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1、,2018.11.16,Dendritic cells,Dendritic cells(DCs)are professional antigen-presenting cells that inform the fight against invasive pathogens while enforcing tolerance to self-and harmless environmental antigens.They capture pathogens and receive signals from pathogens that influence the outcome of imm
2、une responses.On the basis of these signals,DCs orchestrate antigen-specific T-cell differentiation toward Th1,Th2,Th17,or Tfh pathways.They can silence self-reactive T cells by inducing deletion,anergy,or active regulation(via regulatory T Treg cells).This chapter will focus on the function and dev
3、elopment of DCs,and the mechanisms by which they link innate immunity to adaptive immunity.,DISCOVERY,DCs were discovered as part of an effort to understand the initiation of immunity.The mouse as an experimental animal was critical because of a system,developed by Mishell and Dutton,in which mouse
4、spleen cell suspensions could be stimulated to generate antibody responses in culture.,小鼠脾脏悬浮细胞,刺激,产生抗体,One of the early observations was that lymphocytes alone were not sufficient to induce antibody-forming cell responses and that an adherent accessory cell was required.The search for the accessory
5、 cell led to the discovery of DCs.,WhatsTheaccessory cell?,单独的淋巴细胞,刺激,无抗体产生,Identification and Isolation,DCs were discovered by Steinman and Cohn when they examined spleen adherent cells by phase contrast microscopy.（相差显微镜）They then employed physical techniques to fractionate spleencells and purify
6、DCs.These cells were found to adhere to plastic or glass,had low buoyant density,and did not bind to erythrocytes coated with antibody.（粘附性）,Sequential steps of density centrifugation（密度梯度离心）in bovine serum albumin gradients and adherence to glass were originally used to purify DCs.,At the time,macr
7、ophages were thought to be the key accessory cell because they composed a major population of adherent cells and also because their role in innate immunity had been long appreciated.,key accessory cell,purified antigen loaded macrophages induced immune responses when reinjected into their hosts.,ant
8、igen loaded,免疫反应,But,macrophages failed to show robust activity in induction of antibody responses in vitro,and they rapidly degraded ingested antigens suggesting that they would be unable to present it to lymphocytes.,体外,无免疫反应,At the time,the precise function of the MHC in antigen presentation was
9、not known,but it was already clear that the MHC was a key genetic determinant of immune responses and that it encoded many of the antigens involved in graft rejection.The mixed leukocyte reaction(MLR)was considered an in vitro model system to study graft rejection and was used by Steinman and Cohn t
10、o examine the function of DCs.混合白血球反应mixed leucocyte reaction 缩写为MLR。将同种动物两个个体的白血球或淋巴细胞混合一起，培养数日，则这些细胞的成细胞化，DNA的合成变得旺盛。将此反应称混合白血球反应或混合淋巴细胞反应（mixed lymphocyte reaction）,They found that DCs were nearly two orders of magnitude more potent than unfractionated spleen cells,B cells,or macrophages in stimu
11、lating allogeneic T cells in the MLR.On the basis of these experiments,they speculated that DCs were the accessory cells that present antigen to T cells to initiate immune responses.However,their conclusions were not widely accepted by immunologists because unlike traditional immune responses,the ML
12、R did not require priming.,DC刺激能力强,accessory cell,Proof that DCs are antigen-presenting cells came from experiments performed by Nussenzweig and Steinman who cocultured DCs with responding T cells and hapten modified thymocytes.They showed that DCs induced MHC-restricted cytotoxic T cellsspecific to
