1、细胞与分子免疫学课程课件细胞与分子免疫学课程课件（优选）细胞与分子免疫学课程课件3Chapter 7 Lymphocyte Maturation and Expression of Antigen Receptor Genes4nGeneral Features of Lymphocyte Maturation The maturation of B and T lymphocytes consists of:1.Lineage commitment and proliferation2.Expression of antigen receptor genes3.Selection of th
2、e mature repertories5p Early maturation:1.Pluripotent stem cells give rise to all lineages of blood cells1)It is difficult to precisely define the mechanisms by which stem cells become to the lymphoid lineage.2)B cell:bone borrow T cell:thymus3)By the gene mutations in experimental animals or human
3、patients.i.Transcription factors ii.Growth factors iii.Growth factors receptor6Example 1Bubble boy disease 1.Mutated in IL-7 gene and IL-7 receptor gene have profound deficiencies in mature T and B cells.2.Mutations in a chain of the IL-2 receptor g g chain,leads to an immunodeficiency disease:X-lin
4、ked severe combined immunodeficiency disease,bubble boy.7p Antigen Receptor Gene Recombination and Expression1.There may be 107 or more different T and B lymphocyte clones in one individual,each clone produce one specific antigen receptor.2.By somatic recombination,each individual does not need to h
5、ave such enormously genes.3.During the pre-B cell or pre-T cell stage,an immature form of antigen receptor is formed which to transduce signals to induce further maturation4.In more mature lymphocytes,complete antigen receptor expressed,which promote cell maturation.8pSelection Process That Shape th
6、e B and T Lymphocyte Repertoires1.The preservation of useful specificities is called positive selection:T cells in thymus.B cells maturing are not well known.2.Negative selection is the process that eliminates developing lymphocytes with strong binding to self-antigen.9pAcquisition of Functional Com
7、petence1.B cells can secrete antibodies.2.T cells can differentiate into distinct subsets of T cells.3.Expression of a variety of cell surface and intracellular molecules that participate in lymphocyte activation and effector functions.10n Formation of Functional Antigen Receptor Genes in B and T Ly
8、mphocytesElucidation of the mechanisms of antigen receptor gene expression is one of the landmark achievements of mordern immunology1 胚系理论胚系理论(Germline Theory)在胚系基因组中包含了免疫基因库，可对外界的众多抗在胚系基因组中包含了免疫基因库，可对外界的众多抗原发生应答，并能遗传。原发生应答，并能遗传。What are the problems with this theory?2 体细胞突变理论（体细胞突变理论（Somatic Mutati
9、on Model）基因组中的免疫球蛋白基因相对较少，抗体的多样性主基因组中的免疫球蛋白基因相对较少，抗体的多样性主要是由体细胞基因突变引起的。要是由体细胞基因突变引起的。What are the problems with this theory?11胚系理论与体细胞突变理论所遇到的问题胚系理论与体细胞突变理论所遇到的问题1 胚系理论胚系理论1）基因的种类少于抗体的多样性）基因的种类少于抗体的多样性So many specificities so few genes2 体细胞突变理论（体细胞突变理论（Somatic Mutation Model）1）部分编码）部分编码V区基因有众多的变化但部分
10、编码区基因有众多的变化但部分编码C区的区的基因却相对稳定。基因却相对稳定。2）不同类的同型的免疫球蛋白具有相同的）不同类的同型的免疫球蛋白具有相同的V区。区。Same variable regions on different isotypes123 Dreyer Bennett 假说：假说：免疫球蛋白单一的肽链是由两个分隔的基因编码：免疫球蛋白单一的肽链是由两个分隔的基因编码：每个免疫球蛋白类基因可能只有单个每个免疫球蛋白类基因可能只有单个C区基因，在胚区基因，在胚系基因组中与系基因组中与V区基因是分隔开的。区基因是分隔开的。在抗体产生细胞的发育过程中，其中一个独立的在抗体产生细胞的发育过程
