| Clinical organ transplantation has remarkably advanced and is currently a well-established treatment for all sorts of organs'end-stage disease. Short-term survival rates are currently excellent. Unfortunately, long-term survival is still comparatively poor, mainly because of the chronic rejection and the toxicities of nonspecific immunosuppressants. To gain transplantation tolerance between donor organs and hosts is the ultimate goal of all sorts of organ transplantations. Intriguingly, CD4~+CD25~+ regulatory T cells (Tregs), which express a high level of Foxp3, display strong immune suppressive effect. Studies have shown that Tregs transfusion can prevent the graft-versus-host disease (GVHD) and transplantation rejection. Thus, Tregs have been suggested as potential reagents for adoptive cell therapy. However, the amount of naturally occurring Tregs only account for 1% of peripheral blood mononuclear cells, they are narurally anergic and poorly proliferative after TCR stimulation, such that cell numbers are severely limiting. Apparently, Tregs need to be massively expanded in vitro before they could be transfused back to patients and achieve the therapeutic objective. How to expand Tregs for the induction of transplantation tolerance is one of the hot areas where intensive studies are being performed.Interleukin-15(IL-15) is a pleiotropic cytokine of the 4-α-helix bundle cytokine family, which was discovered by Burton and Grabstein in 1994 due to its ability to mimic IL-2-dependent T cell proliferation. A special feature of IL-15 is that it shares with IL-2, another member of the 4-α-helix bundle cytokine family, the IL-2 receptor beta (IL-2Rβ) and IL-2 receptor gamma/gamma common (IL-2Rγ/γc). IL-15 and IL-2 produce similar effects after their interactions with the T cells. IL-15 mainly binds to IL-15Rαchain in high affinity, and it maintains a high affinity (Ka≥1011M-1) to the private chain of itsαreceptor, even in the absence of IL-2Rβ和IL-2Rγ/γc receptor subunits. In contrast, the affinity of IL-2 and IL-2Rαis very low (Ka～108M-1) in the absence of IL-2Rβγ. In vitro study showed that the ability of IL-2 to simulate Tregs expanding is superior to IL-15. However, IL-2 or IL-15 deficient mice display normal or slightly reduced Tregs amount and function, which are significantly decreased in IL-2 and IL-15 double deficient mice. This indicates that IL-15 plays an equally important role in the expanding of Tregs. Nevertheless, how IL-15 performs its function remains to be addressed by further studies.Rachael et al. showed that Tregs do not proliferate in the presence of either IL-15 or dermal fibroblasts, but do significantly proliferate when both IL-15 and dermal fibroblasts are present. This indicates that IL-15 can promote Tregs proliferation, but only in the presence of other types of cells. The underlying reason is that dermal fibroblasts present IL-15 to T cells in the form of membrane-bound IL-15. In the initiation of immune reaction, dendritic cells (DC) are the most potent antigen-presenting cells that activate initial T cells. Like dermal fibroblasts, DC can also present IL-15 to target cells as membrane-bound IL-15; however, it remains unclear whether DC and IL-15 can induce Tregs proliferation. For this reason, we isolated Tregs from human peripheral blood, and utilized IL-15, DC, and DC combined with IL-15 to intervene the proliferation of Tregs, and explored the related molecular mechanisms. Our work aims to improve the in vitro proliferation of Tregs and obtain a sufficient number of Tregs, and to lay a foundation for the induction of immune tolerance in organ transplantation. The main results and conclusions of our study are summarized bellow:A. Isolation and characterization of dendritic cells and CD4~+CD25~+ regulatory T cells1. Human peripheral adherent monocytes were induced with GM-CSF and IL-4 and loaded with the allogeneic antigen on the 6th day. The cells were harvested on the 7th day. The results showed that the cells obtained gradually converted from adherent cells to suspended cells that exhibit irregular morphology, coarse surface, large amount of laminated folds, and spike-like processes. These cells were found to express co-stimulating molecules such as CD80 and CD86, which, as shown in MLR, can stimulated the proliferation of autologous CD4~+CD25- T cells. These results indicate that the cells that we obtained have the morphology, immunophenotype, and functional characteristics of DCs.2. Human peripheral blood mononuclear cells were positively and negatively selected with immunomagnetic beads. The results showed that the viability of the cells was 95. 