| The selective elimination or inhibition of antigen-specific T cells is one of the fundamental strategies for the treatment of transplant rejection and autoimmune diseases because it does not suppress the overall immune systems. Therefore, numerous specific immune therapies have been explored during decades. Killer artificial antigen-presenting cells (KaAPCs), which are genetically- engineered to overexpress the apoptosis-inducing Fas ligand(FasL) onto dendritic cells (DCs) and macrophages, have been studied in animal and human models of allograft transplantation, autoimmunity or allergy by various investigators. However, several major drawbacks associated with their cellular nature limit their clinical uses. The generation of KAPCs is time-consuming and cost-intensive due to the high variability of FasL expression when scaled. To overcotne the limitations related to cell-based treatments, acellular immunotherapies, which utilize the high affinity and specific binding between pMHC polymers and T cell receptors (TCR) to target antigen-specific T cells, has been further developed. But little information for in vivo treatment is available by now.In this report, killer artificial antigen-presenting cells have been generated by covalently co-coupling H-2Kb-Ig dimers and anti-mouse Fas mAbs onto poly lactic-co-glycolic acid (PLGA) microspheres with a diameter of 4.5um, and then administrated into alloskin recipient mice followed by overall immune function inspection. It was found that KAPCs treatment can significantly prolong-the skin allograft survival without impairment of host overall immune function. The results are summarized as following:1. Cell-sized cationic PLGA microparticles are successfully fabricated using a double-emulsion-solvent evaporation method. The PLGA microspheres displayed a heterogeneous size distribution under the SEM. Approximately 61.2% of these microspheres were 4.0-5.0μm in diameter. The mean zeta potential was 36.3±6.11 mV, as detected by the PALS zeta instrument, implying their strong capacity to covalently couple protein molecules. The BSA protein loaded onto 5×106 PLGA microspheres reached up to 54.2μg. The killer PLGA microspheres were prepared by immobilizing H-2Kb-Ig dimers and anti-mouse Fas mAbs onto PLGA microspheres, and displayed correct phenotype as detected by flow cytometry.2. C57BL/6 mice (H-2Kb) and BALB/c mice (H-2Kd) were used as donor and recipient, respectively, for skin transplantation. The recipients were randomly assigned to 1 of 4 groups and injected (i.v. via the tail vein) with KaAPC, anti-Fas PLGA, Blank PLGA or PBS on days 5,7 and 9 after transplantation. Splenocytes were then prepared from recipients on day 12, labeled with CFSE, and then co-cultured with third-party (KM mice) spleen cells for 3 days and 7days. On day 3 post skin transplantation, the recipients were injected subcutaneously in the right groin with SP2/0 myeloma cells followed by treatment with KaAPC or control beads. Tumor size was monitored daily. On day 23, tumor tissue was isolated. Frozen section were prepared and stained with FITC-anti-mouse CD3. The results displayed that KaAPCs treatment prolonged the skin allograft survival for 4.5-7 days, but did not significantly decrease the proliferation response of T cells in a third-party MLR and anti-tumor effects with the similar myeloma tumor growth progression and infiltration of CD3+T cells as compared with control groups.3. C57BL/6 mice (H-2Kb) and bml mice (H-2kbml) were used as donor and receptor, respectively, for skin transplantation. The recipients were randomly assigned to 1 of 4 groups and injected (i.v. via the tail vein) with KaAPC, anti-Fas PLGA, Blank PLGA or PBS on days 7,9 and 11 after transplantation. On day 14 after transplantation, splenocytes were isolated from recipients, labeled with CFSE, and then co-cultured with third-party (BALB/c mice) spleen cells for 3 days and 7days. On day 3, the recipients were injected subcutaneously in the right groin with B16 melanoma cells followed by treatment with KaAPC or control beads. Tumor size was then monitored daily. Finally, tumor tissue was isolated on day 23. Frozen section were prepared and stained with FITC-anti-mouse CD3 and PE-anti-mouse CD8. On day 11, spleen cells were isolated from recipient mice, labeled with CFSE, and then co-cultured with Yac-1 lymphadenoma cells for 4-5 hours, followed by 7-AAD staining and flow cytometry analysis to inspect NK activity. Furthermore, the frequencies of T cells and apoptotic T cells in spleen cell population were detected by,APC-anti-CD3, Annexin V and PI staining in recipient mice on day 9,11 and 13,respectively. Simultaneously peripheral blood-was collected from recipient mice for the enumeration of lymphocytes, monocytes and neutrophils. The results displayed, that KaAPCs treatment prolonged the skin allograft survival for 24.3-27.3 days, but did not significantly decrease the proliferation response of T cells in a third-party MLR, cytotoxicity of NK cells and anti-tumor effects with the similar melanoma tumor growth progression and infiltration of CD3+T cells and CD8+T cells as compared with control groups; On day 13, the significently higher frequency of apoptotic T cells and lower percentage of T cells in spleen were found in KaAPCs treatment group comparing with control groups; Peripheral neurrophils were also’found decreased significently while lymphocytes and monocytes showed no signtficent changes in KaAPCs treatment group.4. Cationic PLGA nanoparticles are successfully fabricated using a double-emulsion-solvent evaporation method. The PLGA nanospheres displayed a heterogeneous size distribution under the SEM. The mean zeta potential was 36.2±5.00mV, as detected by the PALS zeta instrument, implying their strong capacity to eovalently couple protein molecules. The nano-size KaAPCs were prepared by immobilizing H-2Kb-Ig dimers and anti-mouse Fas mAbs onto PLGA nanospheres, and displayed correct phenotype as detected by fluorescence staining. Furthermore, the nano-size KaAPCs were administered intravenously into BALB/c mice that had previously been grafted with skin squares from C57BL/6 mice. The KaAPCs with a diameter of 200nm can significantly delay the alloskin graft rejection while the 450nm-KaAPCs did not display the therapeutic effect. |
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