13、 the hapten and that macrophages and other purified populations of lymphoid cells were nearly inactive as accessory cells.,DC,反应性T细胞,半抗原修饰胸腺细胞,Monoclonal Antibodies to Dendritic Cells,A series of mouse DC-restricted monoclonal antibodies were produced by Steinman and others starting in 1980s,includi
14、ng 33D1(specific for the cell surface receptor DCIR2/Clec4a4).NLDC-145(specific for the adsorptive endocytosis receptor).DEC205/cluster of differentiationCD205).N418(specific for the CD11c integrin整合素).These monoclonal antibodies were used to establish the unique functions of DCs within heterogeneou
15、s mixtures of leukocytes and to identify DCs in situ.,Dendritic Cell Subsets,In the mouse,three major groups of DCs exist in the steadystate:plasmacytoid DCs(pDCs),conventional DCs(cDCs),and migratory DCs(mDCs)（1）pDCs are important mediators of antiviral immunity through their ability to produce lar
16、ge amounts of type I interferons(IFNs)upon viral infection（2）cDCs are composed of two major subsets,namely CD8+and CD8-，Both can process and present antigens to T cells and also secrete cytokines such as interleukin(IL)-12,which can influence the ultimate polarization of the T-cell response to patho
17、gens.（3）mDCs are present in nonlymphoid tissues such as the liver,gut,skin,lung,and aorta(主动脉),and they are composed of two main subsets CD103+and CD103-,pDC,cDC,mDC,cDCs（CD8+and CD8-）,two subsets have unique functions in vivo and are not redundant Some of the differences in cDC subset function may
18、be accounted for differential expression of toll-like receptors(TLRs)or other mediators of cDC activation.Spleen CD8+cDC express TLR3(recognizing doublestranded ribonucleic acid RNA)but lack TLR5(recognizing flagellin(鞭毛蛋白)and TLR7(recognizing single-stranded RNA),whereas CD8-cDCs express TLR5 and T
19、LR7 but have low levels of TLR3.,cDC,CD8+TLR3 dRNA,CD8-TLR5 flagellin/TLR7 sRNA,cDCs（CD8+and CD8-）,antigen processing capacity of the two cDCs types CD8+cDC are specialized for MHCI cross-presentation,and enriched in Tap1,Tap2,calreticulin(钙网织蛋白),calnexin(钙联接蛋白),Sec61,ERp57,ERAAP,as well as cystatin
20、(胱抑素)B and C,all of which are involved in MHCI presentation or inhibition of enzymes that process peptides for MHCII presentation.CD8-cDC,which are biased for MHCII presentation,were enriched in cathepsins C(组织蛋白酶类 c),H,and Z,asparagine endopeptidase(天冬酰胺内切酶),GILT,and H2-Mbeta 1,all of which are imp
21、licated in the MHCII antigen-processing pathway.,ANATOMIC DISTRIBUTION,Lymphoid Organs Peripheral lymphoid organs(ie,spleen,lymph nodes,and mucosal associated lymphoid tissues)are the sites where primary immune responses develop,including activation of helper,killer,and antibody-forming cells.T-cell
22、 area also contains large interdigitating cells(指突状细胞)that were initially thought to be macrophages.However,cytologic and functional studies of spleen and lymph nodes in rat and mice revealed that interdigitating cells lack phagosomes(吞噬体)and lysosomes(溶酶体),and are poorly phagocytic.Mouse interdigit
23、ating cells were later proven to be cDC based on immunohistochemistry using DC-specific monoclonal antibodies.In human lymphoid organs,interdigitating cells in the T-cell area express CD83,a member of immunoglobulin superfamily.,Nonlymphoid Organs DCs are also found in most nonlymphoid organs and in
24、 all epithelial surfaces that contact the environment.In organs such as heart,lung,kidney,the dermal layer of skin,and meninges and choroid plexus in the brain,they are found in interstitial spaces that are drained by lymphatics.Interstitial DCs(间隙DC)can be distinguished from macrophages by abundant
25、 expression of MHCII and low levels of lysosomal hydrolases(水解酶),Afferent Lymphatics(输入淋巴管)Afferent lymphatics in rats,rabbits,guinea pigs,sheep,and humans contain cells with motile processes that were named veiled cells（面纱细胞）because of their large membrane protrusions.In the healthy individual,thes
