1、Innate Immune SystemmonocytemacrophagebacteriaThe most ancient defensePhysical & chemical barriers and cellular lineRecognition by the innate immune system sets the stage for an effective adaptive immune response.機體在種系發生和進化過程中逐漸形成的一種天然免疫防御功能，構成機體抵御病原生物入侵的第一道防線. Innate immuneThe evolution of immune s

2、ystemConserved signalling in innate immunity in Drosophila and mammals. 固有免疫系統的組成屏障細胞分子皮膚黏膜屏障：物理、化學、微生物血-腦屏障、血-胸腺屏障血-胎屏障、氣-血屏障單核-巨噬細胞、中性粒細胞、樹突狀細胞、T 細胞、NK細胞、NKT細胞、B1細胞、肥大細胞、嗜堿性粒細胞、嗜酸性粒細胞、nuocyte等。抗菌肽、溶菌酶、急性期蛋白、補體、細胞因子和黏附分子、1、固有免疫屏障(1) The Epithelial Layer: The initial barrier to infectionOuter body s

3、urfaceKeratin barrierEpithelial cell sheddingDryness epidermis with keratinPhysical (mechanical) defense of skinInner body surfacesMucusViscousProtects underlying cellsContains antimicrobial factorsLysozymeConstant fluid flowTearsSalivaIntestinal peristalticUrine production and urinationVaginal secr

4、etionsMucociliary clearanceEpithelial cell sheddingPhysical (mechanical) defense of MucosaMucosal Tissuesthese surfaces are constantly exposed to bacteria and other organismsSebum, unsaturated fatty acids“antimicrobial lipids” Low pHSkin (pH 3-5)Stomach (pH 1.5 3)Vagina (pH 3 5)Urine (pH 6)9Chemical

5、 defenseTLRMicrobial Products(LPS, PG, etc)Antimicrobial PeptidesCytokinesExpress toll-like receptors that recognize specific PAMPProduce antimicrobial peptides (AMP) : kill microbes Secrete pro-inflammatory cytokines: alert the hostImmune function of epithelial cellsImmune system cells in the lamin

6、a propria and in the epithelial layerIntraepithelial lymphocytes (adaptive immunity)Three places where IgA can neutralize antigensSecretory IgA (adaptive immunity) Commensal species are nonpathogenic microorganisms that protect epithelium by competing with pathogens for nutrients and attachment site

7、s .Epithelium is protected by a flora of nonpathogenic microorganisms (commensal species)Skin Tongue EsophagusRespiratory tract and head outer ear, eye, mouth, oropharynx, nasopharynx Sterile sites: middle ear, brain, lower respiratory tract (trachea, bronchiole, lung)Gastrointestinal tract esophagu

8、s, stomach, small intestine, large intestineGenitourinary system anterior urethra, vagina Sterile sites: bladder, cervix, uterusSkinSites of human body that the normal flora microbes colonizeThere are about 500-1000 microbial species living in healthy human gutThe Gut Microbiome The human intestinal

9、 microbiota is composed of 1013 to 1014 bacteria whose collective genome (microbiome) contains at least 100 times as many genes as our own genome, at least ten times as many cells as human body. Normal flora may aid the host in several waysAid in digestion of foodProtect the host from colonization w

10、ith pathogenic microbes. Help the development of mucosa immunity This may cause inflammation and bleedingAntibiotics can make you sick by killing the commensal bacteria and allowing a opportunistic organism to invadeThe barrier function of the intestinal epithelium.Nature. 2012 Sep 13;489(7415):231-

11、41. Physical barrier of the epithelial layer Mucus/cilia to remove particles (lung, intestines)Acid pHAnti-microial peptides secreted by some epithelial cells (small intestines, small airways of lungs)surfactant-A and -D proteins opsonize pathogens for enhanced phagocytosis (lung)Commensal bacteria

12、(compete with pathogenic bacteria)Secretory IgA (adaptive immunity)Intraepithelial lymphocytes (adaptive immunity) (2) 體內特殊生理屏障血-胎屏障血腦屏障血腦屏障 血-腦屏障由軟腦膜、脈絡叢的腦毛細血管壁和包在壁外的星狀膠質細胞形成的膠質膜共同組成。 血-胎屏障 胎盤屏障由母體子宮內膜的基蛻膜和胎兒絨毛膜共同組成，能夠有效防止母體內的病原體及其毒性代謝產物進入胎兒體內。 其它屏障 人體的胸腺、睪丸等部位存在屏障結構。2、固有免疫細胞 PhagocyteNKILLs（固有樣淋巴細