7± 2.1 %,the purity of the cells was 95.2%,and the expression rate of intracellular factor Foxp3 was 94.2%. The cells we obtained can inhibit the proliferation of sensitized T cells in vitro, indicating these cells have the cellular phenotype and functional characteristics of Tregs. Therefore, the method that we developed can be used to isolate Tregs in vitro, and this laid a foundation for further study on Tregs.B. The proliferation of CD4~+CD25~+ regulatory T cells induced by IL-15 and Dendritic cells1. Tregs were cultured (1×104cells/well) in U-shaped 96-well plates, and were divided into 4 different groups according to the presence of Dynal beads or DCs (1×104 cells/well, inactivated with mitomycin). The results showed that in the presence of the magnetic beads coated with anti-CD3/CD28 antibody, the proliferation of Tregs was not observed with 0-100 U/ml IL-15, but was slightly observed with 200 U/ml IL-15. In contrast, 25 U/ml IL-2 induced significant Tregs proliferation in the presence the magnetic beads coated with anti-CD3/CD28 antibody. Furthermore, in the presence of the DCs loaded with allogeneic antigen, both IL-15 and IL-2 induced similar extents of Tregs proliferation in a dose-dependent manner within 0-200 U/ml cytokine. The magnitudes of the induction of Tregs proliferation by IL-15 and IL-2 were not statistically significant, when the concentration of the cytokine was within the range of 100-200 U/ml. These results indicate that IL-15 can also induce Tregs proliferation when combined with DCs.2. CD4~+ T cells were positively screened using immunomagnetic beads, analyzed for purity, cellular phenotype, and CD62L using flow cytometry, and assayed for the inhibitory of the proliferation of CD4~+CD25- regulatory T cells using 3H-TdR incorporation. The results showed that proliferated Tregs induced by IL-15 presented by DC in trans are of high purity, and maintain the typical CD4~+CD25~+Foxp3~+ phenotype. Tregs have strong inhibitory effect in vitro on the proliferation of Teff activated by specific allogeneic antigen, but not that of Teff activated by third-party allogeneic antigen. Tregs express high level of CD62L. These results indicate that proliferated Tregs, which were induced by IL-15 presented in trans by DCs, display the same phenotypes of naturally produced Tregs. In addition, proliferated Tregs display specific inhibitory effect, and have the capability to migrate to the target location and perform specific functions.C. The related mechanism of the proliferation of CD4~+CD25~+ regulatory T cells induced by IL-15 and Dendritic cells1. Tregs and DCs were incubated separately with 100 U/ml IL-15 at 37℃for 15 min, and were analyzed for IL-15Rαand cell-surface IL-15 using flow cytometry. The results showed that the level of Tregs-surface IL-15Rαwas extremely low and Tregs-surface IL-15 was not detectable. In contrast, we detected high level of IL-15Rαon the surface of DCs, and the level of IL-15 bound to DCs surface was far higher than that bound to Tregs surface (p < 0.05). This indicates that the ability of Tregs to bind IL-15 is much lower than that of DCs. In addition, the presence of IL-15-IL-15Rαcomplex on DCs surface was confirmed with special ELISA.2. Tregs were cultured with DCs loaded with allogeneic antigens at 1:1 ratio in the presence of 100 U/ml IL-15. The cell mixture was harvested, and DCs and Tregs in the mixture were labeled respectively with PE-anti-CD86 antibody and FITC-anti-CD4 antibody. Subsequent confocal microscopy showed that DCs and Tregs form stable cell aggregates, indicating that DCs may present IL-15 in trans to Tregs and hence induce the Tregs proliferation.3. DCs and Tregs were cultured together in plate-bottom 96-well plates in the presence of 100 U/ml IL-15 and divided into 2 groups. The block group was added 40ug/ml anti-IL-15Rαantibody,while the control group was added equal volume of PBS. The results showed that if DCs were blocked by anti-IL-15Rαantibody before they were incubated with IL-15, the concentration of IL-15-IL-15Rαcomplexes on DCs surface decreased, and so was the proliferation of Tregs. These result support the notion that IL-15 was presented in trans by DCs to Tregs and induce Tregs proliferation.4. Analysis of DC surface IL-15 as a function of time: DCs were cultured in medium containing 100 U/ml IL-15 for 4 days, and then were assayed for surface IL-15 using flow cytometry. The cells continued to be cultured in the medium containing Glycine buffer (pH = 3), and analyzed for surface IL-15 10 min later. The cells were again cultured in medium without cytokines for 24 hr, and analyzed for DC-surface IL-15 using flow cytometry. DCs and Tregs were co-cultured in plate-bottom 96-well plates each at a density of 1×105 cells/well. These cells were then divided into 3 groups. The control group contained no IL-15, while the maintenance group contained 100 U/ml IL-15 throughout the course. In the withdrawal group, IL-15 was removed on the 4th day of the co-culture, and continued to be cultured for 24 hr. The cells of each group were harvested on the 5th day of the culture, homogenated and assayed for IL-15-IL-15Rαcomplexes using special ELISA. In a separate experiment, Tregs were cultured with DCs inactivated with mitomycin in U-shaped 96-well plates each at a density of 1×104 cells/well with 100 U/ml IL-15. The samples were divided into 2 groups. In the withdrawal group, the cytokine (IL-15 or IL-2) was removed on the 4th day, and continued to be cultured for additional 24 hr. The