| Although transplantation of tissues to replace diseased organs has emerged as an important medical therapy,rejection caused by immune responses to alloantigens on the grafted tissues and organs are a major impediment.Nonspecific immunosuppressive drugs can delay or prevent rejection to greatly increase the shortterm survival of the transplant by inhibiting the proliferation of effector cells or by killing these rapidly dividing cells.But medical problems arise from their use and antibody-mediated rejection(AMR)remains a lingering problem.Even in cases of a deceased donor,10-year graft failure rates were 49.7%.In recent 20 years,emerging evidence based on the analysis of different populations of T cells with regulatory activity including subsets of CD4+T cells and CD8+T cells provided new insights into the conventional immunesuppressive approaches,and suggested a novel regulatory immune cell-based immune tolerance induction strategy to prolong graft survival and facilitate more specific tolerance to the graft without suppressing other immune function after transplantation.Among subset of CD8+regulatory T cells,CD8+CD28-T suppressor(Ts)cells,were documented to elicit immune tolerance in animal model and transplant patients.Our previous studies have expanded large numbers of human CD8+CD28-Ts cells in a relatively short period of time by stimulating CD8+T cells with alloantigen presenting cells(APCs)when supplemented with triple common yc cytokines IL-2,IL-7 and IL-15 in vitro.With this foundation,we further investigated the principal function of expanded CD8+CD28-Ts cells and the mechanism of yc cytokines in the expansion of CD8+CD28-T cells.Enriched CD8+T cells from PBMCs of responder(A-CD8+T cells)were cocultured with APCs from HLA-A,-B,-DR mismatched donor(B-APCs)to perfrom mixed lymphocyte reaction(MLR)for 9 days.CD28 expression was analyzed on CD8+T cells in a kinetics study over a 9-d period to further evaluate the role of the combination of γ c cytokines IL-2,IL-7,and IL-15 in the CD8+CD28" T cells.It was found that cytokine stimulation yielded a gradually increasing proportion of CD8+CD28-T cells and greater total number of cells with the culture time.On average a frequency of(55.93%±4.98%)CD8+CD28-T cells,compared with APCs stimulation-alone T cells was also observed after treatment with the combination of yc cytokines on 9th day(P<0.001).Meanwhile,we assessed the effect of yc cytokines on the proliferation of CD8+CD28-T cells or CD8+CD28+T cells,respectively.An apparent increase in proliferation in both subsets,with respect to control,was observed in response to treatment with yc cytokines(P<0.001),with an enormous expansion of the CD8+CD28-T cells.An analysis of the apoptosis of CD8+CD28+T cells and CD8+CD28-T cells found that the addition of yc cytokines would result in a decreasing percentage of necrotic cells in all cases,with a prominent role in CD8+CD28-T cells,which further encouraged the expansion of CD8+CD28-T cells(P<0.05).Surprisingly,CD8+CD28+T cells were purified and incubated in coculture system with yc cytokines,as a consequence of generating large amounts of CD8+CD28’ T cells during the culture(P<0.001),indicating a progressively decreasing CD28 expression might come along with CD8+CD28+T cells proliferation.Subsequently,we established a mixed lymphocyte reaction(MLR)system with three HLA completely mismatched individuals(individuals A/B/I whose HLA-A,-B and-DR fully mismatched with each other)as a group to mimic transplant circumstances in vitro,and verified whether the suppressive capacity of these expanded CD8+CD28’ T cells was antigen specific.The CFSE-labeled CD4+T cells from individual A(A-CD4+T cells,responder)were stimulated with APCs from the HLAA,-B,-DR mismatched individual B(B-APCs,stimulator)at a ratio of 1:1 to perform mixed lymphocyte reaction(MLR).CD8+CD28’ T cells priming the B-APCs in the expanded culture served as suppressor cells.Responder CD4+T cells and APCs from individual I(HLA-A,-B and-DR fully mismatched with A and B,I-APCs)were