細胞與分子免疫學課程課件示范課件

1、細胞與分子免疫學課程課件細胞與分子免疫學課程課件（優(yōu)選）細胞與分子免疫學課程課件（優(yōu)選）細胞與分子免疫學課程課件3Chapter 7 Lymphocyte Maturation and Expression of Antigen Receptor Genes3Chapter 7 4General Features of Lymphocyte Maturation The maturation of B and T lymphocytes consists of :Lineage commitment and proliferationExpression of antigen receptor

2、 genesSelection of the mature repertories4General Features of Lymphocyt5 Early maturation:Pluripotent stem cells give rise to all lineages of blood cellsIt is difficult to precisely define the mechanisms by which stem cells become to the lymphoid lineage. B cell: bone borrow T cell: thymus3) By the

3、gene mutations in experimental animals or human patients. i. Transcription factors ii. Growth factors iii. Growth factors receptor5 Early maturation:Pluripotent6Example 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 i

4、n a chain of the IL-2 receptor g chain, leads to an immunodeficiency disease: X-linked severe combined immunodeficiency disease, bubble boy.6Example 1Bubble boy disease 17 Antigen Receptor Gene Recombination and ExpressionThere may be 107 or more different T and B lymphocyte clones in one individual

5、, each clone produce one specific antigen receptor.By somatic recombination, each individual does not need to have such enormously genes.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 maturationIn more mature lym

6、phocytes, complete antigen receptor expressed, which promote cell maturation.7 Antigen Receptor Gene Recomb8Selection Process That Shape the B and T Lymphocyte RepertoiresThe preservation of useful specificities is called positive selection: T cells in thymus. B cells maturing are not well known.2.

7、Negative selection is the process that eliminates developing lymphocytes with strong binding to self-antigen. 8Selection Process That Shape 9Acquisition of Functional CompetenceB cells can secrete antibodies.T cells can differentiate into distinct subsets of T cells.Expression of a variety of cell s

8、urface and intracellular molecules that participate in lymphocyte activation and effector functions.9Acquisition of Functional Com10 Formation of Functional Antigen Receptor Genes in B and T LymphocytesElucidation of the mechanisms of antigen receptor gene expression is one of the landmark achieveme

9、nts of mordern immunology1 胚系理論(Germline Theory)在胚系基因組中包含了免疫基因庫，可對外界的眾多抗原發(fā)生應答，并能遺傳。What are the problems with this theory?2 體細胞突變理論（Somatic Mutation Model）基因組中的免疫球蛋白基因相對較少，抗體的多樣性主要是由體細胞基因突變引起的。What are the problems with this theory?10 Formation of Functional Ant11胚系理論與體細胞突變理論所遇到的問題1 胚系理論1）基因的種類少于抗體的

10、多樣性So many specificities so few genes2 體細胞突變理論（Somatic Mutation Model） 1）部分編碼V區(qū)基因有眾多的變化但部分編碼C區(qū)的基因卻相對穩(wěn)定。2）不同類的同型的免疫球蛋白具有相同的V區(qū)。 Same variable regions on different isotypes11胚系理論與體細胞突變理論所遇到的問題1 胚系理論123 Dreyer Bennett 假說：免疫球蛋白單一的肽鏈是由兩個分隔的基因編碼：每個免疫球蛋白類基因可能只有單個C區(qū)基因，在胚系基因組中與V區(qū)基因是分隔開的。在抗體產(chǎn)生細胞的發(fā)育過程中，其中一個獨立的V

11、區(qū)基因序列將與C區(qū)序列結(jié)合成為完整的VC基因，然后在細胞內(nèi)表達。兩個基因 一條多肽鏈：此模型可以解釋如下問題：1）一個基因的部分片斷是多變的，而另一部分相對不變。2）一個V區(qū)可以與不同類的C區(qū)結(jié)合，產(chǎn)生出不同類型的抗體。123 Dreyer Bennett 假說：免疫球蛋白單134 The genetic foundation of antibody diversityThe Nobel Prize in Physiology or Medicine 1987for his discovery of the genetic principle for generation of antibod