cytokine remained in the medium of the maintenance group. The proliferation of Tregs was quantified using 3H-TdR incorporation. The results showed that IL-15 was detectable on the surface of DCs 24 hours after the removal of IL-15 in the culture system. This indicates the formation of IL-15-IL-15Rαcomplex in the culture system, and thus Tregs could continue to proliferate. This also indicates that IL-15 could be recycled via trans-endosomal recycling, and thus could remain on DC surface for a long period of time. IL-15 could be presented by DCs to Tregs, and induce Tregs proliferation.5. Tregs were co-cultured with DCs each at a density of 1×104 cells/well in U-shaped 96-well plates. The cells were divided into the experimental and control group, with and without the addition of IL-15. The supernatant was sampled and analyzed for IL-2 concentration using ELISA on the 1st, 2nd, 3rd, 4th, and 5th day of the culture. The results showed that the concentration of IL-2 remained unchanged in the control group and was much higher in the experimental group, in which the concentration peaked at 60 pg/ml on the 4th day. We observed no Tregs proliferation in this experiment. The results indicate that IL-15 indeed regulates the secretion of IL-2 in DCs, but this does not play a predominant role in the induction of Tregs proliferation by DCs and IL-15.6. Tregs were resuspended in medium containing 100 U/m IL-15, and 600μl (containing 4×104 cells) cell suspension was added to each well of 24-well plates. The samples were divided into 2 groups. The experimental group utilized TranswellTM chambers equipped with 3.0μm (pore size) filter, and each chamber was loaded with 4×104 suspended DC (100μL). In the control group, Tregs were cultured with 4×104 (100μl) of suspended DCs inactivated with mitomycin in 24-well plates. The cells were harvested on the 5th day, and the cell number was quantified using Trypan blue under light microscopy. Tregs did not proliferate in the experimental group, but did significantly proliferate in the control group (p < 0.05). This indicates that the IL-2 and sIL-15-IL-15Rα, which were produced by the interaction of IL-15 and DCs, do not play an important role in the induction of Tregs proliferation.7. Tregs were cultured in U-shaped 96-well plates at a concentration of 5×104 well/plates. The cells were divided into 5 groups. The control group contained no IL-15 and DCs. The DC group contains only DCs inactivated with mitomycin (5×104 cells/well). The IL-15 group contained only IL-15 (100 U/ml). The combined induction group contained DCs and IL-15 at the above concentrations. In the blocked combined induction group, the addition of IL-15 was preceded by the addition of DCs and 40ug/ml anti-IL-15Rαantibody. After 24 hours, the cells were harvested, and the proteins in CD4~+CD25~+ regulatory T cells were extracted and analyzed for the expression of p-ERK, p-AKT, p-STAT5 and P27kip1 using Western-Blot. The results showed that DC-mediated activation could successfully induce the activation of Akt (target molecules of PI3K), but not the activation of Erk1/2 (the target molecule of MEK1/2) or STAT5, or the degradation of p27kip1 in Tregs. In contrast, when external IL-15 was added alone, IL-15 could not significantly induced the activation of Akt, Erk1/2, and STAT5 or the degradation of p27kip1, because of the low expression of IL-15Rαin Tregs. It was only in the presence of both DCs and IL-15 that significant induction of the activation of Akt, Erk1/2 and STAT5 and the degradation of p27kip1 in Tregs, and hence the proliferation of Tregs. In addition, if DCs and IL-15 were added after the addition of anti-IL-15Rαantibody, the activation of Akt, Erk1/2 and STAT5 and the degradation of p27kip1 were all inhibited, and the proliferation of Tregs was reduced. These results indicate that DCs and IL-15 mainly induce the proliferation of Tregs via the in trans presentation of IL-15.Conclusion: We successfully developed a method to isolate and culture DCs and Tregs from peripheral blood. We demonstrate that IL-15 can induce the proliferation of Tregs in the presence of DCs. Proliferated Tregs induced by DCs and IL-15 are of high purity and have the phenotype of naturally occurring Tregs and perform specific inhibitory effect. They have the capability to migrate to the target location and perform their functions. Tregs have the potential to be applied for the induction of transplantation tolerance in clinics. The induction was mediated by IL-15 presented in trans by DCs. IL-15 can be recycled via trans-endosomal recycling, and remain on the surface of DCs for long-period of time. Therefore, DCs can continue to present IL-15 to Tregs and induce the proliferation. IL-15 may also regulate the secretion of IL-2 derived from DC, which plays a supportive role in the induction of Tregs proliferation by DCs and IL-15. The molecular mechanism of Tregs proliferation induced by DCs and IL-15 may involve the activation of Akt, Erk1/2 and STAT5, and the degradation of p27kip1.
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