11、中，其中一个独立的V区区基因序列将与基因序列将与C区序列结合成为完整的区序列结合成为完整的VC基因，然基因，然后在细胞内表达。后在细胞内表达。两个基因两个基因 一条多肽链：一条多肽链：此模型可以解释如下问题：此模型可以解释如下问题：1）一个基因的部分片断是多变的，而另一部分相对）一个基因的部分片断是多变的，而另一部分相对不变。不变。2）一个）一个V区可以与不同类的区可以与不同类的C区结合，产生出不同类区结合，产生出不同类型的抗体。型的抗体。134 The genetic foundation of antibody diversityThe Nobel Prize in Physiology
12、or Medicine 1987for his discovery of the genetic principle for generation of antibody diversity“in1976.Susumu Tonegawa Japan Massachusetts Institute of Technology(MIT)Cambridge,MA,USA b.193914Proof of the Dreyer-Bennett hypothesisVVVVVVVVVVVVVA mechanism to rearrange V and C genes in the genome so t
13、hat they can fuse to form a complete Immunoglobulin gene.CVCA single C region gene encoded in the germline and separate from the multiple V region genesFind a way to show the existence of multiple V genes and rearrangement to the C gene15Proof of the Dreyer-Bennett hypothesisVVVCVVVVVVSize fractiona
14、te by gel electrophoresisCVVVVVVVVVCVVVVVVVVVCut germline DNA with restriction enzymesVVVVVVVVVCA range of fragment sizes is generatedBlot with a V region probeBlot with a C region probeThe following example describes events on only ONE of the chromosomesC:two C genes in each of the human TCR b and
15、TCR g chain,one C gene in each of TCR a,d chain.The thymus is the major site of maturation of T cells.Tolerance induced in immature lymphocytes by recognition of self antigen in the generative lymphoid organs is also called central tolerance.Vb-to-DJb rearrangements occur在抗体产生细胞的发育过程中，其中一个独立的V区基因序列将
16、与C区序列结合成为完整的VC基因，然后在细胞内表达。The stimuli come from cells including:Maturation of T lymphocytes3)RAG proteins are first expressedSo many specificities so few genesmembrane-bound antigen receptor is not expressed;In three separate loci:在抗体产生细胞的发育过程中，其中一个独立的V区基因序列将与C区序列结合成为完整的VC基因，然后在细胞内表达。Mature B cellIn
17、fluence the specificities of the T cells.AT GTGACAC2 体细胞突变理论（Somatic Mutation Model）其中P碱基与N碱基的加入是导致抗体出现多样性的主要原因：RAG-1 and RAG-2 are first expressed.Organization of Ig Gene LociElucidation of the mechanisms of antigen receptor gene expression is one of the landmark achievements of mordern immunology1
18、6CVVVVVCVVVVVSize fractionate by gel electrophoresisVVVVCVBlot with a V region probeBlot with a C region probeCut mature B cell DNA with restriction enzymesVVVBlot with a V region probeBlot with a C region probeCVVVVVVSize fractionate by gel electrophoresis-compare the pattern of bandswith germline
19、DNAV and C probes detect the same fragmentSome V regions are missingThe C fragment has got largerVVVVCVEvidence for gene recombination17Organization of Ig and TCR Genes in the Germline1.Organization of Ig Gene Loci1)Three separate loci encode the Ig chains.chromosome 14:H chain locus chromosome 2:k
20、k chain chromosome 22:l l chain2)Multiple copies of at least three different types of gene segments:V:300 base,separate by noncoding DNA,at the 5 end of each V region is a nucleotide sequence which is called leader peptide.18C,each Ig locus has a distinct arrangement and number of C gene.In human:Ig
