Induction Of CD4~+T Cell Apoptosis And Promotion Of Memory CD4~+T Cell Generation By Regulatory Dendritic Cells

Dendritic cells(DCs) are the most potent professional antigen presenting cells that play pivotal roles in the initiation of primary immune responses and induction of immunological tolerances.Different functions of DCs may be explained by existence of different subsets of DCs or DCs at different developmental stages.In recent years, identification and characterization of phenotype and cytokine profile of DCs with regulatory properties(so-called regulatory DCs),and,inveatigation of their roles in the immune regulation and the pathogenesis of immune disorders attract much attention. Now,regulatory DCs can be generated in vitro or in vivo by coculture with stromal cells or inhibitory cytokines including IL-10 or TGF-β,or other substances such as Vitamin D receptor ligands,vasoactive intestinal peptide(VIP),thymic stromal lymphopoietin (TSLP).Regulatory DCs can induce regulatory T cells,inhibit T cell proliferation or induce T cell anergy.In our previous study,we found that splenic stroma could drive mature dendritic cells(mDCs) to differentiate into a novel subset of regulatory dendritic cells(diffDCs) which strongly inhibit mDCs-induced CD4⁺T cells proliferation via nitric oxide(NO),however,diffDCs could not induce generation of regulatory T cells.Up to now,the detailed mechanisms for the inhibitory functions of diffDCs remain to be further investigated.Apoptosis of T cells is well regulated to maintain immune homeostasis.After antigen recognition,peripheral naive CD4⁺ T cells proliferate and differentiate into effector T cells to initiate immune responses.When the pathogenic antigen is deleted, effector T cells can go to apoptosis to prevent from disturbing T cell homeostasis and immune diseases such as autoimmunity and leukemogenesis.T cell apoptosis is regulated by several mechanisms:extrinsic cell-death-receptor-mediated and caspase dependent apoptosis,intrinsic mitochondria- and caspase-dependent apoptosis,or caspase-independent cell death.Considering that diffDCs can inhibit T cell proliferation, we wonder whether diffDCs could induce apoptosis of the activated CD4⁺T cells to exert their inhibitory effects.If so,what are the underlying mechanisms?Immunological memory is the hallmark of aquired immune system.It results from clonal expansion and differentiation of antigen-specific lymphocytes that ultimately persist for a lifetime.Memory lymphocytes confer immediate protection in peripheral tissues and mount recall responses to antigens in secondary lymphoid organs.Memory lymphocytes include memory B cells and memory T cells.Memory B cells can proliferate and differentiate to plasma cells that secrete antibody in response to antigenic stimulation. Memory T cells can be divided into effector memory T cells and central memory T cells according to immediate effector function and homing receptor expression.Effector memory T cells migrate to inflamed peripheral tissues and display immediate effector function,whereas central memory T cells expressing CCR7 and CD62L home to T cell areas of secondary lymphoid organs,have little or no effector function,but readily proliferate and differentiate to effector T cells in response to antigenic stimulation.The generation of memory T cells can be divided into three phases including clonal expansion, clonal contraction and memory cell maintainance.The quantity of memory T cells can be regulated during these three phase.The large clonal expansion of CD8⁺ T cells in response to some infections has made the development of CD8⁺T cell memory more amenable to study,giving rise to a model of memory cell differentiation in which a fraction of fully competent effector T cells transition into longlived memory T cells.However,due to the relatively low frequencies of memory CD4⁺ T cells,the studies about memory CD4⁺ T cells are difficult,how memory CD4⁺T cells are generated and then modulated remain to be fully understood.So,we wonder whether diffDCs may affect the generation of memory CD4⁺ T cells.Therefore,we have two parts for our investigation of the effects of regulatory DCs (diffDCs) on the CD4⁺T cells as follows.1.Induction of CD4⁺T cell apoptosis by regulatory dendritic cells and the underlying mechanismsThe primaty aim of this part of our study is to investigate whether diffDCs can induce apoptosis of antigen-specific CD4⁺T cells,and if so,what are the underlying mechanisms.Firstly,we added diffDC to of mDC and OVA323-339 specific CD4⁺ T cells coculture system and observed whether diffDC could induce apoptosis of activtated CD4⁺ T cells. We found that the percentage of late and early apoptotic activated CD4⁺ T cells increased markedly in the diffDC/mDC/CD4T coculture system,indicating diffDCs could