T Cells Induced To Mature And Effect Of Immunosuppressive Agent On Regulatory T Cells

T cells mainly mediate cell immunity and regulate the immunity of body. So T cellshave the important functions on the therapy of tumor, AIDS and autoimmunity diseases,as well as organ transplantation rejection. Thymus provides the highly effectiveenvironment for early T cells to develop into antigen-specific and functional T cells.Common lymphoid precursors which differentiate from HSC migrate into thymus,undertake the positive and negative choice helped with thymic epithelial cells,macrophages, dentritic cells and cytokines produced by them, and develop into mature Tcells to transfer the peripheral lymph organs to exert function.Notch signaling is important on regulating T cells development and differentiation.In mammal, there are four Notch receptors (Notch1,Notch2,Notch3 and Notch4 ) andtwo ligand families (Delta like and Jagged). Several lines of evidence implicate that theDelta-like 1, a number of Delta like family, involved in Notch signaling is essential for thedevelopment of T cells from HSC. Specifically, Notch signaling promotes αβT celldevelopment and the na?ve T cell conversion into regulation T cells.For many years the scientific researcher has devoted to the T cell differentiation invitro, and continue to open out the factors that affect T cell differentiation in vivo and seekthe methods in vitro to have the functional T cells to cure the diseases of human body.First, people chose the fetal thymic organ culture (FTOC) and reaggregate thymic organculture (RTOC) to simulate the development of T cells in vivo and successfully inducedthe functional T cells. But the two culture systems are difficult to do and the singlepositive T cells developed in them is lower. Thomas M and colleagues demonstrated thatBM stromal cell lines, which have been extensively used for the differentiation of Blymphocytes from HSCs, may fail to express specific Notch ligands necessary for T cellcommitment and differentiation. The ectopic expression of Delta-like-1 on OP9 BMstroma cells provides the necessary signals that are responsible for the induction of T celldifferentiation and successfully induce T cell development from hematopoietic progenitorcells by OP9-DLL1 in vitro. This culture system is simple, but the single positive T cells,especially single positive CD4 T cells developed in it is lower.In order to overcome the insufficiency of the above culture systems and establishone kind of simple and effective development culture system of T cells in vitro, weconstructed the S17-DLL1 cell line which ectopically expressed Delta-like 1. CD4+ CD8+thymocytes in C57 mice are sorted and added into the S17-DLL1 to detect the fuction ofthe cell line. It will establish the found to induce HSC development into mature T cells.Sorted CD4+ CD8+ thymocytes in C57 mice were co-cultured with the S17-DLL1cells for three days, and the differentiation and development of former cells weresubsequently evaluated by flow cytometry. The proportion of CD8 single positive T cellsis lower, but there were 50% CD4 single positive T cells in this system. The result isdifferent from that of Schmitt and Zuniga-Pflucker. Owing to different support cells andculture cells, the results are different. Our date imply the addition of the high expressionMHCⅠ cells into the culture system or co-culture cells with OP9-DLL1 can promote theproportion of CD4 and CD8 single positive T cells.As we known, HIV virus mainly attacks the CD4 positive cells. Because the lack offunctional CD4 positive T cells, the immunity in AIDS patients is lower and complicatinginfections in them on later period. Finally, they will dye. If we can use the HSC of thepatients to induce a great quantity of CD4 single positive T cells and to inject them intothe patient body, it will contribute to AIDS patients. Our culture system may producemassive CD4 single positive T cells, which provide the basis of certain experiment forclinical therapy of AIDS.On the condition of DLL1, CD4+ CD8+ T cells can differentiate into massive CD4single positive T cells in our culture system. The result suggests that Notch signaling atvarious stages of lymphocyte development promotes the development of CD4+ and CD8+single-positive (SP) T cells from CD4+ CD8+ (DP) precursor. Moreover, sorted CD4+CD25-T cells in the culture system contained the S17-DLL1 for five days inhibitesignificantly the T cell multiplication which caused by the homogeneous antigen, namelyhave the adjustment ability like regulating T cells. Therefore the DLL1 participation inNotch signal passage also has the important function in the adjustment nature T cellgrowth.Immunological tolerance is the ability of the immune system to discriminate betweenself and non-self, which contains central and peripheral tolerances. Central toleranceeliminates self-reactive T cells by clonal deletion in the thymus. Peripheral tolerance isachieved by the anergy and ignorance of T cells and the effect of regulatory