Ex-vivo Expansion Of Regulatory T Cell For Therapeutic Application In Transplantation & Relative Roles Of B7-1 And B7-2 In Thymic Development And Peripheral Homeostasis Of Regulatory T Cell

PartⅠIsolation of naturally occurring regulatory T cells(Treg)andcompartion of two ex-vivo expansion methods【Objective】To compare efficiency of two major protocols for the robust ex-vivoexpansion of Tregs for adoptive therapy and the qualities of expanded Tregs.【Methods】Tregs were isolated from lymphatic tissues of OVA-specific TCR transgenicmice DO11.10 with one-step positive selection after labeling with biotin-conjugatedanti-CD25,and stimulated by either functional monoclonal antibodies anti-CD3/28 orsyngenic bone marrow derived dendritic cell(BM-DC)loaded with OVA peptide inpresence of high-dose exogenous IL-2 for 1 up to 4weeks.The fold increase,viability,purity,phenotype and immunological characteristics of expanded Tregs were assessed.【Results】Stimulation of freshly purified Tregs with Abs or DCs recovered more than90% CD4~+Foxp3~+ cells with increased level of CD25 and Foxp3.Both Abs- andDCs-expanded Tregs remain hypoproliferative in response to antigen-specific TCR stimulation relative to the conventional CD4~+ T cells and stimulated Tregs secretconsiderable amounts of the immunosuppressive cytokine IL-10,but produce no or smallamounts of IFN-γand IL-2 as compared to conventional CD4~+ T cells.Importantly,DCswere much more efficient than Abs in expanding and maintaining the viability of purerTregs【Conclusion】DCs are more competent in ex vivo expanding and maintaining Tregs for theadoptive therapy with sustaining their viability and natural characteristics ex vivo evenfollowing exposure to antigen-beating DCs.PartⅡComparative analysis of dendritic cells and anti-CD3/28expanded regulatory T cells for suppression of alloreaction in vitro andvivo【Objective】to compare abilities of Abs- or DC-expanded Tregs to abolish direct andindirect alloreaction in vitro and in vivo.【Methods】For assessing the suppressive function of the expanded Tregs in vitro,twodirect [HVGD:B6 DC+Ba CD4+T cells+Ba DC/OVA+DO11.10 Treg and GVHD:BaDC/OVA+B6 CD4+T+DO11.10 Treg] and one indirect [HVGD:Ba DC /OVA(1μM)/B6 Ag+B6 CD4+T+DO11.10 Treg] mixed lymphocyte reaction(MLR)assayswere established.To test the capacity of ex vivo expanded Tregs to regulate recipientresponses to donor alloantigen in vivo,expanded DO 11.10 Tregs and B6 DC were adoptivetransferred to BABL/c mice implanted OVA-osmotic pump,after 5 days,the cells werecollected from the spleen and lymph nodes(LNs)were rechallenged in vitro with donorallogeneic DCs for 3 days.The proliferation of responder cells and the frequency ofIFN-γ-producing effector cells were detected by flow cytometry.The production of cytokines(IL-2 and IFN-γ)was determined using commercial ELISA kit.【Results】Comparing with Abs-expanded Tregs,DCs-expanded Tregs were less potentto contained the alloreactive T-cell proliferation,but more efficient in blocking thedifferentiation of alloreactive T cells into IFN-γ-producing effector cells in vitro.OnlyDCs-expanded Tregs could survive in vivo 5 days after adoptive transfer and suppressedthe host immune system response to allo-stimulation.【Conclusion】DC-expanded Tregs provides a clinically advantageous means of preventingunwanted immune reactions to alloantigen.PartⅢRelative roles of B7-1 and B7-2 in thymicdevelopment and peripheral homeostasis of naturally occurringregulatory T cell【Objective】To clarify relative roles of B7-1 and B7-2 in thymic development andperipheral homeostasis of naturally occurring Tregs for modification of tolerance protocoltargeting CD28/B7 interaction.【Methods】The frequencies of CD4~+CD25~+Foxp3~+ Tregs in the blood,thymus andperipheral lymphatic tissues of mice deficient in B7-1,B7-2 or both B7-1/B7-2 weremeasured by flow cytometry and compared with that in wild type mice.The abilities ofbone marrow derived DCs obtained from WT mice and mice deficient in B7-1,B7-2 orboth to maintain the isolated Treg and to stimulate alloreactive T cells in vitro werecompared side by side.【Results】Deficiency in either B7-1 or B7-2 could reduced the proportion of regulatory Tcell in thymus by around 30%,While B7-2 could maintain about 85% of regulatory T cellin peripheral,comparing with about 65% for B7-1.On the other hand,De deficent in B7-2 other than B7-1 could potently compromise the ability to prime the alloreactive T cells.【Conclusion】Although B7-2 expression is more effective in maintaining peripheral Tregshomeostasis,selective blocking B7-2 could efficiently hamper the alloreaction with relativeless impact on development and homeostasis of regulatory T cell comparing to completelyblocking the CD28/B7 interaction.