21、 k k light chain:a single C geneIg l l light chain:four functional C genesIg H heavy chain:nine different Ig isotypes and subtypes.J segments,30-50 base pairs long.D segments,in the human Ig heavy chain locus.1.Organization of Ig Gene Loci192.Organization of TCR Gene LociIn three separate loci:TCR a
22、,da,d chain:chromosome 14TCR b b chain:chromosome 7TCR g g chain:chromosome 7V:like Ig V geneC:two C genes in each of the human TCR b b and TCR g g chain,one C gene in each of TCR a,da,d chain.J:in all TCR loci,between V and C genes.D:in TCR b b chain and d d chain.20Antigen Receptor Gene Recombinat
23、ionDiversity of antigen receptor genes21Mechanisms of Somatic Recombination of Anitgen Receptor Genes1.有限的有限的DNA是怎样产生无限的专一是怎样产生无限的专一 性的性的?2.V区是怎样找到区是怎样找到J区的？为什么区的？为什么V区不与区不与V区相连？区相连？3.DNA是怎样断裂的？是怎样断裂的？4.DNA是怎样重新组合的？是怎样重新组合的？22RSS(重排信号序列重排信号序列)，recombinase(重组酶重组酶)，12-23 rule(一个一个规则规则)。1、Recombination
24、 Signal Sequences(RSS),重排信号序列：重排信号序列：v基因片断两端存在两个基因片断两端存在两个特异的保守序列：特异的保守序列：7聚体聚体与与9聚体。聚体。v 7聚体与聚体与9聚体之间间隔聚体之间间隔是是23bp或者是或者是12bp。vRSS:7聚体聚体-间隔间隔-九聚九聚体体(The heptamer The heptamer spacer spacer nonamer)nonamer)Mechanisms of Somatic Recombination of Anitgen Receptor Genes23 RSS（重排信号序列（重排信号序列）242、重组酶：、重组酶
25、：一组参与一组参与V、（、（D）、）、J基因片断重组的酶，包括以下几种：基因片断重组的酶，包括以下几种：RAG-1与与RAG-2(recombination-activating genes):一种内切酶，一种内切酶，只表达在只表达在T和和B淋巴细胞淋巴细胞不成熟阶段。不成熟阶段。末端脱氧核苷酸转移酶末端脱氧核苷酸转移酶(terminal deoxynucleotidyl transferase,TdT)：表达于：表达于T、B细胞前体，此酶可将数个核苷酸通过不细胞前体，此酶可将数个核苷酸通过不需要模板的方式加到需要模板的方式加到DNA的断段。的断段。其他其他 切开发夹结构的内切酶，参与修复切开
26、发夹结构的内切酶，参与修复DNA双链断段的双链断段的DNA外切酶、外切酶、DNA合成酶等，如合成酶等，如DNA连接酶连接酶IV，DNA依赖的蛋白依赖的蛋白激酶。激酶。25 重组酶所催化的重排步骤1.RAG 蛋白复合物结合蛋白复合物结合到到RSS。2.蛋白复合物拉近将要蛋白复合物拉近将要相连的相连的DNA片断。片断。3.互补链切断，发夹结互补链切断，发夹结构形成。构形成。4.其他其他DNA修饰蛋白结修饰蛋白结合到发夹结构剪切合到发夹结构剪切RSS末端。末端。5.发夹结构被随机剪切，发夹结构被随机剪切，碱基或者增加或者减碱基或者增加或者减少。少。6.DNA连接酶连接产生连接酶连接产生编码连接点和信
27、号连编码连接点和信号连接。接。2623-mer=two turns12-mer=one turn3、Molecular explanation of the 12-23 ruleIntervening DNAof any length23V9712D J792723-mer12-merLoop of interveningDNA is excised The shape generated by the RSSs acts as a target for recombinases7997V1V2V3V4V8V7V6V5V9D JV1D JV2V3V4V8V7V6V5V9 An appropri
28、ate shape can not be formed if two 23-mer flanked elements attempted to join(i.e.the 12-23 rule)3.Molecular explanation of the 12-23 rule28This rule can explain why in heavy chain,the V can not directly bind to J.AGCTGCAATATA7聚体与9聚体之间间隔是23bp或者是12bp。T cells in thymus.Ig l light chain:four functional
29、C genesThe nicked strand flips outCytokines:thymic stromal cells including epithelial cells,Stimulate the proliferation of immature T cells,especially IL-7.The stimuli come from cells including:The nucleotides that flip out,become part of the complementary DNA strandThe pre-T cell receptor was forme
30、d.TA CACTGTG-compare the pattern of bandsRecombination activating gene products,(RAG1&RAG 2)and high mobility group proteins bind to the RSSIn terms of G to C and T to A pairing,the new nucleotides are palindromic.Thymocytes can not recognize self MHC-restricted will die by apoptosis.Maturation of T
