| PurposeCorneal transplantation is the most successful form of organ transplantation, and the most effective treatment for the irreversible corneal blindness. Despite ocular immune privilege, a significant number of corneal allografts undergo immune rejection which remains the leading cause for the corneal graft failure. Corneal allografts are in direct contact with the anterior chamber, which has been demonstrated that corneal allograft survival correlates closely with the graft's capacity to induce anterior chamber–associated immune deviation (ACAID) to donor alloantigens. Transforming growth factor beta-2 (TGFβ-2) constitutively present in the aqueous humor of the eye, maintains dendritic cells (DCs) in the eye in a immature state. ACAID arises when these immature DC capture intraocular alloantigens and migrate to the spleen, where in the presence of natural killer T cells and marginal zone B cells, a microenvironment is created which activates antigen-specific T cells to differentiate into regulatory T cells that interfere with the induction of delayed hypersensitivity (DTH) as well as its expression. Recently, studies in the immunology have led to new interest in T cell regulation of the immune responses, and scientists recognize these regulatory T cells (Treg) play a crucial role in mediating immune tolerance and protection from autoimmune disease. Two generic types of regulatory T cells are described in the recent literature.One type is natural regulatory T cells (nTreg or innate Treg) which are the prototype and develops spontaneously within the thymus.The other generic type of regulatory T cells called inducible regulator T cells( iTreg or adaptive Tregs) arises as a consequence of diverse experimental manipulations. Very recently, a growing body of data from animal models has provided a compelling evidence that adoptively transferred CD4+CD25+Treg could attenuate even cure many autoimmune and allergic diseases and induce dominant transplantation tolerance. Harnessing naturally arising CD4+CD25+Tregs have emerged as potential reagents for adoptive cell therapy. In doing so, three main obstacles have to be resolved. First, CD4+CD25+Tregs represent less than 10% peripheral blood CD4+T cells, and their numbers are severely limited. Second, few data exist in the literature concerning their antigen-specificity. For an effective cell therapy, antigen-specific CD4+CD25+Treg would be required to minimize potential adverse effect. Third, the mechanism of CD4+CD25+Treg involved in immune tolerance is still unclear. The special anatomy of eye provides a novel way to study the role of Treg in preventing immune IFNlammation locally and systemicallyThe purpose of the study is to detect the change of Treg after corneal allograft transplantation in mouse, to describe the feasibility of the generation and expansion of Tregs with alloantigen-specificity induced by TGF-βDC, to investigate the different effectiveness of innate Treg and adaptive Treg in immunomodulation of murine corneal allograft rejection. It applies an important theary for the therapy of corneal allograft rejection and autoimmune diseases.Methods1. Using a mouse corneal graft model, grafts were evaluated by clinical observation and the gene expression levels of cytokines in allografts were determined by RT-PCR at various times. The number and phenotype of Treg in the submandibular lymph node (SMLN), superficial cervical lymph node (SCLN) and splenic lymphocytes (SPL) were studied by 3-color flow cytometric analysis (FACS) 3 days after isograft or allograft transplantation.2. Immature dendritic cells (iDC) were derived from bone marrow (BM) of mice and cultured in low-dose granulocyte-macrophage colony-stimulating factor (GM-CSF) RPMI-1640 medium with 0.2ng/ml TGF-β2. Antigen-loaded DCs were collected after incubation with soluble antigen (sAg) derived from donor. Three days after PBS or antigen-loaded syngeneic TGF-β2 DCs were injected into the tails vein of mice, the changes of the number and phenotype of Treg in SPL were studied by FACS analysis.3. The phenotype of the 3-subpopulation of CD4+/CD4+CD25+/ CD4+CD25-T cells derived from normal murine SPL were analyzed by FACS. CD+CD25+Tregs were isolated by 2-step magnetic cell sorting (MACS) and the Treg–specific transcription factor Foxp3, intracellular CTLA-4 and surface antigen CD127 were identified by FACS.4. PBS, TGF-βDCs, innate Tregs or adaptive Tregs were injected into the murine tail veins. Three days after the injection, the corneal allograft transplantations were performed and eyes were evaluated by clinical observation at various times. The difference of the number and phenotype of Treg in SPL were analyzed by FACS 3 days after the allograft transplantation in mice of difference groups.Results1.①After transplantation, none of the isografts were rejected, whereas all allografts were rejected by 8 d~24 d and the mean survival time was 7.8±4.66 d.