13、胞）DC MCBasophil Eosinophil T細胞 NKT細胞 B1細胞Monocyte-macrophageNeutrophilRecognition of an infection once it gets past the epithelial barrier單核巨噬細胞 腹腔巨噬細胞粘附于玻璃表面黏附性高爾基體發達，線粒體豐富分化過程中體積增大、細胞器增多、偽足更多、吞噬能力增強、產生更多水解酶、分泌大量可溶性因子。單核細胞是白細胞中體積較大，蹄狀核Professional phagocytic cellsNeutrophilMast cellMonocyteMacropha

14、geDCMonocyte - bloodHistiocytes connective tissueKupffer cells liver Mesangial cells kidney Microglial cells brainAlveolar macrophages/ Dust cells lungPAMPPRR固有免疫細胞病原體吞噬靶細胞裂解炎癥反應天然免疫細胞的效應（1）吞噬殺傷病原體3個階段：識別recognition、吞噬ingestion、消化digestionM細胞功能Opsonins and Phagocytic receptorsOpsonins Complement com

15、ponents (C3b) Collectins (mannose-binding lectin) AntibodiesPhagocytic receptors Receptors for opsonins (complement receptors, Fc receptors) Pattern recognition receptors (mannose receptor, etc.) Receptors for apoptotic cellsM細胞功能Surface receptors of macrophagesToll-like receptorsToll樣受體Mac-1、CR3Fc受

16、體M細胞功能Fc receptors M細胞功能Fc receptors; a cell- and ligand-specific familyFc receptors exist in different “flavors” on the surface of distinct immuno cells. As their names are suggesting they all bind Fc regions of specific immunoglobulin classes and contribute to the elimination of pathogens. NK cell

17、s specifically express FcRIII.Staphylococcal Protein A Inhibits Phagocytosis by Blocking FcM細胞功能Figure 8-21What is happening in phagolysosomes? M細胞功能Oxidative attack via ROS & RNS:generated by NADPH phagosome oxidase (phox) enzyme complexThe oxygen consumed by phagocytes to support ROS production by

18、 the phox enzyme is provided by the respiratory burst (吞噬細胞吞噬病原體后有氧代謝活躍，短時間內耗氧量顯著增加，這種現象稱為呼吸爆發）M細胞功能Reactive Oxygen IntermediatesLipid Peroxidation Membrane, Protein and DNA DamageH2O2O2-OH-M細胞功能NO pathway yield NO L-Aginine + 1/2O2 Citrulline + NO yield Nitric peroxide (過氧化氮) free redical. NO + O2

19、- ONOO-inducible nitric oxide synthetase (iNOS)M細胞功能M細胞功能Mac-1(CD11b/CD18, M2, or CR3) Receptors for apoptotic cellsThe steps of efficient apoptotic cell clearance. Find-me and eat-me signals and some phagocyte receptors前列腺素E白三烯B4PAF磷脂酶過氧化物（2）介導炎癥：分泌炎癥介質，參與炎癥M細胞功能Figure 1-14Macrophages engulf bacter

20、ia and produce inflammatory cytokinesM細胞功能(3)免疫調節功能Activated macrophages secrete numerous pro-inflammatory cytokines and chemokinesM細胞功能(4)抗腫瘤作用膜融合，胞內溶酶體作用蛋白水解酶、溶細胞素、TNF-ADCC腫瘤細胞M細胞功能（1）單核巨噬細胞是專職抗原提呈細胞（APC）。 （2）加工處理外源性抗原和內源性抗原，具有免疫原性的小分子肽段與MHC分子結合形成肽MHC復合物表達于細胞表面，供T細胞識別。 （3）單核巨噬細胞B7分子與活化T細胞表達的CD28作用

