淋巴细胞成熟与抗原受体基因的表达.ppt

*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2007口腔,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,淋巴细胞成熟与抗原受体基因的表达,免疫细胞（,immunocytes,）,凡参与免疫应答或与免疫应答有关的细胞,包括：,淋巴细胞,单核巨噬细胞,抗原递呈细胞,粒细胞、肥大细胞、血小板等,淋巴细胞,T,淋巴细胞,B,淋巴细胞,NK,细胞,免疫活性细胞,(,immunocompetent cell,，,ICC,),T,、,B,细胞均有特异性抗原受体，接受抗原刺激后能发生活化、增殖和分化，产生特异性免疫应答，故称免疫活性细胞,第一节,B,细胞成熟过程,B cell maturation,骨髓内成熟,骨髓外成熟,B,细胞因在骨髓（,b,one marrow,）或禽类法氏囊（,b,ursa of Fabricius,）中分化成熟而得名，简称,B,细胞,The discovery of B cell immunity,1957-,Bruce Glick,Ohio State University,Studies on the function of the a lymphoid organ in the cloacal region of the chicken the,bursa of Fabricius,Bursectomy no apparent effect,None of the bursectomised chickens made anti-Salmonella antibodies,Bursa,was the organ where antibody producing cells developed,Antibody producing cells were thereafter called B cells,Transfer foetal,liver cells,Origin of B cells and organ of B cell maturation,No Mature,B cells,After birth,development continues in the bone marrow,Normal bone marrow,Defective bone marrow,Mature B cells,in periphery,B cell development starts in the foetal liver,起源：,胎儿期,胎肝和骨髓,出生后,骨髓,两阶段,:,骨髓内成熟,(,抗原非依赖期,),骨髓外成熟,(,抗原依赖期,),一、骨髓内成熟过程,1,、骨髓微环境和,B,细胞成熟,2,、,B,细胞成熟的分期,3,、未成熟自身反应性,B,细胞的阴性选择,4,、,Ig,基因重排,1,、骨髓微环境和,B,细胞成熟,B,细胞成熟四阶段,祖,B,细胞（,pro B cells,）,前,B,细胞（,pre B cells,）,未成熟,B,细胞（,immature B cells,）,成熟,B,细胞（,mature B cells,）,1,、骨髓微环境和,B,细胞成熟,骨髓基质细胞提供微环境,VCAM-1,SCF,IL-7,基质细胞,VLA-4,c-Kit,IL-7R,祖,B,细胞,Early pro-B,c-Kit,SCF,VLA-4,pro-B,Cytokines and cell-cell contacts at each stage of differentiation are different,Stromal cell,CAM,VCAM-1,Early pro-B,IL-7R,Stromal cell,Late pro-B,Pre-B,IL-7,Cytokines and cell-cell contacts at each stage of differentiation are different,Bone marrow stromal cell,Maturing B cells,B,B,Stromal cell,2,、,B,细胞成熟的分期,分期,重链,轻链,表面标志,祖,B,细胞,(pro-B),（重链基因重排）,胞质,前,B,细胞,(pre-B),（轻链基因重排）,替代轻链,类,Ig,未成熟,B,细胞,(immature-B),（轻链基因重排、阴性选择）,或,IgM,成熟,B,细胞,(mature-B),+,或,IgM,IgD,四阶段：,祖,B,细胞,前,B,细胞,未成熟,B,细胞,成熟,B,细胞,主要事件：,Ig,基因重排,阴性选择,3,、阴性选择,（,negative selection,）,发生于未成熟,B,细胞,若,mIgM,与基质细胞上的多价自身抗原结合，发生交联而致未成熟,B,细胞,凋亡,（,apoptosis,）,若,mIgM,与单价可溶性自身抗原结合，引起自身反应性,B,细胞,无能,(anergy),通过阴性选择的未成熟,B,细胞继续发育为,成熟,B,细胞,（表达,mIgM,和,mIgD,）,Negative selection-Apoptosis,Immature,B