significantly induce apoptosis of the activated CD4⁺ T cells.Then we wondered whether diffDCs could induce apoptosis of T cells that had been already primed and activated by antigen-pulsed mDCs.To determine this,naive antigen-specific CD4⁺ T cells were stimulated by mDCs and antigen peptide for 24 hours,and then diffDCs or mDC were added into the culture system.It was found that diffDCs could increase the percentage of late and early apoptosis of the activated CD4⁺ T cells,whereas mDCs failed.The data indicated that diffDC could directly induce apoptosis of the activated CD4⁺ T cells.Next,we went further to investigate the detailed mechanisms.We first determined whether soluble factors from diffDCs or cell-cell contact between diffDCs and activated CD4⁺ T cells is required for the apoptosis induction of the activated CD4⁺ T cells by diffDCs.So we performed Transwell assays.It was demonstrated that cell-cell contact between diffDCs and activated CD4⁺ T cells or cell-ceU contact dependent soluble factors was required for diffDCs-induced apoptosis of activated CD4⁺ T cells.Meanwhile, we found that higher levels of IFN-γand NO existed in diffDC/mDC/CD4T supernatants as compared to that in mDC/CD4T supematants,and their production was dependent on cell-cell contact between diffDCs and activated CD4⁺ T cells.The data indicated IFN-γand NO might be involved in diffDCs-induced apoptosis of the activated CD4⁺ T cells.As cell-cell contact between diffDCs and the activated CD4⁺ T cells was required for diffDCs-induced apoptosis of the activated CD4⁺ T cells,we wondered what membrane molecule(s) on diffDCs triggered apoptosis of the activated CD4⁺ T cells.It is well known that various death ligands such as Fas ligand,TRAIL,B7-Hland B7-DC can trigger apoptosis of activated CD4⁺ T cells.Therefore we first detected whether diffDCs expressed these molecules.We found that diffDCs expressed high level of FasL and B7-H1,but did not express TRAIL and B7-DC.To prove whether FasL on diffDC plays vital role in the apoptosis induction of the activated CD4⁺ T cells,we prepared diffDCs or mDCs from wild-type C57BL/6J mice or FasL-mutant gld mice,and then used them in DCs-CD4⁺ T cells coculture system.It was shown that diffDCs from gld mice (gld-diffDC) failed to induce apoptosis of activated CD4⁺ T cells and lost their ability to inhibit CD4⁺ T cell proliferation almost completely.However,gld-mDCs could prime CD4⁺ T cells proliferation normally.These results demonstrated that FasL on diffDCs played vital role in diffDCs-induced apoptosis of activated CD4⁺ T cells and could trigger apoptosis of activated CD4⁺ T cells effectively.Further in vivo results demonstrated that gld-diffDCs was not able to inhibit mDC-induced CD4⁺T cell proliferation in vivo.Due to the importance of FasL for diffDC's function,we want to know the mechanism of high expression of FasL on diffDCs and whether the in vivo counterparts of diffDCs in the spleen also highly express FasL.As stromal cell-derived TGF-βplays a vital role in the differentiation of diffDCs,we observed the effect of TGF-βon FasL expression of diffDC,and found that stromal cell-derived TGF-β contributed to the high expression of FasL on diffDCs through activation of ERK in diffDCs.Meanwhile,we found that the in vivo counterpart of diffDC in spleen (CD11b^hiCD11c^lowIa^low cells) expressed FasL higher than CD11c^hiIa^hi cells(mDC). Furthermore,during immune response,in addition to the increased percentage of diffDCs, FasL expression on diffDCs was up-regulated significantly,further indicating the importance of FasL for diffDC's function.As soluble factor(s) produced by cell-cell contact between diffDC and mDC/CD4T was also involved in diffDCs-induced apoptosis of CD4⁺ T cells,we want to know what these soluble factors were.Recent studies indicate that IFN-γplays important roles in inducing apoptosis or loss of CD4⁺ T cells during infectious diseases and antitumor responses.In our previous study,we found that the inhibiting function of diffDCs was mediated by nitric oxide(NO).Furthermore,our previous results showed that the level of IFN-γand NO in the supernatants in diffDC/mDC/CD4T coculture system was higher than that in mDC/CD4T coculture system which depended on cell-cell contact between diffDCs and activated CD4⁺ T cells.So we wondered whether IFN-γor nitric oxide played a role in diffDCs-induced apoptosis of CD4⁺ T cells.Using anti-IFN-γblocking antibody and the inhibitor of NO production,we found that both IFN-γand nitric oxide were partially involved in diffDCs-induced apoptosis of activated CD4⁺ T cells.As FasL on diffDCs triggered apoptosis of CD4⁺ T cells and both IFN-γ,and NO were involved in diffDCs-induced apoptosis of CD4⁺ T cells,we wondered whether high level of IFN-γand NO in diffDC/mDC/CD4T coculture system