21、，提供T細胞活化第二信號。(5)加工處理提呈抗原，啟動特異性免疫應答M細胞功能分泌 IL-1 IL-6 IL-12 TNF-a IL-8 GM-CSF細胞因子酶其它因子 殺傷腫瘤細胞 抗原呈遞作用前列腺素白三烯補體成分纖維蛋白結合蛋白凝血因子溶菌酶酸性水解酶賴氨酸酶酯酶膠原蛋白酶彈性纖維蛋白酶免疫調節作用吞噬并殺傷病原微生物 巨噬細胞的功能M細胞功能.Mantovani A Blood 2006;108:408-409M2s refer to diverse forms of M2 activation. M細胞亞群Macrophage subsetsKey properties and fu

22、nctions of polarized macrophages Macrophage recruitment. Distinct macrophage subsets regulate inflammation and wound healing. In situ macrophage proliferation. A recent discovery has shown that, contrary to previous thinking, macrophages can enter the cell cycle and proliferate locally. Thus far, in

23、 situ proliferation has been shown to be specific for T helper 2 (TH2)-type responses to worms. Cell surface markers commonly used in macrophage research中性粒細胞 (Neutrophil)在普通光學顯微鏡下觀察Giemsa染色血涂片，中性粒細胞呈淡藕荷色，多葉核，胞漿中含有大量的細胞顆粒。透射電鏡下其胞漿顆粒清晰可辨。一般特性 1. 具有較強的趨化作用和明顯的吞噬能力,主要對抗胞外菌的感染。 2.無需激活即能夠發揮強大的殺菌作用,產生的過氧化

24、氫又可與鹵化物、髓過氧化物酶（myeloperoxidase，MPO）組成MPO殺菌系統。 3. 在局部引發感染時，可迅速發揮吞噬殺傷和清除作用。無抗原提呈作用。Neutrophil receptors that are important for phagocytosisPresence of neutrophils in tissue sites (not blood) is a sign of acute inflammation中性粒細胞主要膜分子補體受體：CR1、CR3 和 CR4Fc受體：lgG Fc受體（FcR/）其他膜分子： LFA-1、IL-8R等The inflammati

25、on中性粒細胞是首先到達感染部位的效應細胞The phases of various leukocytes to the infection siteLeukocyte recruitment to sites of inflammationor DC2、固有免疫細胞 PhagocyteNKILLs（固有樣淋巴細胞）DC MCBasophil Eosinophil T細胞 NKT細胞 B1細胞Monocyte-macrophageNeutrophil過敏性疾病抗原的處理與提呈(2)NK 細胞 ( Natural Killer cells )大顆粒淋巴細胞：占外周血淋巴細胞總數的10-15%.體

26、積較大，胞漿含有大的嗜苯胺顆粒。不表達特異性抗原識別受體TCR（TCR OR TCR OR CD3）或BCR( OR CD19) 。NK細胞基本概念FcR: recognize antibody covered cell -ADCCThe biological functions of NK cells.Science.2011 Jan 7;331(6013):44-9. NK細胞基本概念Multiple sites for NK-cell development.Nat Rev Immunol.2007 Sep;7(9):703-14. Gut, Brain, UterusNK來源于骨髓造血

27、干細胞。主要分布于外周血和肝臟。NK細胞的來源和分布Factors that influence the development, homeostasis and survival of NK cellsNat Rev Immunol.2011 Aug 26;11(10):645-57. IL-15在NK細胞發育分化的整個過程中起重要作用.NK細胞的分化及亞群Requirements for transcription factors in NK cell development and function. Adv Immunol.2011;109:45-85. NK細胞的分化及亞群Pheno

28、typic markers of developing NK cells in the mouseCD16 FcR NK1.1 in mice or CD56 in humans NK細胞的分化及亞群Murine NK cells subset CD27/CD11bCD11blo CD27loCD11blo CD27hiCD11bhi CD27hiCD11bhi CD27lo NK1.1/DX5(1)CD56CD56bright :占外周血NK 10%，為中間過渡分化細胞，以分泌細胞因子為主。CD56dim : 占外周血NK 90%，為終末分化細胞，以殺傷功能為主。Human NK cells

29、 subsetNK細胞的分化及亞群CD56bright natural killer (NK) cells are the major producers ofNK-derived cytokines following activation of monocytes.NK細胞的分化及亞群Human NK cells subset(2)CD27人外周血NK細胞多為CD27lo ,這些均為CD56dim 。CD56bright 是CD27hi NK 細胞主體. (3)NKp46人和鼠NK細胞分化及亞群的統一:前體階段: CD122+ NKp46- iNKR-未成熟階段: CD122+NKp46+