26、e veiled cells continually carry and present self-and harmless antigen to the adaptive immune system,thereby contributing to the induction of peripheral tolerance.During infection,they carry pathogen-derived antigens and induce specific T-cell activation.Thus veiled cells correspond to mDCs.,Dendrit
27、ic Cells in the Skin and Other Body Surfaces Several types of DCs,monocytes,and macrophages exist in the skin and at other body surfaces.Professional antigen presenting cells in the skin include Langerhans cells(LCs)in the epidermis,and CD103+and CD103-DCs in the dermis,DENDRITIC CELL DEVELOPMENT,Or
28、igin of Conventional Dendritic Cells in the Lymphoid Organs DCs,like all other leukocytes,develop from bone-marrow-derived hematopoietic stem cells.Monocytes,macrophages,granulocytes,megakaryocytes(巨核细胞),and erythrocytes differentiate from a common myeloid progenitor(CMP)whereas B,T,and natural kill
29、er(NK)cells differentiate from a common lymphoid progenitor.Although early cell transfer and genetic experiments were interpreted to indicate that DCs originated from lymphoid and myeloid progenitors,subsequent work showed that all DCs are derived from CMP.,Relationship between Dendritic Cells and M
30、onocytes In the steady state,DCs and monocytes can readily be distinguished based on cell surface marker expression.DCs are CD11c+CD115-/lo MHCII+monocytes are CD11b+CD115+F4/80+Under inflammatory conditions or in tissues such as lung and intestine,where they can be exposed to pathogenic and nonpath
31、ogenic microbes,monocytes develop many of the characteristic features associated with DCs,and the distinction becomes far more difficult.,experiments in humans and mice show that monocytes can develop some of the features of DCs under conditions of inflammation in vivo,or when cultured with cytokine
32、s in vitro,but they are not precursors of cDCs.Monocytes undergoing reverse transmigration,which simulates their entry into lymphatic vessels from tissues,lose expression of monocyte markers CD14 and CD64 and upregulate human leukocyte antigen DR and CD54.Phagocytosis further stimulates expression o
33、f CD80,CD86,human leukocyte antigen-DR,DC-LAMP,and CD83,all of which are expressed by DCs.,DENDRITIC CELL DEVELOPMENT,Dendritic Cell Progenitors in the Bone Marrow a common precursor for monocytes,macrophagesm and classical DCs(macrophage-DC progenitors MDPs).MDP are Lin-CX3CR1+CD11b-CD115+cKit+CD13
34、5+and account for 0.5%of all bone marrow mononuclear cells in mice.When cultured with granulocyte-macrophage-colony stimulating factor(GM-CSF)in vitro or adoptively transferred into mice,these cells produced macrophages and DCs,but not neutrophil granulocytes,B-/-T lymphocytes,or NK cells.Therefore,
35、MDPs are more restricted than CMPs,from which they are derived.,DENDRITIC CELL DEVELOPMENT,A DC-restricted progenitor that produces cDCs and pDCs but not monocytes in vitro or in vivo was then identified based on reduced cKit(CD117)and residual Flt3 expression and named common-DC progenitor(CDP;Lin-
36、CD115+Flt3+CD117lo).The CDP is downstream of CMP and MDP because adoptive transfer of either CMP or MDP gives rise to CDP and monocytes.More importantly,these experiments showed that the split between the monocyte and DC lineages occurs in the bone marrow between the MDP and CDP stages of developmen
37、t.,Migratory Pre-Dendritic Cells cDC precursors must migrate from the bone marrow to the lymphoid organs through the blood MDPs and CDPs are restricted to the bone marrow.The identity of the DC precursor was suggested by the finding that low-density CD11c+MHC II-SIRPlo(信号调节蛋白)cells isolated from blo