31、 lymphocytes蛋白复合物拉近将要相连的DNA片断。5)Ig a and Ig b of BCR complex starts to express.一组参与V、（D）、J基因片断重组的酶，包括以下几种：Find a way to show the existence of multiple V genes and rearrangement to the C geneGrowth factors receptorTCR a gene recombination commences.29V7239D7129JV723972397129D7129J72397129VDJRecombina
32、tion activating gene products,(RAG1&RAG 2)and high mobility group proteins bind to the RSSThe two RAG1/RAG 2 complexes bind to each other and bring the V region adjacent to the DJ region 互补链断裂，发夹结构形成互补链断裂，发夹结构形成4.Steps of Ig gene recombination30VDJ72397129A number of other proteins,(Ku70:Ku80,XRCC4
33、and DNA dependent protein kinases)bind to the hairpins and the heptamer ends.VDJThe 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 regionVDJ7 2397129DNA ligase IV joins the ends of the V and D re
34、gion to form the coding joint and the two heptamers to form the signal joint.4.Steps of Ig gene recombination31V1V2V3V4V9D JLooping out works if all V genes are in the same transcriptional orientationV1V2V3V9D J缺失性重排缺失性重排D J7129V47239V17239D7129JHow does recombination occur when a V gene is in oppos
35、ite orientation to the DJ region?V45.重排方式以重链重排为例非非缺失性重排缺失性重排LL32D J7129V47239V4 and DJ in opposite transcriptional orientationsD J7129V472391.D J7129V472393.D J7129V472392.D J7129V472394.Non-deletional recombination33D J7129V472391.D JV4712972393.V to DJ ligation-coding joint formationD J7129V472392
36、.Heptamer ligation-signal joint formationD JV471297239Fully recombined VDJ regions in same transcriptional orientationNo DNA is deleted4.347D129J6.Generation of Diversity of the B and T Cell Repertoires Junctional diversity:P nucleotide additions7V239D7129JV7239TC CACAGTGAG GTGTCACAT GTGACACTA CACTG
37、TGThe recombinase complex makes single stranded nicks at random sites close to the ends of the V and D region DNA.7D129J7V239CACAGTGGTGTCACGTGACACCACTGTGTCAGATTADJVTCAGATTAUUThe 2nd strand is cleaved and hairpins form between the complementary bases at ends of the V and D region.35V2V3V4V8V7V6V5V972
38、39CACAGTGGTGTCAC7129GTGACACCACTGTGVTCAGUDJATTAUHeptamers are ligated by DNA ligase IVV and D regions juxtaposedVTCAGUD JATTAU36VTCAGUD JATTAUEndonuclease cleaves single strand at random sites in V and D segmentVTCGAAGD JATTATAThe nucleotides that flip out,become part of the complementary DNA strand6
39、.1 Generation of the palindromic sequenceIn terms of G to C and T to A pairing,the new nucleotides are palindromic.The nucleotides GA and TA were not in the genomic sequence and introduce diversity of sequence at the V to D join.VTCAGUD JATTAURegions to be joined are juxtaposedThe nicked strand flip
40、s out 376.2 Junctional Diversity N nucleotide additionsVTCGAAGD JATTATATerminal deoxynucleotidyl transferase(TdT)adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNACACTCCTTATTCTTGCAAVTCGAAGD JATTATACACACCTTATTCTTGCAAComplementary bases annealVD JDNA polymera