cell recognises,MULTIVALENT,self Ag,B,Clonal deletion by apoptosis,Y,Y,B,Immature,B,Y,B,Y,Y,Y,B,Anergic B cell,IgD normal IgM low,Immature,B cell recognises,soluble self Ag,No cross-linking,Y,Y,B,Immature,B,IgM,IgD,IgD,IgD,Negative selection-Anergy,Y,Y,Y,Y,Y,Y,Mature B cell,exported to the,periphery,Y,Y,IgD and IgM normal,IgM,IgD,IgD,IgD,IgD,IgM,IgM,IgM,Immature,B cell doesnt recognise any,self Ag,Y,Y,B,Immature,B,B,B,Negative selection-Mature B cells,Binding to self molecules in the bone marrow can lead to the death or inactivation of immature B cells,4,、,Ig,基因重排,（,gene rearrangement,）,Ig,的基因结构,(,胚系基因,),链基因,-2,号染色体,链基因,-22,号染色体,H,链基因,-14,号染色体,轻链基因分为,V,、,J,、,C,三组基因片段,重链基因分为,V,、,D,、,J,、,C,四组基因片段,无功能，不表达,The numbers of functional gene segments for the V regions of human heavy and light chains,Ig,的基因重排（,Ig gene rearrangement,）,B,细胞成熟过程中，,Ig,基因片段经重排后方能表达为功能性的,Ig,重链基因重排,轻链基因重排,等位排斥和同种型排斥,V,D,J,C,D,J,V,C,D,J,V,C,D,J,V,Germline,D,H,-J,H,joining,V,H,-D,H,J,H,joining,V,J,C,V,C,J,V,Germline,V,L,-J,L,joining,Heavy chain rearrangement,C,V,D,J,V,H,D,H,J,H,-C,H,joining,V,C,J,V,L,J,L,-C,L,joining,Light chain rearrangement,DJ,编码重链,VDJ,(V,基因,),VDJC,Splicing of IgM and IgD RNA,C,m,1,C,m,2,C,m,3,C,m,4,C,d,1,C,d,2,C,d,3,pA1,V,D,J,C,m,1,C,m,2,C,m,3,C,m,4,C,d,1,C,d,2,C,d,3,V,D,J,AAA,RNA cleaved and polyadenylated at pA2,V,D,J,C,m,IgM mRNA,RNA cleaved and polyadenylated at pA1,AAA,V,D,J,C,d,IgD mRNA,C,g,1,C,g,3,C,d,C,m,V,D,J,C,m,1,C,m,2,C,m,3,C,m,4,C,d,1,C,d,2,C,d,3,DNA,pA1,pA2,V,D,J,Two types of mRNA can be made simultaneously in the cell by differential usage of alternative polyadenylation sites and splicing of the RNA,VJ(V,基因,),VJC,编码轻链,等位排斥（,allelic exclusion,）,Ig,基因重排时，一对同源染色体上的两个等位基因中只有一个能表达,使得一个,B,细胞只能表达一种轻链和一种重链,同种型排斥（,isotypic exclusion,）,一个,B,细胞中，,链基因和,链基因只有其中的一种能够表达,使得一个,B,细胞只能表达,或,链,中的一种,Ig,基因重排机制,Ig,基因重排机制,重组识别信号序列和重组酶,重组酶,RAG1,和,RAG2,识别信号序列,RSS,12/23,原则与,VDJ,基因片段重组,V,7,23,9,J,7,12,9,D,H,7,12,9,7,12,9,V,H,7,23,9,J,H,7,23,9,RSS,间隔序列相同的基因片段之间不能发生重组,只有,RSS,间隔序列不同的基因片段之间能发生重组,12merRSS,间隔序列的基因片段与,23merRSS,间隔序列的基因片段才有可能发生重组,Recognition signal sequences(RSS)and,12/23 Rule,V,7,23,9,D,7,12,9,J,V,7,23,9,7,23,9,7,12,9,D,7,12,9,J,7,23,9,7,12,9,V,D,J,RAG1&RAG 2 and high mobility group proteins bind to the