12、y diversity“in1976.Susumu Tonegawa Japan Massachusetts Institute of Technology (MIT) Cambridge, MA, USA b. 1939134 The genetic foundation of 14Proof of the Dreyer - Bennett hypothesisVVVVVVVVVVVVVA mechanism to rearrange V and C genes in the genome so that they can fuse to form a complete Immunoglob

13、ulin 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 gene14Proof of the Dreyer - Bennet15Proof of the Dreyer - Bennett hypothesisVVVCVVVVVVSize fractionate by gel elect

14、rophoresisCVVVVVVVVVCVVVVVVVVVCut 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 chromosomes15Proof of the Dreyer - BennetC: two C genes in each of the

15、human TCR b and 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在抗體產(chǎn)生細胞的發(fā)育

16、過程中，其中一個獨立的V區(qū)基因序列將與C區(qū)序列結(jié)合成為完整的VC基因，然后在細胞內(nèi)表達。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:在抗體產(chǎn)生細胞的發(fā)育過程中，其中一個獨立的V區(qū)基因序列將與C區(qū)序列結(jié)合成為完整的VC基因，然后在細胞

17、內(nèi)表達。Mature B cellInfluence the specificities of the T cells.AT GTGACAC2 體細胞突變理論（Somatic Mutation Model）其中P堿基與N堿基的加入是導致抗體出現(xiàn)多樣性的主要原因：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

18、mordern immunology16CVVVVVCVVVVVSize 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

19、 bandswith germline DNAV and C probes detect the same fragmentSome V regions are missingThe C fragment has got largerVVVVCVEvidence for gene recombinationC: two C genes in each of the17Organization of Ig and TCR Genes in the Germline1.Organization of Ig Gene LociThree separate loci encode the Ig cha

20、ins. chromosome 14: H chain locus chromosome 2: k chain chromosome 22: 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.17Organization of Ig and

21、TCR G18C , each Ig locus has a distinct arrangement and number of C gene.In human:Ig k light chain: a single C geneIg 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.Or

22、ganization of Ig Gene Loci18C , each Ig locus has a dist192. Organization of TCR Gene LociIn three separate loci:TCR a, d chain: chromosome 14TCR b chain: chromosome 7TCR g chain: chromosome 7V: like Ig V geneC: two C genes in each of the human TCR b and TCR g chain, one C gene in each of TCR a, d c

23、hain.J: in all TCR loci, between V and C genes.D: in TCR b chain and d chain.192. Organization of TCR Gene 20Antigen Receptor Gene RecombinationDiversity of antigen receptor genes20Antigen Receptor Gene Recomb21Mechanisms of Somatic Recombination of Anitgen Receptor Genes有限的DNA是怎樣產(chǎn)生無限的專一 性的?V區(qū)是怎樣找到J

24、區(qū)的？為什么V區(qū)不與V區(qū)相連？DNA是怎樣斷裂的？DNA是怎樣重新組合的？21Mechanisms of Somatic Recomb22RSS(重排信號序列) ， recombinase(重組酶) ，12-23 rule(一個規(guī)則) 。1、 Recombination Signal Sequences (RSS), 重排信號序列：基因片斷兩端存在兩個特異的保守序列：7聚體與9聚體。 7聚體與9聚體之間間隔是23bp或者是12bp。RSS: 7聚體-間隔-九聚體(The heptamer spacer nonamer) Mechanisms of Somatic Recombination o

