancer and Its Clinical Significance

B7-H1, a recently described member of the B7 family of costimulatory molecules, is thought to be involved in tumor immune escape by inducing T-cell apoptosis. In order to investigate the relationship between B7-H1 and immune escape of bladder cancer, B7-H1 expression in 50 cases of bladder cancer was detected by using immunohistochemical method. Survival curves were con- structed using the Kaplan-Meier method and independent prognostic factors were evaluated using the Cox regression model. Our results showed that the positive rate of B7-H1 immunostaining in normal bladder tissue and bladder cancer was 0 and 72% respectively. The expression of B7-H1 was strongly associated with the pathological grade, clinical stage and recurrence （P〈0.05）. The survival rate was significantly lower in patients with B7-H1 positive group than in those with B7-H1 negative group and multi-variable analysis revealed that B7-H1 could be regarded as an independent factor in evaluating the prognosis of bladder cancer. It is concluded that the expression of B7-H1 is strongly associated with neoplastic progression and prognosis of bladder cancer. The manipulation of B7-H1 may become a beneficial target for immunotherapy in human bladder cancer.

Expression of Costimulatory Molecules B7/CD28 in Systemic Lupus Erythematosus

Summary: The expression of the costimulatory molecules B7/CD28 in peripheral blood mononuclear cells (PBMC) of the patients with systemic lupus erythematosus (SLE) and its relation to the pathogenesis of SLE were studied. The expression of the costimulatory molecules in PBMC in 30 patients with active SLE and 20 cases of healthy controls was detected by using the techniques of immunofluorescence and flow cytometer. The result showed that the expression percentage of CD28+, CD4+CD28+ in T cells of PBMC from the patients with SLE decreased significantly as compared with that in healthy control group, while the expression percentage of CD80+, CD19+CD80+ in B cells was significantly increased than that in healthy control group (P<0.01). It suggested that the abnormal expression of costimulatory molecules B7/CD28 played a role in the pathogenesis of SLE.

Immune response induced by hematoporphyrin derivatives mediated photodynamic therapy:Immunogenic cell death and elevated costimulatory molecules

Photodynamic therapy(PDT)not only destroys tumor cells directly but also induced anti-tumor immune response through damage-associated molecular patterns(DAMPs).It is reported that anti-tumor response was associated with light dose and photosensitizer used in PDT.In this study,4T1 tumor cells were implanted on both the right and left flanks of mice.Only the right tumor was treated by HpD-PDT,while the left tumor was not irradiated.The anti-tumor immune response induced by HpD-PDT was investigated.The expression of DAMPs and costimulatory molecules induced by HpD-PDT were tested by immuno fluorescence and flow cytometry in vivo.Different light doses of PDT were designed to treat 4T1 cells.The killing effect was assessed by CCK-8 kit and apoptosis kit.The expression of DAMPs on 4T1 cells after HpDPDT were evaluated by flow cytometry,western blot and ATP kit.This study showed that CD4^(+)T,CD8^(+)T and the production of IFN-γwere increased significantly on day 10 in righttumor after PDT treatment compared with control group.HpD-PDT enhanced the expression of calreticulin(CRT)on tumor tissue.Importantly,co-stimulatory molecular OX-40 and 4-1BB were elevated on CD8^(+)T cells.In vitro,immunogenic death of 4T1 cells was induced after PDT.Besides,the expression of DAMPs increased with the increasing of energy density.This study indicates that anti-tumor immune effect was induced by HpD-PDT.The knowledge of the involvement of CRT,ATP and co-stimulatory molecules uncovers important mechanistic insight into the anti-tumor immunogenicity.It was the first time that co-stimulatory molecules were investigated and found to elevate after PDT.