was related to FasL on diffDCs.Our results indicated that high level secretion of IFN-γ,and NO in diffDC/mDC/CD4T supernatants was dependent on FasL on diffDCs because the production of IFN-γand NO decreased in the coculture system of FasL-deficient diffDCs and mDC/CD4 T cells. Further results demonstrated that FasL-dependent high level of IFN-γ,derived from the activated CD4⁺ T cells was responsible for high level of NO produced by diffDCs and activated CD4⁺ T cells.In addition,we found that diffDCs could upregulate Fas expression of activated CD4⁺ T cells,the process was dependent on cell-cell contact and FasL on diffDCs but independent on IFN-γand NO.The data suggest that diffDCs may sensitize the activated CD4 T cells to FasL-induced apoptosis.All together,the results demonstrate that regulatory dendritic cells(diffDCs) can induce apoptosis of the activated CD4⁺ T cells via IFN-γ-induced NO in a FasL-dependent manner,thus inhibiting the proliferation of the activated CD4⁺ T cells and exerting negative immune regulation.2.Promotion of memory CD4⁺T cell generation by regulatory dendritic cellsThe primaty aim of this part of our study is to investigate whether diffDCs can affect the generation of memory CD4 T cells.First,we observed whether diffDCs could survive and keep their function after long-term culture in vitro.We found that diffDCs could be cultured in the presence of ESSC supernatant for a long period,survived at least 30 days in vitro and still kept the the function of inhibiting mDC-induced activated CD4⁺ T cell proliferation.Meanwhile, the in vivo counterpart in the spleen of diffDCs exists in na(?)ve mice and the number of diffDCs increases during immune response.These data provide the time and space evidence for the possibility that diffDCs may modulate the generation of memory T cells.As the studies about memory T cells especially memory CD4⁺ T cells are difficult for long time-span and the relatively low frequencies of memory CD4⁺ T cells,how memory CD4⁺ T cells are generated and then modulated remain to be fully understood. Some recent studies showed that after antigen clearance CD4⁺ T effector cells rested and transited to functional memory cells in vitro and in vivo.So we used similar experimental system to study how diffDCs modulate the transition of CD4⁺ T cell effectors to memory cells.In vitro results showed that,after resting,most cells of CD4⁺ T cell effectors would die via apoptosis,however,while being cocultured with diffDCs,the rested CD4⁺ T cell effectors(diffDC-rested effector CD4⁺T cells) grew better,died less and survived longer than the rested CD4⁺ T cell effectors alone beyond at least 21 days in Vitro.Using adoptive transfer model,we found that adoptive transfer of diffDCs also prolonged the survival of rested effector CD4⁺T cells in vivo.Furthermore,by detecting CFSE division and cell cycle,we found that both diffDC-rested effector CD4⁺T cells and the rested effector CD4⁺T cells alone underwent no active cell division,indicating diffDC prolonged the survival of the rested effector CD4⁺T cells without further dvision.Next,we analyzed the characteristics of the surviving diffDC-rested effector CD4⁺T cells.As compared with cytokine production by naive CD4⁺T and effector CD4⁺T cells, diffDC-rested effector CD4⁺T cells produced IL-2 and IFN-γmore rapidly and effectively after TCR stimulation,which is one of the major features of memory CD4⁺T cells,diffDC-rested effector CD4⁺T cells showed similar phenotypcs of the effector CD4⁺T cells rested for 3 days in that FSC^low SSC^low CD25^-/low CD69^- CD45RA^- CD122^-FasL^- CD44^hi CD127⁺.However,diffDC-rested effector CD4⁺T cells expressed lower Fas and had more CD62L^hi and CD45RB^hi cells.These data indicate that diffDCs prolong the survival of rested effector CD4⁺T cells and promote their transition to memory-like CD4⁺T cells in vitro.In vitro results showed that the enhanced survival of rested effector CD4⁺T cells by diffDCs required cell-cell contact between diffDCs and effector CD4⁺T cells.As to what are the underlying mechanisms by which diffDCs promote the transition of effector CD4⁺T cells to memory-like CD4⁺T cells remain to be further investigated.In summary,diffDC can induce apoptosis of the activated CD4⁺T cells through IFN-γ-induced NO in a FasL-dependent manner,providing another manner for the negative regulation of T cell response by regulatory DC.Also,regulatory DC can prolong the survival of the rested effector CD4⁺T cells and promote the transition of effector CD4⁺T cells to memory-like CD4⁺T cells in cell-to-cell contact manner.Our results will contribute to the beeter understanding of the immune regulation and the pathogenesis of the immunological disorders,and also may be useful to the design of immunotherapy for the immunological diseases.