38、od,bone marrow,and periphery had the potential to produce cDCs.Although this group of cells was heterogeneous,the combination of these markers and persistent expression of Flt3 defined pre-DCs and allowed for their isolation in mice.,Pre-DCs migrate from the bone marrow to the blood and then to peri
39、pheral lymphoid organs and nonlymphoid tissues.These cells comprise 0.5%of all leukocytes in bone marrow,0.02%in blood,0.05%in the spleen,and 0.03%in the lymph nodes.Pre-DCs have a short-half-life in the blood of 1 hour;this,together with the small number of these cells in blood and tissues,may expl
40、ain why previous efforts to identify pre-DCs failed and why human pre-DCs have yet to be identified.,Pre-cDCs isolated from the bone marrow,blood,or spleen give rise to both CD8+and CD8-cDCs in lymphoid and nonlymphoid organs.Thus,the pre-DC is a progenitor with significant plasticity.In conclusion,
41、the DC and monocyte lineages split in the bone marrow,where MDPs give rise to both monocytes and CDP;the latter produce pre-DCs,which migrate from bone marrow through the blood to the periphery to give rise to DCs.,Origin of Nonlymphoid Tissue Dendritic Cells,Skin The skin contains LCs in the epider
42、mis and CD103+and CD103-DCs in the dermis.Experiments using bone marrow chimeras（嵌合体）and parabiotic mice demonstrated that LCs are long-lived cells that divide in situ in the skinThese cells originate from fetal liver-derived progenitors in an Flt3L-independent manner,and they are only replaced by b
43、one marrow derived hematopoietic cells during inflammation.,Gut,Kidney,Lung,and Liver Similar to the skin,CD103+and CD103-DCs are found in nearly all mouse nonlymphoid tissues.CD103+nonlymphoid tissue DCs express Flt3,a marker expressed on most cells in the DC lineage but not on monocytes.Only after
44、 depletion of endogenous CD11c+cells were monocytes able to produce some CD103-DCs.CD103-DCs appear to be more heterogenous;some of these cells are derived from pre-DCs while others may be of monocyte origin.,Plasmacytoid Dendritic Cells,pDCs were described by pathologists as a unique population of
45、cells in the peripheral lymphoid organs that resemble plasma cells and monocytes and were referred to as plasmacytoid monocytes（浆细胞样单核细胞）.In 1990s,Svensson et al.described a population of cells in human blood that produces large amounts of type I IFN upon exposure to herpes simplex virus,and they na
46、med these cells IFN-producing cells.pDCs were first purified from human tonsil and blood on the basis of being CD4+CD3-CD11c-.They were then shown to develop DC-like morphology upon culture with IL-3 and anti-CD40.Liu and colleagues discovered that purified pDCs produced 100 to 1,000 times more type
47、 I IFN than the other blood cell types following viral activation.,Plasmacytoid Dendritic Cells,pDCs were identified and isolated from lymphoid tissues in mice,and later in rats,monkeys,and pigs.In humans,the pDCs express CD4,MHCII,CD68,CD123,and blood DC antigen 2(BDCA2),but do not express other li
48、neage marker such as CD3(T cell),CD14(monocyte),CD19(B cell),CD16 and CD56(NK cell),or CD11c,BDCA1,and BDCA3(cDC).They are identified as CD4+,CD11c-,and Lin-(CD3,CD14,CD16,CD19,CD56)cells.In mice,pDCs express CD11c(lower levels than cDCs),Gr1,B220,and PDCA1;low levels of MHCII;do not express CD11b o
49、r CD19;and they are frequently isolated by virtue of CD11c and B220 expression.,Plasmacytoid Dendritic Cells,pDCs are closely related to cDCs;they originate from the same progenitor,the CDP.Development of pDCs is also critically dependent on Flt3L.pDCs do not express high levels of MHC II in the ste
50、ady state and therefore,their primary function is not antigen presentation to CD4 T cells.pDCs focus on sensing infection and producing type I IFN.,Growth Factors for Dendritic Cells and Monocytes,GM-CSF,M-CSF and Flt3L are essential growth factors for myeloid cell development in vitro and in vivo.D