41、ses fill in the gaps with complementary nucleotides and DNA ligase IV joins the strandsTCGAAGATTATACACACCTTATTCTTGCAAD JTATAExonucleases nibble back free endsVTCGACACACCTTATTCTTGCAAVTCDTAGTT AT ATAG C38VDJTCGACGTTATATAGCTGCAATATAJunctional DiversityNNNNNNNNNNNNNNNGermline-encoded nucleotidesPalindro
42、mic(P)nucleotides-not in the germlineNon-template(N)encoded nucleotides-not in the germlineCreates an essentially random sequence between the V region,D region and J region in heavy chains and the V region and J region in light chains.39Junctional DiversityIg k light chain:a single C geneFor the non
43、productive one,the second allelic chromosomes can recombine.Immature B cellThe coexpression of IgM and IgD make the B cell acquire the functional competence.Maturation of T lymphocytesMHC molecules in thymus are important.V and C probes detect the same fragmentThymus donorImmature B cell蛋白复合物拉近将要相连的
44、DNA片断。Mature B cell1）部分编码V区基因有众多的变化但部分编码C区的基因却相对稳定。5)Ig a and Ig b of BCR complex starts to express.In terms of G to C and T to A pairing,the new nucleotides are palindromic.Maturation of T lymphocytesAT GTGACACRecombination activating gene products,(RAG1&RAG 2)and high mobility group proteins bind
45、to the RSS3)RAG proteins are first expressed40Maturation of B Lymphocytes1.Pro-B cell 2.Pre-B cell3.Immature B cell4.Mature B cell41Maturation of B Lymphocytes42Stages of B Lymphocyte Maturation:1.Pro-B cell:1)not produce Ig 2)Maker:CD19 and CD10 3)RAG proteins are first expressed 4)TdT enzyme is ex
46、pressed most abundantly 5)Ig a a and Ig b b of BCR complex starts to express.6)First recombination of Ig genes occurs in heavy chains43Recombination in Pro-B cell:442.Pre-B cell A primary transcript that includes the rearranged VDJ complex and the proximal C genes is produced;Functional mRNA for the
47、 m m heavy chain is produced;m m protein in pre-B cells are translated;membrane-bound antigen receptor is not expressed;Pre-B cell receptors are expressed on the cell surface at low levels.Only in hematopoietic tissues;45468)The pre-BCR regulates further somatic recombination of Ig genes in two ways
48、:8.1)For the productive rearrangements:allelic exclusion exsits.For the nonproductive one,the second allelic chromosomes can recombine.47刘老师：刘老师：您好！上节课讲到内切酶在您好！上节课讲到内切酶在V和和D上随机切开增加了上随机切开增加了VD间连接间连接的多样性。那么这个区域将来编码成蛋白质即抗体后，并不位的多样性。那么这个区域将来编码成蛋白质即抗体后，并不位于于V片段的片段的CDR区，如何能够增加抗体的多态性？区，如何能够增加抗体的多态性？还有一个问题是
49、还有一个问题是TdT随机增加几个碱基从而增加了基因的多态随机增加几个碱基从而增加了基因的多态性，那么在重排机制中如何保证随机增加碱基后使得重排的基性，那么在重排机制中如何保证随机增加碱基后使得重排的基因的碱基数依旧是因的碱基数依旧是3的倍数而不发生编码错误？如果编码的不是的倍数而不发生编码错误？如果编码的不是3的倍数，是否是在转录水平上随机添加碱基的倍数，是否是在转录水平上随机添加碱基U来使编码恢复正来使编码恢复正常。常。谢谢老师！来自崔雪晶谢谢老师！来自崔雪晶48C,each Ig locus has a distinct arrangement and number of C gene.I
50、n human:Ig k k light chain:a single C geneIg l l light chain:four functional C genesIg H heavy chain:nine different Ig isotypes and subtypes.J segments,30-50 base pairs long.D segments,in the human Ig heavy chain locus.1.Organization of Ig Gene Loci492.Organization of TCR Gene LociIn three separate