Effect of Dihydrotestosterone on Costimulatory Molecules in a Mouse Model of Graves’ Disease

Objective Graves’disease is the most common autoimmune thyroid disease and its prevalence and clinical manifestations are disparate between females and males.Costimulatory molecules play an essential role in regulating autoimmune responses.The objective of this study was to determine if expression of inhibitory molecules was correlated with treatment by dihydrotestosterone(DHT)in an in vivo BALB/c mouse model of experimental autoimmune Graves’disease.Methods Female BALB/c mice were immunized three times with thyroid stimulating hormone receptor A-subunit encoded by adenovirus to establish a Graves’disease model.Three different doses of DHT or a matching placebo were administered by implantation of slow-release pellets a week before the first immunization.Four weeks after the third immunization,the mice were euthanatized,and then the spleen and thymus were removed.Total thyroxine and free thyroxine levels in serum of mice were detected using a radioimmunoassay kit.Real-time polymerase chain reaction was performed to estimate the expression of costimulatory molecules in lymphocytes from the spleen and thymus.Flow cytometry was used to analyze the percentage of CD4^+T cells in splenic lymphocytes.Quantitative data were compared with unpaired t-tests.Correlation between two variables was analyzed using Analysis of Variance.Results Treatment with DHT can dramatically reduce total thyroxine and free thyroxine levels.Higher expression of programmed death-1 was found in the spleen of Graves’disease mice receiving 5 mg of DHT treatment(0.635±0.296 vs.0.327±0.212;t=2.714,P=0.014),similarly,T-cell immunoglobulin domain and mucin domain 3(TIM-3)in both the spleen(1.004±0.338 vs.0.646±0.314;t=2.205,P=0.022)and the thymus(0.263±0.127 vs.0.120±0.076;t=3.221,P=0.004)also increased after 5 mg of DHT treatment compared with the parallel placebo model mice.Moreover,the percentage of CD4^+T cells declined in the splenic lymphocytes of Graves’disease mice treated with 5 mg of DHT(19.90%±3.985%vs.24.05%±2.587%;t=2.804,P=0.012).A significant negative association was observed between expression of TIM-3 in the spleen and serum levels of total thyroxine(r=-0.7106,P=0.014)as well as free thyroxine(r=-0.6542,P=0.029).Conclusion This study demonstrates that DHT can ameliorate experimental autoimmune Graves’disease,which may occur by up-regulating expression of programmed death-1 and TIM-3 and inhibiting development of CD4^+T cells.

CD16+ Cells and Costimulatory Molecules of Lymphocyte Activation Present inside Human Kidney Grafts and in Blood Circulation

Background: We studied the expression of important costimulatory molecules of lymphocyte activation and the presence of CD16+ cells on aspiration biopsies of kidney transplants, measured three soluble factors and when indicated tested their robustness in diagnosing acute rejection. Methods: Fine-needle aspiration biopsies were performed either on days seven or 14 - 30 post-transplantation among stable kidney transplants and on the day of acute rejection diagnosis, while a sample of peripheral blood was collected simultaneously. The cyto preparations were studied by the enzymatic avidin biotin complex staining. The immunocytochemistry was directed to CD16, CD28, CD152, ICOS, CD40, CD154, CD26 and CD27. We performed the analysis in the peripheral blood by ELISA for soluble(s) CD16, CD26, and CD154. Results: The group of acute rejection cases showed a significant up-regulated expression of CD16, CD26, ICOS and CD40 as compared to the group of stable cases. Both sCD16 and sCD154 were significantly higher in the blood samples of the group with acute rejection. Thymoglobulin down-regulated CD154 and sCD16. CD16, CD26 and ICOS exhibited very high sensitivity and specificity for acute rejection diagnosis. Conclusions: The presence of CD16+ cells inside the graft emerged as a distinct player in acute rejection, confirming other previous reports whereas we first document that in human kidney transplants, ICOS and CD26 are significantly up-regulated and both reached positive predictive values for acute rejection ≥ 80%. The other costimulatory molecules, with the exception of CD40, though widely known, did not show robust association with immune events.