T cells (Treg).CD4+ CD25+ Treg cells, which constitutively express high-affinity IL-2 receptor (IL-2R)α-chain (CD25), have been demonstrated to be an essential component for immunetolerance. CD4+ CD25+ Treg cells can develop in the thymus, and comprise 5% ~ 10%peripheral CD4+ T cells in mice and humans. Importantly, the forkhead transcriptionfactor Foxp3 is recently identified to be a functional marker of CD4+ CD25+ Treg cells,playing an essential role in their generation.Early studies have shown that the adoptive transfer of CD4+ CD25+ Tregcell-depleted CD4 single-positive (SP) T cells produced autoimmune diabetes insyngeneic T-cell-deficient mice. The production of the disease can be prevented by theadoptive transfer of CD25+ thymocytes. Naturally occurring CD4+ CD25+ Treg cells canprevent the development of muti-organ-specific autoimmunity induced by thymectomyfor 3 days. In several autoimmune diseases in mice and humans, the function or numbersof Treg cells decreased. In addition, it has been demonstrated that Treg cells play a criticalrole in the transplant immune tolerance in some models or inducing protocols. Thefunctional characteristics of CD4+ CD25+ Treg cells are naturally anergic and suppressive.They are non-proliferative (i.e. anergic) to in vitro antigenic stimulation, and the anergicstate is closely linked with their suppression. CD4+ CD25+ Treg cells in na?ve micesuppress the activation or proliferation of other T cells in a dose-dependent manner. It isshown that CD4+ CD25+ Treg cells can regulate antigen-presenting cells (APCs, includingDCs), na?ve T cells, B cells and NK cells directly or indirectly via cell-to-cell contact orvia soluble factors, although how CD4+ CD25+ Treg cells alter the functions of these cellsin details is unclear.CsA could form high-affinity complexes with intracellular cyclophilin (40, 41). Thecomplex associates with the catalytic subunit of calcineurin to inhibit its phosphataseactivity and interaction with various substrates, including transcription factor NFAT(42-44). Therefore CsA blocks the dephosphorylation and nuclear translocation of NFATs,subsequently prevents IL-2 transcription and T cell activation (42-45). CsA is animportant immunosuppressive agent that is used widely in organ transplantation andvarious autoimmune diseases across the world (19-21). While CsA significantly decreasedthe levels of CD4+ and CD8+ T cells.Accumulating evidence has shown that the thymus is important on the generation ofthe peripheral CD4+ CD25+ Treg cells (33). CD25 is expressed by 5% – 10% CD4 SPthymocytes in mice, rats and humans. These CD4 SP thymocytes (CD25+) couldefficiently inhibit T cell proliferation in vitro and can prevent the development ofautoimmune diseases in vivo (7). To determine the reason for the reduced levels of CD4+CD25+ Treg cells in the periphery of CsA-treated mice, we detected the changes of CD4+CD25+ Treg cells in the thymus using three-color FCM. With the reduced size of thethymus after injection with CsA, the total cell numbers of CD4+ CD8-CD25+ Treg cells inthe thymus of CsA-treated mice decreased markedly as compared with those in thecontrol mice. The results suggest that thymus might input much less Treg cells to theperiphery in CsA-treated mice. However, the percentages of CD4+ CD25+ Treg cells inCD4+ CD8-thymocytes in CsA-treated mice were significantly higher than those incontrol mice. Whereas the percentages of CD4+ CD25+ Treg cells in the spleens ofCsA-treated mice were significantly lower than those in control mice. These datacollectively indicated that CD4+ CD25+ T cells were resistant to CsA in the thymus andwere sensitive to CsA in the spleens as compared with the non-CD4+ CD25+ Treg cells.It has reported that the differentiation, acquisition of functional capacity andformation of a sizeable pool of Treg cells in the thymus were independent on IL-2signaling, but that IL-2 signaling seemed to be critically required for maintaining thehomeostasis and competitive fitness of Treg cells in the periphery in vivo (22, 23). It waspossible that CsA not only inhibites the development of CD4+ CD25+ Treg cells in thethymus but also influences the homeostasis of them in the periphery. Owing to theblockade of IL-2 transcription by CsA, the development of CD4+ CD25+ Treg cells wasinhibited so the number of peripheral Foxp3+ CD4+ CD25+ Treg cells reduced. Furtherstudy showed that CsA could remarkably impair the immunosuppressive function ofCD4+ CD25+ Treg cells. CD4+ CD25+ Treg cells in CsA-treated mice inhibited theproliferation of CD4+ CD25-T cells stimulated by mitogen (Con A) or allogeneic antigens,but it was much less efficient than those in control mice. Therefore, CsA couldsignificantly reduce both the numbers and the function of Treg cells in mice.Thus, our data indirectly showed that NFAT pathway might play an important role inthe development, survival and activity of Treg cells in the primary and secondary immunesystems. This study might be significant on the clinical usage of CsA.