30、 iNKR-成熟階段: CD122+NKp46+ iNKR+CD107ahi NKCD107aLo NKNK細胞的分化及亞群Recognition mechanism of NK cellsKAR & KIR識別非HLA-I類分子配體是殺傷活化性受體（killer activating receptor, KAR）識別HLA-I類分子的受體，在生理狀態下抑制性作用為優勢，為殺傷抑制性受體（Killer inhibitory receptor, KIR）NK細胞的的識別受體ITIM：immunoreceptor tyrosine-based Inhibitory motif ITAM：immun

31、oreceptor tyrosine-based activation motif NK Receptors bind with class MHC molecules:-KIR (killer immunoglobin-like receptors):Number of immunoglobin-like domain: KIR2D/KIR3DCytoplastic region: longer-KIR2DL/KIR3DL(ITIM), inhibitory receptor shorter-KIR2DS/KIR3DS, non-covalent combination with DAP-1

32、2(ITAM), activating receptor-KLR (killer lectin-like receptor): Heterodimer of CD94 & NKG2 (C type lectin) CD94: short cytoplastic region, no signal transmission NKG2A: ITIM in cytoplastic region -CD94/NKG2A, inhibitory receptor NKG2C: no signal transmission, bind with DAP-12(ITAM) -CD94/NKG2C, acti

33、vating receptorNK細胞的的識別受體NK receptors bind with non class MHC molecules NKG2D: Express mainly on the surface of NK and T No signal transmission Non-covalent binding with DAP-10(ITAM) MHC class chain-related molecules A/B(MIC A/B) Natural cytotoxic receptor(NCR): NKp46,NKp30,NKp44 IgSF Bind with othe

34、r molecules(ITAM)NK細胞的的識別受體Killer activatory receptor(KAR)Killer inhibitory receptor(KIR) KIR: KIR2DS, KIR3DS KLR: CD94/NKG2C NKG2D NKp46 NKp30 NKp44NCRKIR2DL, KIR3DLCD94/NKG2ABind class I HLAmoleculesFunctionBind non-class I HLA molecules Receptors associated with killer activation and killer inhib

35、ition on NK cellsNK細胞的的識別受體NK細胞對靶細胞的識別模式 1. “丟失自我”識別模式 是指殺傷抑制受體與MHC I類分子結合可以抑制NK細胞的殺傷功能，并將任何缺乏MHC I類分子的細胞作為靶細胞處理。被病毒感染的細胞或腫瘤細胞缺失MHC I類分子，NK細胞由此被激活而發揮殺傷功能。 NK細胞的的識別機制NK cells express “sensing” receptors for self-MHC class I NK細胞的的識別機制Target killing by NK cellsNK細胞的的識別機制 2. “壓力誘導”識別模式 是指NK細胞活化性受體NKG2D

36、分子能識別多種腫瘤細胞表面的MICA分子。 MICA分子在轉錄時需要熱休克因子（HSF）的參與，而HSF通常需要在病毒感染、惡性轉化和炎性反應等壓力誘導下才能發揮轉錄表達作用，一旦機體受到此類壓力誘導，HSF將促進MICA等分子的表達，從而被NKG2D所識別，激活NK細胞，發揮殺傷功能。 NK細胞的的識別機制Nat Rev Immunol.2012 Mar 22;12(4):239-52. NK細胞的的識別機制 1.釋放穿孔素/顆粒酶（1）穿孔素：在鈣離子存在的條件下，可在靶細胞的細胞膜上形成多聚穿孔素“孔道”，使水和電解質迅速進入細胞內，導致靶細胞崩解死亡。（2）顆粒酶（絲氨酸蛋白酶）：可循

37、穿孔素在靶細胞上形成的孔道進入靶細胞，通過激活凋亡相關的酶系統而導致靶細胞凋亡。NK細胞的殺傷機制NK細胞的殺傷機制2. 表達FasL 活化后NK細胞（FasL）靶細胞（Fas） 形成Fas三聚體 胞漿內的死亡結構域相聚成簇 與Fas相關死亡結構域蛋白結合 募集和激活caspase8 caspase8級聯反應導致靶細胞凋亡。FADD (Fas-Associated Death Domain protein) NK細胞的殺傷機制3.分泌TNF- TNF- 靶細胞（I型TNF受體）結合形成TNF-R三聚體 胞漿內的死亡結構域相聚成簇 激活caspase8 導致靶細胞凋亡。DED (Death ef

