Effect Of Anti-B7 Monoclonal Antibody With Immunosuppressants On T Lymphocytes Proliferation After Infecting With Cytomegalovirus And Their Molecular Mechanisms

Graft versus host disease (GVHD) is the major complication of post allogeneic hematopoietic stem cell transplantation. It is immune response reacted by the donor cell recognizing the surface antigen of the host cell, resulting in multi-organ damage in host. How to overcome the malpractice of existent immunosuppressants or to improve the methods of using immunosuppressants, or to create an ideal, high performance, low poisonous strategy to prevent and weaken GVHD, has become problem that need to be resolved urgently.Mature T lymphocytes play a core role in the generation and development of GVHD. The activation of T lymphocyte not only requires the signal stimulus of antigenic specificity, but also needs the requisition of costimulatory signal. CD28/B7 is just the major system of costimulatory signal. B7-1 and B7-2, which belong to the superfamily of immunoglobulin, are two important members of B7 molecular family. The co-ligands of B7-1 and B7-2 are CD28 and CTLA-4. CD28 exists on the surface of T lymphocyte, while CTLA-4 exists on the surface of activating T lymphocyte. Combination of B7 and CD28 produces the second activation signals. When the first activation signals are present, CD4 positive and CD8 positive clone cells will be activated, start to proliferate, increase the expression of kinds of adhesion molecules on cell surface, strengthen the intercellular interaction. Meanwhile, they promote geneexpressions of many kinds of cytokines, and that will facilitate the generation and development of immune response. Prevoius research has confirmed that blocking the interaction of B7 on antigen presenting cell and CD28 may induce the tolerance of T lymphocyte. B7-1 and B7-2 show different biochemistry character, although the structures of them are similar. So far, there are many researches on the roles of B7-1 and B7-2 molecules, but rare on differences between the effects of B7-1 and B7-2.Severe GVHD may be related to the overbalance of cytokines secreted by Thl subgroup and Th2 subgroup. CD4~+ Th cells in graft are activized by heterology major and secondary histocompatibility antigens expressed in host, and then secrete dissolvable cytokines to promote the antigenic specificity and cytotoxicity of T lymphocytes' activity and tardive hypersensitiveness. According to the differences of secreting cytokines spectra , CD4~+ Th cell clones divides into two subpopulations: Thl and Th2. Thl subpopulation produces IL-2, INF-γ, LT, IL-3 and GM-CSF, which can promote CTL activity and DTH reaction. Thl subpopulation promotes cell immune response. It is the major subpopulation to initiate GVHD. However, Th2 subpopulation chiefly secretes IL-4,IL-5,IL-6 and IL-10 and promotes humoral immune reaction, immunological tolerance and mucous membrane immune reaction. According to above knowledge, the interreaction and relationship between Thl and Th2 subpopulation have been caused major concern in the occurrence and development of GVHD.Mixed lymphocyte reaction (MLR) is an important in vitro model to study the antigen recognition reaction of graft in GVHD. In this reaction system, to some extent, proliferation of lymphocytes and releasing of cytokines represent the graft-versus-host reaction in vivo.Mycophenolate mofetil (MMF) and cyclosporine (CsA) are immunosuppressants used to prevent GVHD in recent years. Mycophenolic acid (MPA) is an active immunosuppressant and MMF is the precursor of MPA. MPA is a reverse inhibitor of inosine monophosphate dehydrogenase, which is critically involved in purine synthesis resulting in ceasing the lymphocyte proliferation. CsA is a lipophilic cyclicundecapeptide isolated from the fungus. CsA binds to an intracellular immunophilin (cyclophilin) and inhibits calcineurin phosphatase, which is required in gene transcription for T-cell activation.The human cytomegalovirus (HCMV), a member of the beta-herpesvirinae subfamily, is a widespread pathogen responsible for