RSS,The two RAG1/RAG 2 complexes bind to each other and bring the V region adjacent to the DJ region,The recombinase complex makes single stranded nicks in the DNA,The nicks are sealed to form a hairpin structure at the end of the V and D regions,The recombinase complex remains associated with the break,Steps of Ig gene recombination,V,D,J,7,23,9,7,12,9,A number of other proteins,(Ku70:Ku80,XRCC4 and DNA dependent protein kinases)bind to the hairpins and the heptamer ends,.,V,D,J,The hairpins at the end of the V and D regions are opened,and exonucleases and transferases remove or add random nucleotides to the gap between the V and D region,V,D,J,7,23,9,7,12,9,DNA ligase IV joins the ends of the V and D region to form the coding joint and the two heptamers to form the signal joint.,Steps of Ig gene recombination,Ig,的表达（成熟,B,）,重链与轻链的表达,链和,链,链或,链,重链与轻链的装配,链和,链分别与轻链配对，形成,IgM,和,IgD,（,BCR,）,特异性相同，恒定区不同,Bursectomy no apparent effect,成熟B细胞在外周遇到抗原，产生免疫应答，最终分化成为浆细胞和记忆细胞,Transfer foetal,A number of other proteins,(Ku70:Ku80,XRCC4 and DNA dependent protein kinases)bind to the hairpins and the heptamer ends.,重组酶RAG1和RAG2,Heavy chain rearrangement,DH-JH joining,Mature peripheral,祖T细胞（pro T cells）,链基因-2号染色体,Steps of Ig gene recombination,B cell doesnt recognise any,class switching,Steps of Ig gene recombination,胸腺内发育中的T细胞,IgD and IgM normal,Early pro-B,二、骨髓外成熟过程,成熟,B,细胞在外周遇到抗原，产生免疫应答，最终分化成为浆细胞和记忆细胞,两个主要变化,:,体细胞高频突变,Ig,转类（同种型转换）,B cell recognises,non-self antigen,in periphery,Ig-secreting plasma cell,Differentiation in the periphery,Y,Y,Y,Y,Y,Y,Y,Y,Y,B,Y,Y,Y,Y,Y,Y,Y,B,Y,Y,Y,Y,Y,Y,Y,B,Mature peripheral,B cell,Y,Y,Y,Y,Y,Y,Y,B,memory cell,1,、体细胞高频突变,(somatic hypermutation),初次免疫应答后期和再次免疫应答,生发中心活化增殖的,B,细胞,轻链和重链,V,基因发生随机点突变,抗原选择,表达高亲和力,Ig,的,B,细胞被选择存活,表达低亲和力,Ig,的,B,细胞凋亡,抗体亲和力提高,抗体亲和力成熟（,affinity maturation,）,B,B,B,B,B,B,B,B,Mutation,B,B,B,B,B,2,、,重链转类,(class switching),Ig,转类（,class switching,）,同种型转换（,isotype switching,）,B,细胞受抗原刺激后，最先产生,IgM,，然后再转成产生其它类别的,Ig,，这种现象称为,Ig,转类,机制：转换重组，即,VDJ,基因片段与下游的另一个,C,基因重组,特点：,Ig Fab,不变，抗体特异性不变,意义：,Ig Fc,的改变使抗体效应机制发生变化，以适应消灭病原菌的需要,class switching,C,a,2,C,e,C,g,4,C,g,2,C,a,1,C,g,1,C,g,3,C,d,C,m,C,m,C,d,C,g,3,VDJ,S,g,3,C,m,C,d,C,g,3,VDJ,C,g,1,S,g,1,C,a,1,C,g,3,VDJ,C,a,1,C,g,3,VDJ,IgG3 