25、f Anitgen Receptor Genes22RSS(重排信號序列) ， recombinase(重組23 RSS（重排信號序列）23 RSS（重排信號序列）242、重組酶：一組參與V、（D）、J基因片斷重組的酶，包括以下幾種：RAG-1與RAG- 2 (recombination-activating genes): 一種內(nèi)切酶，只表達在T和B淋巴細胞不成熟階段。末端脫氧核苷酸轉(zhuǎn)移酶 (terminal deoxynucleotidyl transferase,TdT) ：表達于T、B細胞前體，此酶可將數(shù)個核苷酸通過不需要模板的方式加到DNA的斷段。其他 切開發(fā)夾結(jié)構(gòu)的內(nèi)切酶，參與修復

26、DNA雙鏈斷段的DNA外切酶、DNA合成酶等，如DNA連接酶IV，DNA依賴的蛋白激酶。242、重組酶：25 重組酶所催化的重排步驟RAG 蛋白復合物結(jié)合到RSS。蛋白復合物拉近將要相連的DNA片斷?；パa鏈切斷，發(fā)夾結(jié)構(gòu)形成。其他DNA修飾蛋白結(jié)合到發(fā)夾結(jié)構(gòu)剪切RSS末端。發(fā)夾結(jié)構(gòu)被隨機剪切，堿基或者增加或者減少。DNA連接酶連接產(chǎn)生編碼連接點和信號連接。25 重組酶所催化的重排步驟RAG 蛋白復合物結(jié)合到RSS。2623-mer = two turns12-mer = one turn3、Molecular explanation of the 12-23 ruleIntervening D

27、NAof any length23V9712DJ792623-mer = two turns12-mer = o2723-mer12-merLoop of interveningDNA is excisedThe shape generated by the RSSs acts as a target for recombinases7997V1V2V3V4V8V7V6V5V9DJV1DJV2V3V4V8V7V6V5V9An appropriate shape can not be formed if two 23-mer flanked elements attempted to join

28、(i.e. the 12-23 rule)3.Molecular explanation of the 12-23 rule2723-mer12-merLoop of interven28This rule can explain why in heavy chain, the V can not directly bind to J.28This rule can explain why inAGCTGCAATATA7聚體與9聚體之間間隔是23bp或者是12bp。T cells in thymus.Ig l light chain: four functional C genesThe ni

29、cked 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 formed.TA CACT

30、GTG- 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 ly

31、mphocytes蛋白復合物拉近將要相連的DNA片斷。5) Ig a and Ig b of BCR complex starts to express.一組參與V、（D）、J基因片斷重組的酶，包括以下幾種：Find a way to show the existence of multiple V genes and rearrangement to the C geneGrowth factors receptorTCR a gene recombination commences.29V7239D7129JV723972397129D7129J72397129VDJRecombinati

32、on 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互補鏈斷裂，發(fā)夾結(jié)構(gòu)形成4. Steps of Ig gene recombinationAGCTGCAATATA29V7239D7129JV723930VDJ72397129A number of other protei

33、ns, (Ku70:Ku80, XRCC4 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 regionVDJ72397129DNA ligase IV joins th

34、e ends of the V and D region to form the coding joint and the two heptamers to form the signal joint.4. Steps of Ig gene recombination30VDJ72397129A number of other31V1V2V3V4V9DJLooping out works if all V genes are in the same transcriptional orientationV1V2V3V9DJ缺失性重排DJ7129V47239V17239D7129JHow doe

35、s recombination occur when a V gene is in opposite orientation to the DJ region?V45.重排方式以重鏈重排為例非缺失性重排LL31V1V2V3V4V9DJLooping out work32DJ7129V47239V4 and DJ in opposite transcriptional orientationsDJ7129V472391.DJ7129V472393.DJ7129V472392.DJ7129V472394.Non-deletional recombination32DJ7129V47239V4 an

36、d DJ in opp33DJ7129V472391.DJV4712972393.V to DJ ligation - coding joint formationDJ7129V472392.Heptamer ligation - signal joint formationDJV471297239Fully recombined VDJ regions in same transcriptional orientationNo DNA is deleted4.33DJ7129V472391.DJV4712972393.347D129J6.Generation of Diversity of