Reciprocal costimulatory molecules control the activation of mucosal type 3 innate lymphoid cells during engagement with B cells

Innate lymphoid cells(ILCs)are the counterpart of T helper cells in the innate immune system and share multiple phenotypes with T helper cells.Inducible T-cell costimulator(ICOS)is recognized on T cells and participates in T-cell activation and T and B-cell engagement in lymphoid tissues.However,the role of ICOS in ILC3s and ILC3-involved interactions with the immune microenvironment remains unclear.Here,we found that ICOS expression on human ILC3s was correlated with the activated state of ILC3s.ICOS costimulation enhanced the survival,proliferation,and capacity of ILC3s to produce cytokines(IL-22,IL-17A,IFN-γ,TNF,and GM-CSF).Via synergistic effects of ICOS and CD40 signaling,B cells promoted ILC3 functions,and ILC3-induced T-cellindependent B-cell IgA and IgM secretion primarily required CD40 signaling.Hence,ICOS is essential for the nonredundant role of ILC3s and their interaction with adjacent B cells.

Inhibiting the expression of CD28 costimulatory molecule on human lymphocytes by special siRNA

Background The B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.Methods According to CD28 gene sequence, we designed and synthysized three different siRNAs ( siRNA-1,siRNA-2, siRNA-3 ) containing 21 bases using SilencerTM siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay.Results Three siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22. 10% ± 1.63% ,73.50% ± 1.02% and 42.90% ± 0.89% respectively compared with the control ( P < 0. 001 ). Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15% ± 0.75% and 4. 55% ±0. 80% ) (P >0. 05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07% ± 0. 96% ) (P > 0. 05 ). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P < 0. 001 ). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P <0.01 ). The control groups showed no marked reduction in cell viability ( P > 0.05 ).Conclusions Three different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level, siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT).

PD-L1 is increased in the spinal cord and infiltrating lymphocytes in experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis is a mouse model of human multiple sclerosis with similar pathology and pathogenesis. Thl cells play an important role in the pathogenesis of experimental allergic encephalomyelitis. This study determined the potential effect of programmed cell death 1 ligand 1 in the pathogenesis of experimental allergic encephalomyelitis induced by injecting myelin oligodendrocyte glycoprotein, complete Freund＇s adjuvant and Bordetella pertussis toxin into C57BL/6J mice. Experimental allergic encephalomyelitis mice developed disease and showed in- flammatory changes in the central nervous system by hematoxylin-eosin staining of spinal cord pathological sections, demyelination by Luxol fast-blue staining and clinical manifestations. The expression of programmed cell death 1 ligand 1 in mice was detected by immunohistochemistry, flow cytometry and western blot anatysis. The expression of programmed cell death 1 ligand 1 in the spinal cord and splenocytes of mice was significantly increased compared with normal mice. Our findings suggest the involvement of programmed cell death 1 ligand 1 in the pathogenesis of ex- perimental allergic encephalomyelitis and suggest this should be studied in multiple sclerosis.

IMMUNOLOGICAL CHARACTERISTICS OF THE LEUKEMIA CELLS TRANSFECTED WITH ONCOSTATIN M GENE BY RECOMBINANT ADENOVIRUS

In the present study, FBL3 murine erythroleukemia cells were transfected with human OSM(hOSM) gene by recombinant adenovirus, then the immunological properties of hOSM genetransfected FBL3 cells(FBL3OSM+) were investigated. 4 hours after transfection with hOSM gene, hOSM could be detected in the supernatant of FBL3OSM+ cells and hOSM secretion peaked at 24 h. The proliferation of FBL3OSM+ cells was inhibited markedly. The clonal formation of FBL3OSM+ cells was suppressed more obviously in comparison with wildtype FBL3 cells when analysed in clonal argar culture. Flow cytometry analysis showed that FBL3OSM+ cells expressed higher levels of Fas protein, B7 and ICAM1 molecules.FBL3OSM+ cells also expressed higher level of MHC class I molecules(H2Kb) but remained unchanged in expression of MHC class II molecules (Ia). CD14, which is a specific marker of monocyte/macrophage and not expressed on the wildtype FBL3 cells, was also detected on the surface of FBL3OSM+ cells. The results suggested that OSM gene transfer could increase the immunogenicity of FBL3 cells and promote their differentiation into macrophagelike cells. The data outline a promising approach to OSM gene therapy of leukemia mediated by recombinant adenovirus.

B7-H3:Another Molecule Marker for Mo-DCs?