produced.,Switch from IgM,VDJ,C,a,1,IgA1 produced.,Switch from IgG3,VDJ,C,a,1,IgA1 produced.,Switch from IgM,三、,Ig,多样性产生机制,多个,V,（,D,）,J,基因片段,重组多样性（,VDJ,、,VJ,随机重组）,连接的多样性（连接的不精确性、,N,区多样性）,N,区插入,Ig,重链基因重排时，在,V-D,之间和,D-J,之间额外加入新的核苷酸，这些核苷酸序列不是依赖模板而加入的，称为非模板区,H,、,L,链的随机组合,体细胞高频突变,Junctional DiversityN nucleotide additions,V,TC,GA,AG,D,J,AT,TA,TA,Terminal deoxynucleotidyl transferase(TdT)adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNA,CACTCCTTA,TTCTTGCAA,V,TC,GA,AG,D,J,AT,TA,TA,CACACCTTA,TTCT,T,GCAA,Complementary bases anneal,V,D,J,DNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strands,TC,GA,AG,AT,TA,TA,CACACCTTA,TTCT,T,GCAA,D,J,TA,TA,Exonucleases nibble back free ends,V,TC,GA,CACACCTTA,TTCT,T,GCAA,V,TC,D,TA,GTT,AT,AT,AG,C,第一节,T,细胞成熟过程,胸腺微环境与,T,细胞成熟分期,T,细胞胸腺内成熟过程,T,淋巴细胞因在胸腺中分化成熟，所以称为胸腺依赖性淋巴细胞（,thymus-dependent lymphocytes,），简称,T,细胞,起源：,胚胎期：卵黄囊、肝脏,成人：骨髓,发育,:,胸腺,胸腺微环境：,哺育细胞、胸腺上皮细胞、,DC,、,M,胸腺激素、细胞因子,胸腺细胞（,thymocytes,）,胸腺内发育中的,T,细胞,一、胸腺微环境与,T,细胞成熟分期,骨髓,胸腺,皮质浅层,皮质深层,髓质,T,细胞成熟分期,T,细胞胸腺内成熟四阶段：,祖,T,细胞（,pro T cells,）,前,T,细胞（,pre T cells,）,未成熟,T,细胞（,immature T cells,）,成熟,T,细胞（,mature T cells,）,T,细胞成熟分期,分期,TCR,CD4/CD8,CD3,祖,T,细胞,（,Pro T cell,）,TCR,CD4-CD8-,DN,CD3-,前,T,细胞,（,Pre T cell,）,TCR pT,:,CD4+CD8+,DP,CD3+,未成熟,T,细胞,（,Immature T cell,）,TCR,CD4+CD8+,DP,CD3+,成熟,T,细胞,（,Mature T cell,）,TCR,CD4+/CD8+,SP,CD3+,胸腺细胞三阶段,:,DN,（,double negative cells,）：,CD4-CD8-,DP,（,double positive cells,）：,CD4+CD8+,SP,（,single positive cells,）：,CD4+/CD8,+,二、,T,细胞在胸腺内成熟过程,三事件：,TCR,基因重排,阳性选择,阴性选择,1、骨髓微环境和B细胞成熟,VL-JL joining,Origin of B cells and organ of B cell maturation,成熟B细胞在外周遇到抗原，产生免疫应答，最终分化成为浆细胞和记忆细胞,重组识别信号序列和重组酶,Ig-secreting plasma cell,Early pro-B,B cell recognises,IgD and IgM normal,祖B细胞(pro-B),未成熟T细胞（Immature T cell）,A number of other proteins,(Ku70:Ku80,XRCC4 and DNA dependent protein kinases)bind to the hairpins and the heptamer ends.,1,、,TCR,受体基因重排,DN,DP,阶段,（祖,T,细胞、前,T,细胞、未成熟,T,细胞）,TCR,基因重排,pT,/CD3,CD4;CD8,TCR,基因重排,TCR/CD3,CD4;CD8,2,、阳性选择,（,positive selection,）,DPSP,（未成熟,T,细胞）,凡所表达,TCR,能够识别结合胸腺上皮细胞上自身,MHC,分子的胸腺细胞被选择而存活,凡所表达,TCR,不能识别结合胸腺上皮细胞上自身,MHC,分子的胸腺细胞则自行凋亡,识别结合,MHC,类分子的胸腺细胞，发育为,CD8+T,细胞,识别结合,MHC,类分子的胸腺细胞，发育为,CD4+T,细胞,结果：,获得自身,MHC,限制性,CD8+T,细胞具有识别,MHC,类分子复合物的能力,CD4+T,细胞具有识别,MHC,类分子复合物的能力,谢谢观看,谢谢观看,