37、the B and T Cell Repertoires Junctional diversity: P nucleotide additions7V239D7129JV7239TC CACAGTGAG GTGTCACAT GTGACACTA CACTGTGThe recombinase complex makes single stranded nicks at random sites close to the ends of the V and D region DNA.7D129J7V239CACAGTGGTGTCACGTGACACCACTGTGTCAGATTADJVTCAGATTAU

38、UThe 2nd strand is cleaved and hairpins form between the complementary bases at ends of the V and D region.347D129J6.Generation of Divers35V2V3V4V8V7V6V5V97239CACAGTGGTGTCAC7129GTGACACCACTGTGVTCAGUDJATTAUHeptamers are ligated by DNA ligase IVV and D regions juxtaposedVTCAGUDJATTAU35V2V3V4V8V7V6V5V97

39、239CACAGTG736VTCAGUDJATTAUEndonuclease cleaves single strand at random sites in V and D segmentVTCGAAGDJATTATAThe nucleotides that flip out, become part of the complementary DNA strand6.1 Generation of the palindromic sequenceIn terms of G to C and T to A pairing, the new nucleotides are palindromic

40、.The nucleotides GA and TA were not in the genomic sequence and introduce diversity of sequence at the V to D join.VTCAGUDJATTAURegions to be joined are juxtaposedThe nicked strand flips out 36VTCUDJATUEndonuclease cleave376.2 Junctional DiversityN nucleotide additionsVTCGAAGDJATTATATerminal deoxynu

41、cleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNACACTCCTTATTCTTGCAAVTCGAAGDJATTATACACACCTTATTCTTGCAAComplementary bases annealVDJDNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strands

42、TCGAAGATTATACACACCTTATTCTTGCAADJTATAExonucleases nibble back free endsVTCGACACACCTTATTCTTGCAAVTCDTAGTT AT ATAG C376.2 Junctional DiversityN38VDJTCGACGTTATATAGCTGCAATATAJunctional DiversityNNNNNNNNNNNNNNNGermline-encoded nucleotidesPalindromic (P) nucleotides - not in the germlineNon-template (N) enc

43、oded 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.38VDJTCGACGTTATATAGCTGCAATATAJ39Junctional Diversity39Junctional DiversityIg k light chain: a single C geneFor the no

44、nproductive 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蛋白復合物拉近將要相

45、連的DNA片斷。Mature B cell1）部分編碼V區(qū)基因有眾多的變化但部分編碼C區(qū)的基因卻相對穩(wěn)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 protei

46、ns bind to the RSS3) RAG proteins are first expressed40Maturation of B Lymphocytes1.Pro- B cell 2.Pre- B cell3. Immature B cell4. Mature B cellIg k light chain: a single C g41Maturation of B Lymphocytes41Maturation of B Lymphocytes42Stages of B Lymphocyte Maturation:1.Pro- B cell : 1) not produce Ig

47、 2) Maker: CD19 and CD10 3) RAG proteins are first expressed 4) TdT enzyme is expressed most abundantly 5) Ig a and Ig b of BCR complex starts to express. 6)First recombination of Ig genes occurs in heavy chains42Stages of B Lymphocyte Matur43Recombination in Pro- B cell :43Recombination in Pro- B c

48、el442.Pre- B cellA primary transcript that includes the rearranged VDJ complex and the proximal C genes is produced;Functional mRNA for the m heavy chain is produced;m protein in pre-B cells are translated;membrane-bound antigen receptor is not expressed;Pre-B cell receptors are expressed on the cel

49、l surface at low levels. Only in hematopoietic tissues;442.Pre- B cellA primary trans4545468) The pre-BCR regulates further somatic recombination of Ig genes in two ways:8.1) For the productive rearrangements: allelic exclusion exsits. For the nonproductive one, the second allelic chromosomes can re