generally asymptomatic and persistent infections in healthy people. It may, however, reactivate and cause severe disease in the absence of an effective immune response, as in immunologically immature and immunocompromised individuals, such as organ allografting, malignant, and human immunodeficiency virus (HIV)-infected patients. Recently, due to the clinical application of haemopoietic stem cell transplant generally, the severe complications of HCMV were increased, which is one of the high risk factors of HCMV infection.The regimen including MMF and CsA has been successfully used as an effective method in the prophylaxis of acute graft versus host disease. It has been reported that the anti B7-1 mAb affiliating CsA can induce T lymphocyte tolerance. MMF plusing short-range MTX sucsessfully prevent GVHD of patients who accepting allogenic bone marrow transplantation. Whether the anti B7-1 mAb and anti B7-2 mAb have different contributions on the costimulate pathway and expressions of cytokines? Whether they inhibit T lymphocyte activation and proliferation? Whether they have synergistic effects with CsA or MPA and what is the influence of cytomegalovirus infection on the effects of them. In this study we will establish the allogeneic lymphocyte reaction system and observe the influence of anti B7-1 and anti B7-2 monoclonal antibody to the cell proliferation, especially uniting MPA or CsA, as well as cytomegalovirus infection. We hope to provide penetrating basis for clinical applications.There are four parts in our research. (1) Effects of anti B7-1 mAb on the proliferation of peripheral blood T lymphocytes and expressions of cytokines. (2) Effects of anti B7-1 mAb uniting MMF on the proliferation of T lymphocytes and expressions of cytokines. (3) Effects of anti B7-1 mAb uniting CsA to the proliferationof T lymphocytes and expressions of cytokines. (4) Preliminary research on the infuence of HCMV cells to effects of the anti B7-1 mAb and immunosuppressants.Part I : In this part, we established T lymphocyte reaction system in vitro. The proliferation of T lymphocytes was detected by BrdU incorporation method; The expression of IL-2, INF-γ and IL-10 mRNA and protein level were detected by RT-PCR and ELISA, respectively. Results: (1) Anti B7-1 mAb (0.5mg/L) and anti B7-2 mAb (0.5mg/L) inhibited the T lymphocyte proliferation significantly as compared with control (P <0.01). Combination of anti B7-1 mAb with anti B7-2 mAb enhanced the inhibitory effect. (2)Anti B7-1 mAb increased expression of IL-10 significantly as compared with control (676.66 ng/L±23.09 ng/L vs 545.71 ng/L±22.45 ng/L, P < 0.01; Anti B7-2 mAb inhibited expression of IL-2, INF-γ, IL-10 significantly as compared with control (44.54 ng/L± 11.02 ng/L vs 99.70 ng/L±9.15 ng/L, P <0.01; 11.51 ng/L±3.19 ng/L vs 82.42 ng/L±25.55 ng/L, P <0.01; 478.31 ng/L± 14.75 ng/L vs 545.71 ng/L±22.45 ng/L, P <0.05. Combination of anti B7-2 mAb with anti B7-2 mAb decreased the expression of IL-2 futher. The expression of INF-γ in combination group also decreased as compared with anti B7-1 mAb alone. The expression of IL-10 in combination group decreased as compared with the use of anti B7-1 mAb alone, but increased as compared with the anti B7-2 mAb alone. (3)Anti B7-2 mAb inhibited expressions of IL-2 and IL-10 mRNA , but increased the expression of INF-γ mRNA significantly as compared with control (0.0 ± 0.0vs 1.17 ± 0.15, P<0.01, 0.66 ± 0.09 vs 0.89 ± 0.05, P<0.05; 2.26 ± 0.39 vs 0.75 ± 0.12, P<0.01. Conclusions: Both anti B7-1 mAb and anti B7-2 mAb inhibite the proliferation of T lymphocyte in vitro. Anti B7-1 mAb and anti B7-2 mAb have different contribution on the expression of cytokines.Part II: The methods were similar to that in part. Results: (1) MPA (50 μmol/L) inhibited T lymphocyte proliferation significantly as compared with control, P <0.01. (2) MPA inhibited the protein levels of IL-2 and INF-γ significantly as compared with control (42.73 ng/L± 14.64 ng/L vs 99.70 ng/L ± 9.15 ng/L, P <0.01; 7.87 ng/L±4.22 ng/L vs 82.42 ng/L±25.55 ng/L, P <0.05), while increased expression of IL-10 (770.95ng/L± 126.85 ng/L vs 545.71 ng/L±22.45 ng/L, P <0.05. Compared with MPAalone, MPA in combination with anti B7-1 mAb (0.5mg/L) increased the expression of IL-10 significantly (941.90ng/L±56.61ng/L vs 770.95 ng/L±126.85 ng/L, P <0.05). (3)Expression levels of IL-2 and INF-γ mRNA in the MPA group was significantly lower than that in the control (0.74±0.10vs 1.17±0.15, 0.52 ± 0.05 vs 0.75 ± 0.12, P <0.01). Combination of MPA with anti-B7-1 mAb showed a significant increase in IL-10 mRNA expression as compared with MPA alone (1.28±0.06 vs 0.84±0.09, P <0.01). Conclusions: MPA inhibited the T lymphocyte proliferation significantly, perhaps duo to its inhibition on expression of IL-2 and INF-γ and increasement of the IL-10 expression; Combination with anti-B7-l mAb enhanced the inhibitory effects of MPA.Part III: The methods were similar to that in part I . Results: (1) CsA (400μg/L) inhibited T lymphocyte proliferation significantly. CsA in combination with anti B7-1 mAb(0.5mg/L) had a much stronger inhibitory effect on proliferation (P<0.01). (2) CsA inhibited protein expressions of IL-2 and INF-γ, while increased the expression of IL-10 . Compared with CsA alone, the protein expression of IL-2 in the group treated with CsA in combination with anti B7-1 mAb decreased significantly (29.39 ng/L±6.82 ng/L vs 48.19 ng/L±8.67 ng/L, P<0.05). CsA decreased protein expression of INF-γ significantly as compared with the control (6.81 ng/L±5.24 ng/L vs 82.42 ng/L±25.55 ng/L, P<0.05). CsA in combination with anti B7-1 mAb decreased protein expression of INF-γsignificantly as compared with CsA alone group (0.30 ng/L±0.52 ng/L vs 6.81 ng/L±5.24 ng/L, P<0.05). CsA increased the protein expression of IL-10. CsA in combination with anti B7-1 mAb even more increased the expression of IL-10. (3 Relative expression levels of IL-2 and INF-γ mRNA in the CsA group are significantly lower than that in the control (0.20 ± 0.18 vs 1.17±0.15, P<0.01; 0.59±0.02 vs 0.75 ± 0.12, P <0.05. The relative expression level of INF-γ mRNA in group of CsA with anti B7-1 mAb decreased significantly as compared with CsA alone(0.38±0.05 vs 0.59±0.02, P<0.01). On the other hand, CsA increasedthe relative expression level of IL-10 mRNA(P<0.01). Combination of CsA with anti B7-1 mAb increased IL-10 mRNA expression even more significantly (1.80±0.13 vs 1.38±0.06, P<0.01). Conclusions: CsA inhibited T lymphocyte proliferation. Combination with anti-B7-1 mAb enhanced its effects might through the inhibition on IL-2 and INF-γ expression and increasement of the IL-10 expression.Part IV: Methods: Peripheral blood mononuclear cells (PBMNCs) were isolated from heparinized venous blood on Ficoll density gradients. After washing by RPMI-1640 culture medium, the cells were resuspended and infected with 50 TCID50 of HCMV. One-way MLR was performed with mitomycin C-treated PBMNCs that infected with HCMV as stimulators and nylon wool-purified allogeneic T cells as responders at a 1:5 responder-to-stimulator ratio. The other methods were similar to that in part I . The results revealed: 1. The groups treated with MPA and anti-B7-2 mAb:(1) In HCMV-uninfected groups: the proliferation of T lymphocyte in trial group treated with MPA (100μmol/L) or anti-B7-2 mAb (0.5mg/L)alone was lower significantly than that in controls (p<0.01), and that in group treated with MPA in combination with anti B7-2 mAb was lower than that in groups using MPA or anti-B7-2 mAb alone (p<0.01). The expression of INF-γ in experimental groups treated with MPA or anti-B7-2 mAb alone were very lower than those of controls; and that in group treated with MPA in combination with anti-B7-2 mAb lower than that in group using anti-B7-2 mAb alone significantly (p<0.01), contrasted with group using MPA alone, the expression level of INF-γ was just decreased a little (p>0.05). (2) In HCMV-infected groups: the proliferation level of T lymphocyte in group treated with MPA alone was lower than that in controls significantly (p<0.05), and that in group united with MPA and anti-B7-2 mAb was lower than that in group using anti-B7-2 mAb alone (p<0.01). The INF-γ expression levels in experimental groups treated with MPA or anti-B7-2 mAb alone were very lower than those of controls; and that in group united with MPA and anti-B7-2 mAb lower than that in group using anti-B7-2 mAb alone significantly(p<0.01). Contrasted with group using MPA alone, the expression level of INF-γ was just decreased a little (p>0.05). (3) Each HCMV-infected groups contrasted with corresponding to HCMV-uninfected groups, the proliferation levels of T lymphocyte were all decreased obviously (p<0.01). The expression levels of INF-γ were all elevated obviously (p<0.01)2. The groups treated with CsA and anti-B7-2 mAb:(1) In HCMV-uninfected groups: the proliferation levels of T lymphocyte in experimental groups treated with CsA (100μg/L) or anti-B7-2 mAb (0.5mg/L)alone were lower than those in control groups significantly (p<0.01); and those in groups treated with CsA in combination with anti-B7-2 mAb were lower than those in groups using CsA or anti-B7-2 mAb alone significantly (p<0.01). The INF-y expression levels of in experimental groups treated with CsA or anti-B7-2 mAb alone were lower than those of controls obviously (p<0.01); and that in group treated with CsA in combination with anti-B7-2 mAb was lower than those in groups using CsA or anti-B7-2 mAb alone (p<0.01).(2)In HCMV-infected groups, the proliferation level of T lymphocyte in group treated with CsA alone was a little lower than that in control (p>0.05), that in group treated with anti-B7-2 mAb was lower than control (p<0.05); that in group treated with CsA united anti-B7-2 mAb was higher than that in groups treated with CsA or anti-B7-2 mAb alone (p<0.01). The INF-γ expression levels in experimental groups treated with MPA or anti-B7-2 mAb alone were very lower than those of controls (p<0.01); and that in group united with MPA and anti-B7-2 mAb was lower than that in groups using CsA or anti-B7-2 mAb alone (p<0.01). (3) Each HCMV-infected groups contrasted with HCMV-uninfected groups, the proliferation levels were decreased significantly (p<0.01). In HCMV-infected groups treated with CsA in combination with anti-B7-2 mAb contrasted with corresponding to HCMV-uninfected groups, the INF-γ expression levels were elevated (p<0.05); the other HCMV-infected groups contrasted with corresponding to HCMV-uninfected groups, the expression levels of INF-γ were all elevated significantly (p<0.01).In summary, some conclusions could be drawn from our results:1. Both anti B7-1 mAb and anti B7-2 mAb could inhibit the proliferation of T lymphocyte in vitro, which induce to immune tolerance of T lymphocyte. Using anti B7-1 mAb or anti B7-2 mAb alone could partly blocking T lymphocyte reaction, the latter was stronger. Combination of these two mAb could block the proliferation of T lymphocyte more completely in unilateral MLR. Anti B7-1 mAb and anti B7-2 mAb on the expression of cytokine have different contribution.2. Certain concertrations of MPA and CsA inhibited the T lymphocyte proliferation significantly, perhaps duo to its inhibition of IL-2 and INF-γ expression and increasement of the IL-10 expression. Immunosuppressants changeed cytokine expressive spectrum and induced shifting the Th1 to Th2, which might be involved in the immunosuppressive effect. The combination of immunosuppressants with anti B7-1 mAb might have a synergic effect on T lymphocyte function and cytokine expression.3. In each HCMV-infected groups, the proliferation of T lymphocyte was decreased obviously as compared to each according HCMV-uninfected groups .The expression of INF-y was increased significantly after infecting HCMV.4. MPA decrease the INF-γ expression after infecting HCMV. MPA in combination with anti-B7-2 mAb further decreased the INF-γ expression. These results showed that the combination of MPA with anti-B7-2 mAb had synergy on expression of cytokines.5. CsA decreased the expression of INF-γ after infecting HCMV. CsA in combination with anti B7-2 mAb further decreased the INF-γ expression. These also showed that CsA and anti-B7-2 mAb had synergy on expression of cytokines.