38、fector domain) NK細胞的殺傷機制Molecules involved in apoptosisTumor necrosis factor receptor (TNFR) superfamily: Fas (CD95)TNFR (I and II)Ligands: Induce apoptosisFasL (CD96L)TNF a and b (Lymphotoxin LT a+b)TRAIL (TNF-related apoptosis inducing ligand) Bcl-2 family Bcl-2, Bcl-xL Inhibit apoptosisBax, Bad P

39、ro-apoptoticNK細胞的殺傷機制4. 抗體依賴性細胞介導的細胞毒作用 （antibody-dependent cell-mediated cytotoxity, ADCC） NK細胞（IgG Fc受體、FcR ），識別殺傷與IgG抗體特異性結合的腫瘤或病毒感染細胞。NK細胞的殺傷機制. ADCC as a therapeutic means to kill tumor cells2、固有免疫細胞 PhagocyteNKILLs（固有樣淋巴細胞）DC MCBasophil Eosinophil T細胞 NKT細胞 B1細胞Monocyte-macrophageNeutrophil過敏性

40、疾病抗原的處理與提呈Innate-like lymphocytes (ILL) are at the X-road of innate and adaptive immunity; they express receptors commonly found on the surface of NK cells, such as members of the NKG2 family and the NK lineage receptor NK1.1, as well as rearranged BCR or TCR (but with very little diversity). ILL do

41、 not undergo clonal expansion before responding effectively to antigen. Characteristics of B-1 and B-2 cells B-1 B細胞B-1細胞抗原受體種類很少，抗原識別譜狹窄。它們識別的抗原主要包括：某些細菌表面共有的TI-2型多糖抗原，如肺炎球菌莢膜多糖(磷酸膽堿)和葡聚糖等；G-菌表面共有的TI-1型多糖抗原，如脂多糖；某些變性的自身抗原，如變性Ig、ssDNA。B-1細胞接受上述抗原刺激后產生的抗體，可對多種細菌和多種變性自身抗原起作用，缺乏嚴格特異性，這在機體抗感染免疫和維持自身穩定過程

42、中具有重要作用。抗原識別譜B-1細胞應答特點B-1細胞可通過表面抗原受體，直接與相應多糖抗原配體交聯結合而被激活，IL-5、IL-9等細胞因子作為細胞活化第二信號，可協助和增強TI-2型多糖抗原對B-1細胞的激活作用和分泌功能；B-1細胞在接受相應多糖抗原刺激后，48小時之內即可產生以IgM為主的低親和性抗體，這對機體早期抗感染免疫和清除變性自身抗原具有重要作用；B-1細胞在增殖分化過程中不發生Ig類別轉換，每個B-1細胞克隆只能產生一種類型Ig；B-1細胞不產生免疫記憶，再次接受相同抗原刺激后，其抗體效價與初次應答時無明顯改變。Comparison of B-1 and B-2 B cell

43、 propertiesPropertyB-1 cellsB-2 cellsN regionsFewExtensiveV region repertoireRestrictedDiverseLocationPeritoneum/pleuraEverywhereRenewalSelf renewal in situBone marrowSpontaneous Ig productionHighLowIsotypesIgMIgM/G/A/D/ECarbohydrate specificityYesRarelyProtein specificityRarelyYesNeed T cell helpNo

44、YesSomatic hypermutation of IgNoHighMemory developmentNoYesYes RarelyRarely YesNo YesCarbohydrate specificityProtein specificityNeed T cell helpSpecificity & requirement for T cell help suggests strikingly different typesof antigens are seen by B-1 and B-2 B cellsT細胞組織分布表達TCR-CD3復合物的T細胞稱為T細胞。主要分布于黏膜

45、及皮下組織，在粘膜免疫過程中可能起重要作用表面標志和特征T細胞表面標志與T細胞大致相同，但多為CD4-、CD8-雙陰性，少部份 為CD8單陽性T細胞，其表面抗原受體缺乏多樣性，抗原識別譜狹窄。就功能而言，T細胞應屬非特異免疫細胞，而不是特異性免疫細胞。 T cells mature in the thymusDN cells include precursors that will generate a minority of T cells that express the TCR (never express CD4 or CD8 co-receptors even as mature T

