Effect And Mechanism Of Tim-1-Fc In The Suppression Of Allograft Rejection

Activation, proliferation and differentiation of CD4⁺ T helper cell are crucial elementsof antigen-specific immune response. This process needs not only the specific binding of Tcellreceptors with antigenic peptide/MHC complexes provided by antigen-presenting cells(APCs), but also needs co-stimulatory molecules such as immunoglobulin family members(CD80, ICOSL, etc.), TNF family members (CD40, OX40, 4-1BB, CD27, LIGHT, etc.)and a variety of inflammatory cytokines. With the in-depth research, new co-stimulatorymolecules have being discovered to be involved in T cells activation. Thus, the activationand regulation of T cells remain major scientific issues to basic immunology research andhot spots to transplantation immunology research.T cell immunoglobulin mucin (Tim) is a newly discovered molecular expressed on thesurface of T cells and APCs and is associated to T-cell activation. Mouse TIM genes arelocated on chromosome 11B1.1, encoding protein Tim-1⁴, and four hypothetical proteinTim-5⁸. Human TIM genes are located on chromosome 5q33.2, encoding protein Tim-l, 3,4, but not Tim-2. Tim represents a group of transmembrane glycoproteins with commonmotifs: immunoglobulin (Ig)-like domain, mucin-like domain, transmembrane region andintracellular domain. In addition to Tim-4, Tim-1, 2 and 3 all contain an intracellulartyrosine kinase phosphorylation site, which is directly involved in the intracellular signaltransduction.Tim-1 is expressed on all activated CD4⁺ T cells, and highly expressed on Th2 cellsafter differentiation, while lowly expressed on Th1, Th17, CD11c⁺ bone marrow-deriveddendritic cells (DCs) and CD19⁺ B cells. Tim-1 is the natural ligand for Tim-4, which isonly expressed on APCs and is involved in T cell activation, proliferation, and apoptosisinhibition. Tim-3 is mainly expressed on Th1 cells and DCs, and after cross-linking withGalectin-9, it can lead to the apoptosis of Th1 cells.However, the mechanisms for immune regulation by Tim-1 and Tim-4 are morecomplex than initially expected. Recent studies showed that Tim-4-Ig may either stimulateor inhibit T cell proliferation depending on its concentration. Moreover, Tim-4-Ig wasdemonstrated to inhibit naive mouse CD4⁺ T cell activation through a novel ligand otherthan Tim-1, as such an inhibitory effect of Tim-4-Ig was specific to naive T cells that do notexpress Tim-1, and the effect disappeared in pre-activated T cells. This suggests thepossibility that the opposite effect of Tim-4-Ig on T cell activation observed in the previous studies could be resulted from its engagement with different receptors expressed on T cells.On the other hand, anti-Tim-1 mAbs (3B3 and IRM-10) were also found to mediate either astimulatory or an inhibitory effect on T cell activation depending on their binding affinity toTim-1 or their influence on the cytoskeleton. Thus, further elucidation of the role of Tim-1in regulating T cell responses is highly important for developing novel therapeuticstrategies targeting Tim-1 for the treatment of autoimmune diseases and allograft rejection.Based on the above studies, further efforts of this study include: (1) Synthesization ofhuman Tim-1 excellular domain and IgG₁ Fc domain fusion protein (Tim-1-Fc). To analyzethe biding activity of Tim-1-Fc with different kinds of mouse cell types such asunstimulated or stimulated dendritic cells (DCs), CD4⁺CD25^- T cells and CD4⁺Foxp3+regulatory T cells (nTregs) to identify the cross-reactive, and to test whether this bindingwas Tim-4 dependent; (2) Exploration of the influence of Tim-1-Fc on allogeneic mixedlymphocyte response, activation and proliferation of CD4⁺ T cells, cells cycle and apoptosisevoked by anti-CD3 and anti-CD28 mAbs. Investigation of the intracellular signalingpathways in CD4⁺ T cells after ligation with Tim-1-Fc. Observation of its effect on theproliferation and Foxp3 expression of nTregs. (3) Intraperitoneal injection of Tim-1-Fc tomice that receive heterotopic cardiac transplantation, while PBS and hIgG₁ treatment wereused as controls. The mechanisms of Tim-1-Fc in suppressing allograft acute rejection areto be explored. Through this research, we could get a better understanding of human Tim-1in mouse T cell-mediated immune responses and allograft rejection suppression, which mayprovide a new target for clinical immunosuppressive therapy.Objective: To synthesize humanized excellular domain of Tim-1 and IgG₁ Fc domainfusion protein (Tim-1-Fc). To analyze the biding activity of Tim-1-Fc with different kindsof mouse cell types such as DCs, CD4⁺CD25^- T cells and nTregs, and to test whether thisbinding activity was Tim-4 dependent. Methods: cDNA sequences of human Tim-1extracellular domain and IgG₁ Fc fragment were got from the gene bank and cloned intothe mammalian cell expression vector pcDNA3.1, the recombinant plasmids weretransformed into CHO cells using transfection reagent. Tim-1-Fc expression was detected by SDS-PAGE and Western-blot, and purified