②The expression levels of cytokines in allografts reached peak at 3 d～1 wk after the allograft transplantation, and the level TNF-αincreased greater than any other cytokines including IL-4, IL-10 and IFN-γduring the period.③Three days after the allograft transplantation, the number of CD4+CD25+Treg was raised up significantly in SCLN and SPL (P<0.01), the percentage of CD4+CD25+ Foxp3+ T cells was all increased in SMLN, SCLN and SPL (P<0.01). The CD4+CD25+ T cells expressed higher levels of CTLA-4 and lower levels of CD28 than those cells in isograft mice.2. TGF-β2 DC increased the number and function of CD4+CD25+Treg. After induced by TGF-β2 DC, the percentage of CD4+CD25+T cells in SPL was increased from 5.52% to 16.19% (P<0.01), and the number of CD4+CD25+ Foxp3+ T cells was also increased significantly (P<0.01). The expression of CTLA-4 of CD4+CD25+ T cells was increased from 69.49% to 84.29%, whereas the expression of CD28 was decreased from 89.71% to 67.13% (P<0.01).3.①These three CD4+T cell populations defined by varying levels of CD25 expression exhibited marked differences in a number of some intracellular or surface antigens. The CD4+CD25+T cells expressed higher levels of Foxp3, CTLA-4, GITR and CD69 (P<0.01) and lower levels of CD28 (P<0.05) than the CD4+ or CD4+CD25- subsets. These innate CD4+CD25+ Tregs were typically confined within the CD45RBlow T cell subset.②After CD4+CD25+ T cells were isolated from murine splenocytes by 2-step magnetic cell sorting, 90.19% (range, 87.29%～92.04%) of the cells expressed both CD4 and CD25, and the yield was 1%～1.2%. Foxp3 was preferentially expressed by CD4+CD25+T cells (66.6±1.033%), whereas the CD4+ or CD4+CD25- subpopulation contained low level of Foxp3 (23.97±1.997%, 16.78±3.24%). CTLA-4 was expressed by a significantly higher proportion of cells in the CD4+CD25+ population (77.37±1.67%) as compared to the CD4+ (21.97±2.39%) or CD4+CD25- (18.04±3.16) subsets. The isolated CD4+CD25+ T cells also expressed the low intensity of the CD127.4.①The corneal allograft rejection was suppressed notably in adaptive Treg injection group. In PBS injection group, all the allografts were rejected by 10～22 days after transplantation. In TGF-βDC injection group, the first allografts appeared obvious opacity by 34 days and all the allografts were rejected by 38 days after transplantation. In innate Treg injection group, the allografts remained clear within 38 days and all the allografts were rejected by 46 days after transplantation. In adaptive Treg injection group, all the allografts remained clear until 60 days after transplantation. The mean allograft survival time of the four groups was 16.50±4.3d, 37.8±2.04, 41.5±2.59 and >60d respectively, and there was statistical difference between the four groups analyzed by NPar test(P<0.05). Corneal opacification appeared to go through a phase of transient rejection in TGF-βDC, innate Treg or adaptive Treg group, but not in PBS injection group.②After PBS,TGF-βDC, innate Treg or adaptive Treg injection, there was an upregulation in the percentage of CD4+CD25+, CD4+CD25+Foxp3+, CD4+CD25+CTLA-4+T cells in SPL 3 days after corneal allograft transplantation, and there was statistical difference between the four groups (P<0.01). Adaptive Tregs were more potent in suppressing effector than innate Treg (P<0.01).Conlusions1. Alloantigen was the main cause of corneal allograft rejection. Regulatory T cells might be involved in the induction of ACAID which contribute to the inhibition of allograft rejection, through a way in which the delayed-type hypersensitivity (DTH) is suprressed. TNF-αwas a main factor among the variety of cytokines that could IFNluence corneal allograft rejection locally, but the mechanism of TNF-αinvolved in ACAID is still unclear.2. TGF-βDC could increase the number and function of Foxp3+CD4+CD25+Treg. Combination with the results in Part 4, Tregs were also involved in the mechanism of DC in immunomodulation of corneal allograft rejection. This has not been reported so far.3. These three innate CD4+T-cell populations defined by varying levels of CD25 expression in mice exhibited marked differences in a number of other intracellular or surface antigens, such as Foxp3, CTLA-4, GITR, CD45RB,CD69,CD28. Cells isolated from murine SPL products by 2-step magnetic cell separation protocol (negative selection and positive selection) were highly enriched in CD4+T cells with phenotypic and functional characteristics of innate CD4+CD25+Treg. The similar studies have not been reported domestically.4. CD4+CD25+Tregs with indirect allospecificity were more potent than antigen-loaded TGF-βDCs or innate CD4+CD25+Tregs in suppressing effector in allograft rejection specific for the same antigen. Our results add a new important perspective for Treg-based immunotherapeutic strategies. None of the pervious reports investigated the different effectiveness of innate Treg and adaptive Treg induced by DC in vivo in immunomodulation of allograft rejection.
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