Using a newly generated monoclonal antibody （2E6） against human B7-H3, we explored the expression of the molecule on dendritic cells derived from monocytes （Mo-DCs）. Its expression was examined by means of immunostaining and flow cytometric （FCM） analysis. The results showed that B7-H3 was expressed in the course of Mo-DC maturation induced with interleukin 4 （IL-4） and granulocyte/macrophage colony-stimulating factor （GM-CSF）. The expression could be detected at all the stages of Mo-DC differentiation, and remained at a quite stable level. Interestingly, B7-H3 was not expressed by T cells and B cells, even these cells were activated respectively by PHA or PWM. A weak expression could be detected on resting monocytes. These data showed that constitutive expression of B7-H3 at a high level was found on imDCs and mDCs derived from monocytes. Due to no expression on T cells and B cells, we speculate that B7-H3 might be another valuable molecule marker for Mo-DCs.

Alterations in lymphocyte subset patterns and co-stimulatory molecules in patients with Alzheimer disease

Alzheimer disease （AD） is characterized by the .presence of β-amyloid （Aβ） plaques in the brain.More evidence of inflammatory parameters, such as, complement factors, proinflammatory cytokines and lymphocytes has been found to be co-localized with Aβ plaques. The research in the past decades has demonstrated abnormalities of both the humoral and cellular immune responses, suggesting an association of immunological aberration and AD. The total percentage of lymphocytes was not found to be altered,

A dimeric structure of PD-L1: functional units or evolutionary relics?

PD-L1 is a member of the B7 protein family,most of whose members so far were identified as dimers in a solution and crystalline state,either complexed or uncomplexed with their ligand(s).The binding of PD-L1 with its receptor PD-1(CD279)delivers an inhibitory signal regulating the T cell function.Simultaneously with the Garboczi group,we successfully solved another structure of human PD-L1(hPD-L1).Our protein crystallized in the space group of C222_(1) with two hPD-L1 molecules per asymmetric unit.After comparison of reported B7 structures,we have found some intrinsic factors involved in the interaction of these two molecules.Based on these results,we tend to believe this uncomplexed hPD-L1 structure demonstrated its potential dimeric state in solution,althougt it could just be an evolutionary relic,too weak to be detected under present technology,or still a functional unit deserved our attentions.

Anti-CD137 Monoclonal Antibody Promotes the Direct Anti-Tumor Effect Mediated by Peripheral Blood-Derived Human Dendritic Cells in vitro

CD137,a costimulatory factor of TNFR family,is expressed on activated T cells and freshly isolated mouse dendritic cells (DCs).To date,there are only limited data dealing with the expression and the effect of CD137 on human DCs.We report in this work that CD137 can coexpress with CD137L on immature peripheral blood-derived human DCs (9.77%).The mature DCs stimulated by LPS showed a much higher level of CD137 expression (36.06%).Ligation of CD137 on the surface of DCs with anti-CD137 monoclonal antibody (mAb) could enhance the direct anti-tumor effect mediated by human DCs in vitro.The agonistic anti-CD137 mAb was able to elevate by about 20% of the DC-mediated inhibition of tumor growth in five tumor cell lines.These results indicate that the appliance of anti-CD137 mAb might be used as a new strategy for tumor immunotherapy.Cellular & Molecular Immunology.2004;1(1):71-76.

Gene Therapy of Cancer:Induction of Anti-Tumor Immunity

Many malignancies lack satisfactory treatment and new therapeutic options are urgently needed.Gene therapy is a new modality to treat both inherited and acquired diseases based on the transfer of genetic material to the tissues.Different gene therapy strategies against cancers have been developed.A considerable number of preclinical studies indicate that a great variety of cancers are amenable to gene therapy.Among these strategies, induction of anti-tumor immunity is the most promising approach.Gene therapy with cytokines has reached unprecedented success in preclinical models of cancer.Synergistic rather than additive effects have been demonstrated by combination of gene transfer of cytokines/chemokines,costimulatory molecules or adoptive cell therapy.Recent progress in vector technology and in imaging techniques allowing in vivo assessment of gene expression will facilitate the development of clinical applications of gene therapy,a procedure which may have a notorious impact in the management of cancers lacking effective treatment.Cellular & Molecular Immunology.2004;1(2):105-111.