50、combine.468) The pre-BCR regulates fur47劉老師：您好！上節(jié)課講到內(nèi)切酶在V和D上隨機切開增加了VD間連接的多樣性。那么這個區(qū)域?qū)砭幋a成蛋白質(zhì)即抗體后，并不位于V片段的CDR區(qū)，如何能夠增加抗體的多態(tài)性？還有一個問題是TdT隨機增加幾個堿基從而增加了基因的多態(tài)性，那么在重排機制中如何保證隨機增加堿基后使得重排的基因的堿基數(shù)依舊是3的倍數(shù)而不發(fā)生編碼錯誤？如果編碼的不是3的倍數(shù)，是否是在轉(zhuǎn)錄水平上隨機添加堿基U來使編碼恢復正常。謝謝老師！來自崔雪晶47劉老師：48C , each Ig locus has a distinct arrangement an

51、d number of C gene.In human:Ig k light chain: a single C geneIg 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 Loci48C , each Ig locus has a di

52、st492. Organization of TCR Gene LociIn three separate loci:TCR a, d chain: chromosome 14TCR b chain: chromosome 7TCR g chain: chromosome 7V: like Ig V geneC: two C genes in each of the human TCR b and TCR g chain, one C gene in each of TCR a, d chain.J: in all TCR loci, between V and C genes.D: in T

53、CR b chain and d chain.492. Organization of TCR Gene 508) The pre-BCR regulates further somatic recombination of Ig genes in two ways:8.1) For the productive rearrangements: allelic exclusion exsits. For the nonproductive one, the second allelic chromosomes can recombine.508) The pre-BCR regulates f

54、ur51上堂課回顧：1 Ig與TCR基因在染色體基因座位上的排列特點2 重排機制 1）一個信號序列 2）一個原則 3）一組酶其中P堿基與N堿基的加入是導致抗體出現(xiàn)多樣性的主要原因：VDJTCGACGTTATATAGCTGCAATATAVTCAGUDJATTAU51上堂課回顧：VDJTCGACGTTATATAGCTGCA524）缺失性重排5）非缺失性重排3、B 細胞的成熟：祖B細胞，前B細胞，非成熟B細胞，成熟B細胞524）缺失性重排3、B 細胞的成熟：祖B細胞，前B細胞，非534、B細胞表面受體的重排：重鏈：等位基因排斥輕鏈：同種型的排斥534、B細胞表面受體的重排：54Pro-B cellP

55、re-B cell54Pro-B cellPre-B cell558.2) the pre-BCR regulates somatic recombination is by stimulating light chain gene rearrangement.3. Immature B cellk chain and l chain gene are rearranged.Ig M is expressed.In a similar manner as in the Ig heavy chain locus. k chain rearranges after heavy chain rear

56、rangement and before l chain.Light chain isotype exclusion.The assembled IgM molecules are expressed on the cell surface in association with Iga and Igb.Immature B cells do not proliferate and differentiate in response to antigens.558.2) the pre-BCR regulates s5656574. Mature B cellCoexpressed m and

57、 d heavy chains in association with the k and l light chain to produce IgM and IgD.The coexpression of IgM and IgD make the B cell acquire the functional competence.It suggests that IgD is the essential activating receptor of mature B cells.Can response to antigens.Without antigens, they will die in

58、 a few days or weeks.The majority of B cells are IgD+ IgM+. 574. Mature B cellCoexpressed 585859Selection of the Mature B cell RepertoireThe repertoire of mature B cells is positively selected from the pool of immature cells before leaving the bone borrow. is different from the T cells selection, pe

59、rhaps serve to preserve all the cells with the capacity to recognize antigens, regardless of specificity.59Selection of the Mature B ce602) Negative selection: Self antigens deliver strong signals to IgM expressing immature B lymphocytes that happen to express receptors specific for these self antig

60、ens.Antigen recognition leads to apoptotic death of immature B cells.Some immature B cells that recognize self antigens may be induced to change their specificities by a process called receptor editing.602) Negative selection: 61B-1 CellsB cells described to-date: B-2 cellsA second population of B c