also cocultured to perform MLR,which acted as controls.After 7 or 11 days of coculture,the percentage of proliferating CD4+T cells was detected for CFSE dilution by flow cytometry.The results showed that compared with the group exposing to I-APCs,ACD8+CD28-T cells can significantly inhibit the proliferation of A-CD4+T cells under the stimulation condition of B-APCs,indicating that in vitro-expanded CD8+CD28-T cells possessed a remarkable ability to quell CD4+T cells proliferation in an alloantigen specific manner.In order to investigate the stability of the allogeneic suppressive capacity in in vivo circumstances for in vitro-expanded CD8+CD28-Ts cells,we then proceeded to perform in vivo experiments.Corresponding to cell components from in vitro experiments,a mixture of human cells(from three different individuals A/B/I whose HLA-A,-B and-DR fully mismatched)was administered into NOG mice by intraperitoneal injection.After 11 days of injection,mice were sacrificed and human CD4+T cells were investigated in the spleen of mice by flow cytometry and immunohistochemistry,determining if CD8+CD28-T cells could confer a local state of immunosuppression.Flow cytometry showed that relative to the group just along with B-APCs,the absolute number of human CD4+T cells(CD45+CD4+cells)from spleen,as an indicator of inhibition of APCs-induced T cell proliferation by CD8+CD28-T cells,was markedly reduced in the group with CD8+CD28-T cells,whereas the suppression was not seen when I-APCs acted as stimulator.The tendency was in agreement with observations from in vitro suppression assay.Moreover,the immunohistochemical images demonstrated human cells,both CD4+cells and CD8+(CD8+CD28-)T cells,were recurrently homed to spleen and kept alive till 11 days after injection.In addition,the result visually revealed a small number of human CD4+T cells infiltration at the site of the group exposed to B-APCs and CD8+CD28-T cells.These result suggested the suppression by A-CD8+CD28-T cells was antigen specific.The existence of immune inhibitory T cells was first proposed in the early 1970s by scientists who identified this activity within the CD8+subset and called these cells CD8+suppressor T cells.However,a lack of available and unique markers for characterizing these cells meant that many decades passed without sufficient supporting evidence of their existence.Then we also performed phenotypic markers of in vitro-expanded CD8+CD28-Ts cells to provide a basis for the phenotypic and functional analysis of CD8+CD28-Ts.The detection of phenotypic markers for CD8+CD28-Ts cells showed that the expression of CD25,CD 132,CD27,CD62L,CCR7,Tim-3,Foxp3 was up-regulated,and cytotoxicity related molecule perforin,granzyme B was down-regulated compared with freshly isolated CD8+CD28-T cellsIn conclusion,rapid enrichment of CD8+CD28-T cells has been induced by stimulating CD8+ T cells under conditions of allogeneic APCs plus the combination ofγ c cytokines IL-2,IL-7,and IL-15 in vitro,which exhibited significantly elevated proportion and greater total cell number.Furthermore,in vitro expanded CD8+CD28Ts cells kept steady allospecific suppressive capacity in in vivo circumstances.The possible mechanism of in vitro expansion of CD8+CD28-Ts cells was discussed,including directly enhancing the proliferation of freshly isolated CD8+CD28-T cells,inducing the transformation of CD28+cells to CD28-cells,decreasing the necrosis of CD8+CD28-T cells.In addition,in vitro expanded CD8+CD28-Ts cells up-regulated the level of CD25,CD 132,CD27,CD62L,CCR7,Tim-3,Foxp3,and down-regulated cytotoxicity related molecule perforin,granzyme B,which might give clues for further investigation of their suppressive mechanisms in future.These findings may provide the necessary experimental basis for the basic and clinical research of CD8+CD28-Ts cells,and lay the foundation for exploring the possibility of CD8+CD28-Ts cell-based therapeutic strategies to apply to clinical transplantation in the future. |
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