by affinity column. The binding activity ofTim-1-Fc (5μg/ml) with fresh isolated or activated DCs, CD4⁺CD25^- T cells and nTregswas labeled using mouse anti-human IgG₁(Fc)-FITC and measured by FCM. Anti-Tim-4mAb (10μg/ml) was added to block the possible Tim-4-Tim-1-Fc engagement. ResultsResults:Western-blot test revealed that Tim-1-Fc molecular weight was about 62KD, purity was>92%. Tim-1-Fc could not bind to unstimulated or stimulated DCs or nTregs, but couldselectively bind to activated CD4⁺CD25^- T cells (Teffs), and this effect could not bereversed by anti-Tim-4 mAb. Conclusion: Tim-1-Fc was constructed successfully. Theremust be a novel ligand for human Tim-1 expressed on mouse Teffs other than Tim-4.Objective: To test the influence of Tim-1-Fc on allo-MLR and on the activation,proliferation, cell cycles, apoptosis and Fxop3 expression of CD4⁺ T cells; to explore theTim-1-Fc-triggered intracellular signaling pathways in CD4⁺ T cells. Methods: Differentconcentrations of Tim-1-Fc (1, 5, 10μg/ml) and different proportions (1:40, 1:20, 1:10) ofDC:T-cell were added to test the influence of Tim-1-Fc on allo-MLR. 3H-TdR, CFSE andELISA were used to assay the proliferation and cytokines production of CD4⁺ T cells coculturedwith Tim-1-Fc. FCM was used to test the influence of Tim-1-Fc on the apoptosis(PI/Annexin V) and Foxp3 expression of CD4⁺ T cells. Western blot was used to explore thephosphorylation of intracellular signal moleculars. Results: Tim-1-Fc significantlyinhibited CD4⁺ T cells activation, proliferation and allo-MLR but not the apoptosis. Theseeffects were Tim-4-independent. Also, Tim-1-Fc reduced IL-2,IFN-γwhile promoted IL-10production, and it negatively regulated Cdk2 and Cdk4 expression while increased p27^kipexpression. Moreover, the phosphorylation of AKT and ERK1/2 in CD4⁺ T cells was alsoinhibited, but the proliferation of nTregs or Foxp3 expression were not inhibited by Tim-1-Fc. Conclusion: Tim-1-Fc was able to inhibit the activation, proliferation, differentiation,Th1 cytokines production and allo-MLR of CD4⁺ T cells, and to suppress thephosphorylation of AKT, ERK1/2 in CD4⁺ T cells, but has not influence on the proliferation and Foxp3 expression of nTregs in vitro.Objective: To observe the influence of Tim-1-Fc on cardiac allograft survival andallograft acute rejection and to explore the in vivo mechanisms. Methods:BALB/c→C57BL/6 mouse model of cervical heterotopic heart transplantation wasperformed, the recipients were divided into 3 groups according to the different treatments(for each group, n=10): Group 1, transplantation + PBS treatment group; Group 2,transplantation + human IgG₁ treatment group (10μg/g/day, intraperitoneal injection, 0-6days post-operatively); Group 3, transplantation+Tim-1-Fc treatment group (10μg/g/day,intraperitoneal injection, 0-6 days post-operatively). The survival time of cardiac allograftwas recorded. At the 7th day after transplant, the mice were killed, the bloods, hearts andspleens were collected. ELISA test for blood cytokines (IL-2, IFN-γ, IL-4 and IL-10) levelsand intragraft RT-PCR detection for inflammatory cytokines production, lymphocytessurface molecules (CD3 and CD11b) and Foxp3 expression were conducted. Recipients'splenic CD4⁺ T cells were isolated by magnetic activated cell sorting and stimulated withallogenetic DCs, the proliferation was detected by using ³H thymidine incorporation using aliquid scintillation counter. The ratio of Tregs in recipient mice spleens was also assessed.CD4⁺CD25^- T cells were isolated and cultured with Tim-1-Fc and/or recombinant humanTGF-β1,then the expression of Foxp3 were determined by FCM. Results: Recipientstreated with Tim-1-Fc showed significantly prolonged allograft survival (17.1±1.1 days)compared to those treated with PBS (6.5±0.5 days; P<0.01) or control hIgG₁ (6.7±0.4 days;P<0.01). Histological analysis of heart grafts at POD 7 revealed severe mononuclear cellsinfiltration and myocardial necrosis in the control groups, whereas almost normal tissuestructure with minimal mononuclear cell infiltration was seen in Tim-1-Fc-treated mice.Tim-1-Fc significantly reduced the intragraft transcription for IL-2, IFN-γ, CD3 and CD11b. The proliferation of CD4⁺ T cells in response to allogenetic DCs was significantlyinhibited in Tim-1-Fc treated group. Moreover, the proportion of CD4⁺Foxp3⁺ Tregs insplenic CD4⁺ T cells was significantly increased, although Tim-1-Fc did not induce Foxp3expression in CD4⁺CD25^- T cells. ConclusionConclusion: Tim-1-Fc was able to prolong mouse heartallograft survival though reducing the inflammatory cells infiltration and Th1 cytokinesproduction in the graft and decreasing the proliferative capability of donor reactive T cellsto allogenetic antigens, while increasing the proportion of Tregs in the spleen, thus shiftingthe balance of Th1/Th2 towards Th2 and the balance of Tregs/Teffs towards regulators,although it did not induce Foxp3 expression in CD4⁺CD25^- T cells directly. These madeTim-1-Fc become a promising drug for anti-rejection therapy.