46、 cells in the periphery) and a majority of cells that will express a TCR together with either CD4 or CD8. T cells do not undergo the self-MHC restricted selection process.DNDPSPAg recognition by -T cells usually does not require MHC presentation, occurs directly (similar to antibody); detailed 3-D s

47、tructure of -TCR more closely resembles antibody than -TCRDirect recognition of TCR-ligand without requirement of antigen processing allows for rapid responseT細胞抗原識別譜感染后產生的熱休克蛋白或表達于受感染細胞表面的熱休克蛋白；感染后異常表達于受感染細胞表面的脂類抗原-CD1分子復合物；某些病毒蛋白或表達于受感染細胞表面的病毒蛋白，如皰疹病毒和牛痘病毒糖蛋白等；分枝桿菌產生的小磷酸化非肽分子，如磷酸糖和核苷酸衍生物。 T細胞生物學功能

48、具有抗感染、抗腫瘤作用，可識別殺傷某些病毒和胞內寄生菌感染的靶細胞，及表達熱休克蛋白和異常表達CD1分子的靶細胞。免疫調節：激活后分泌多種細胞因子參與免疫調節，介導炎癥反應。維持免疫耐受natural killer T cells介于NK和T細胞之間，共表達NK細胞表面分子NK1.1和TCR-CD3復合體分子的T細胞，亦稱NK1.1+T細胞（1987年）。CD1d-restricted natural killer T NKT細胞 發育途徑：NKT細胞具有胸腺內和胸腺外兩種發育途徑，肝臟可能是胸腺外主要的發育場所。成熟NKT細胞主要分布于骨髓和肝臟，此外在小腸固有層、脾臟和淋巴結也有少量的分布

49、。NKT cell are mainly generated in the thymusDNDPSPNK T cells are generated in the thymus from CD4 CD8 DN precursor thymocytes. While most of the thymocytes of this lineage never express CD4 or CD8, their differentiation is CD1d/lipid ligand restricted.NKTNKT細胞亞群：所有NKT細胞均是CD1d限制性的. 是否表達TCR：Va l 4 -Ja

50、 l 8+ I 型 NKT細胞和 Va 14 - Ja l 8 - II型NKT細胞 是否表達NK1.1：NK1.1+ NKT 和NK1.1- NKT小鼠NK1.1+ NKT中少數為 CD4+CD8-NKT細胞；大多數為CD4-CD8-NKT細胞。人NK1.1+ NKT中少數為 CD4+NKT細胞和CD8+NKT細胞；大多數為CD4-CD8-NKT細胞。1）NKT細胞表面TCR缺乏多樣性，識別抗原譜窄，主要識別靶細胞表面由CD1d分子提呈的糖脂類抗原。2）不受MHC限制。Cellular location of CD1a, CD1b, CD1c, CD1d and corresponding

51、CD1-restricted T-cell subsets and lipid antigens. CD1 molecules 半乳糖神經酰胺NKT在CD1d提呈抗原的刺激下被激活，并迅速產生IL-4、IFN、IL-10、IL-13等細胞因子，從而在抗腫瘤、抗感染、抑制自身免疫性疾病及移植免疫中發揮重要的作用。半乳糖神經酰胺But, paradoxically, in other models2、固有免疫細胞 PhagocyteNKILLs（固有樣淋巴細胞）DC MCBasophil Eosinophil T細胞 NKT細胞 B1細胞Monocyte-macrophageNeutrophilR

52、ecognition of an infection once it gets past the epithelial barrierNuocytes represent a new innate effector leukocyte that mediates type-2 immunity.Type-2 immunity: responsible for protective immune responses to helminth parasites and the underlying cause of the pathogenesis of allergic asthma.Type-

53、2 cytokines: interleukin IL-4, IL-5 and IL-13.Nuocytes expand in vivo in response to the type-2-inducing cytokines IL-25 and IL-33, and represent the predominant early source of IL-13 during helminth infection. In the combined absence of IL-25 and IL-33 signalling, nuocytes fail to expand, resulting in a severe