| The global side effects, which are due to systemically administrating immunosuppressants, lead to the development of specific immunotherapies for treatment of allograft rejections. The strategy of selectively eliminating or suppressing alloreactive T cells is one of the promising immunotherapies of treating allograft rejections. Thus, the acellular agents using peptide/major histocompatibility complex (p/MHC) to target antigen-specific T cells have been investigated extensively for decades. Killer artificial antigen presenting cells (KaAPCs), which generated by coupling p/MHC multimers and Fas ligand (FasL) onto acellular scaffold, is one of the latest researches. The KaAPCs based on magnetic and latex beads have been shown the ability to selectively deplete antigen-specific T cells in vitro. However, magnetic and latex beads are absent of biocompatibility. Moreover, the research using the KaAPCs to deplete antigen-specific T cells in vivo and in allograft transplant model is rare.In this research, poly (lactic-co-glycolic acid) (PLGA) based KaAPCs are generated by covalently coupling H-2Kb-Ig dimer and anti-Fas monoclonal antibodies (mAbs) on PLGA microparticles (MPs) surfaces.The H-2Kb-Ig dimer onto KaAPCs are been able to load with peptideds and target to T cell receptor (TCR) of antigen-specific T cells, meanwhile the anti-Fas mAbs are capable to induce the apoptosis of the T cells. In allograft transplant, the H-2Kb-lg dimer are capable of selectively interacting with TCR of H-2Kb alloantigen-specific T cells, and induce apoptosis of these cells. Therefore, KaAPCs are able to specifically treat allograft rejections.Objectives:The ability of the KaAPC to selectively eliminate antigen-specific T cells in vitro and in vivo has been investigated in an OT-1 transgenic mice model. In addition, the ability of the KaAPCs to deplete alloreactive T cells and treat alloskin graft rejections in an alloskin graft transplant model has been studied.Methods and Results:1. Fabrication and characterization of PLGA MPs:PLGA MPs with diameter of 5μm were prepared using a double-emulsion solvent evaporation method. The surface of PLGA MPs was modified with chemistry method.The shape, size distribution and zeta potential of PLGA MPs were characterized through scanning electron microscopy (SEM), dynamic light scattering technique and PALS zeta instrument, respectively. The flow cytometry was used to detect the ability of PLGA MPs to couple protein.The results showed that the MPs displayed a spherical shape under the SEM, the diameter was range from 1 to 10 um and 80% of these MPs were 5-6μm in diameter, the mean zeta potential was 65.2±6.7 mV and a strong capacity to couple protein (2×107 PLGA MPs can be loaded with about 80μg of protein).2. Generation and Phenotypic analysis of KaAPCs:The KaAPCs were generated by coupling H-2Kb-Ig and anti-Fas mAbs onto PLGA MPs. Flow cytometry and immunofluorescence staining were used to detect phenotype of the KaAPCs. The results revealed that the KaAPCs displayed both H-2Kb-Ig and anti-Fas mAbs immobilized to the surface of PLGA MPs.3. KaAPCs selectively deplete antigen-specific T cells:in vitro, KaAPCs loaded with OVA257-264 peptides (Kb/OVA-KaAPC) were cocultured with splenocytes from OT-1 transgenic mice (whose CD8+T cells recognize OVA257-264 peptides in the context of H-2Kb) for 24 hours. The apoptosis and frequency of OVA257-264-specific CD8+T cells were detected by flow cytometry. Coculture of Kb/OVA-KaAPC resulted in a apoptosis of about 80% CD8+T cells and remarkable reduce of OVA257-264-specific CD8+ T cells by about 83% in vitro. In contrast, control cocultures did not lead to significant increase of CD8+T cells apoptosis and decrease frequency of OVA257-264-specific CD8+T cells. The killing effect of KaAPCs is mainly dependent on anti-Fas mAbs, not the activation induction cell death (AICD). In vivo, Kb/OVA-KaAPC were injected (via tail vein) into OT-1 transgenic mice, and the apoptosis and proportion of OVA257-264-specific CD8+ T cells in peripheral blood were analyzed by flow cytometryat different time points. Similarly, injections of Kb/OVA-KaAPC caused a strong apoptosis (about 83% of CD8+ T cells) and significant decrease of OVA257-264-specific CD8+ T cells by 84% in vivo.These results demonstrated that the KaAPCs were able to selectively deplete antigen-specific T cells in vitro and in vivo.4. The study of effective regimen for suppressing allograft rejections by KaAPCs:using C57BL/6 mice (H-2Kb) and BALB/c mice (H-2Kd) as donor and recipient, respectively, to establish the alloskin transplant model. After injection (via tail vein or local of alloskin graft) of KaAPC (generated by coupling in-house H-2Kb monomer and commercial anti-Fas mAbs) into recipients, alloskin graft survival was assessed, CD4+ and CD8+ T cells infiltrated into alloskin graft were detected by immunofluorescence, and general immune functions of recipients was evaluated. The effective regimen for suppressing allograft rejections were determined by comparing some treatment regimen including the dosage, route and times of injection. In compare to control groups, treatment of KaAPCs was able to prolong the allograft survival for 4-6 days, significantly reduce the CD8+ T cells infiltrated into alloskin graft but not CD4+ T cells, and did not impair the general immune functions of recipients.5. The study of optimizing regimen and mechanism of allograft rejections suppression by KaAPCs:using C57BL/6 mice (H-2Kb) and bml mice (H-2Kbml) as donor and recipient, respectively, to establish the alloskin transplant model. After injection (via tail vein) of KaAPC (generated by coupling commercial H-2Kb-Ig dimer and anti-Fas mAbs) into recipients, alloskin graft survival was assessed, the percentage of H-2Kb alloreactive CD8+ T cells in peripheral blood and spleen of recipients was detected by flow cytometry, the H-2Kb alloreactive CD8+T, CD4+T and CD8+T cells infiltrated into alloskin graft was analyzed with immunohistochemical (IHC), the apoptosis of T cells was detected by flow cytometry, the proliferation of host CD3+ T cells was evaluated in a host-versus-donor mixed lymphocytes reaction (MLR), the B, NK and regulatory T cells was detected by flow cytometry, the lymphocytes, monocytes and neutrophils in peripheral blood were detected by automated hematology analyzer, the host antitumor effects and proliferative response in a third-party MLR were evaluated. The results exhibited that KaAPCs were able to enter into spleen and lymphoid node (LN) and interact with CD8+ T cells after three intravenous injections of KaAPCs. Treatment of KaAPCs resulted in a dramatic depletion of alloreactive CD8+T cells in the spleen, peripheral blood and infiltrating into the alloskin graft, and prolonged alloskin graft survival for 42.5 days. Furthermore, KaAPCs were able to contract the proliferation of host CD3+ T cells in a host-versus-donor MLR. A significant increase of CD4+CD25+Foxp3+ regulatory T cells were induced in LN, but not spleen, by KaAPCs. Additionally, KaAPCs did not markedly suppress the immune cells and general immune function of host.In conclusion, the PLGA based KaAPCs are capable of depleting antigen-specific CD8+ T cells in vitro and in vivo, and suppressing the allograft rejections by targeting alloreactive T cells without impairment of general immune functions of host. Thus, the strategy of KaAPCs represents a potential specific immunotherapy for treating allograft rejections and immune disorders. |
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