中科院细胞与分子免疫学课程Chapter7

1,T 细胞表面的分子1 TCR 复合体： CD3分子的 g , e , d链， z 链， TCR a, b链， g , d链。2 辅助受体：CD4与CD8分子3 第二信号：CD2，CD28，CD152， B74 起黏附作用的分子：Integrin5 起游走作用的分子： SelectinTCR复合体与辅助受体组成T细胞激活的第一信号共刺激信号组成第二信号,体内识别抗原的分子：抗体、MHC分子、TCR分子,2,Section IIIMaturation, Activation, and Regulation of Lymphocytes,3,Chapter 7 Lymphocyte Maturation and Expression of Antigen Receptor Genes,4,General Features of Lymphocyte Maturation,The maturation of B and T lymphocytes consists of :Lineage commitment and proliferationExpression of antigen receptor genesSelection of the mature repertories,5,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 gene mutations in experimental animals or human patients. i. Transcription factors ii. Growth factors iii. Growth factors receptor,6,Example 1,Bubble 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 chain, leads to an immunodeficiency disease: X-linked severe combined immunodeficiency disease, bubble boy.,7,Antigen Receptor Gene Recombination and Expression,There may be 107 or more different T and B lymphocyte clones in one individual, 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 lymphocytes, complete antigen receptor expressed, which promote cell maturation.,8,Selection Process That Shape the B and T Lymphocyte Repertoires,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.,9,Acquisition of Functional Competence,B cells can secrete antibodies.T cells can differentiate into distinct subsets of T cells.Expression of a variety of cell surface and intracellular molecules that participate in lymphocyte activation and effector functions.,10,Formation of Functional Antigen Receptor Genes in B and T Lymphocytes,Elucidation of the mechanisms of antigen receptor gene expression is one of the landmark achievements of mordern immunology,1 胚系理论(Germline Theory)在胚系基因组中包含了免疫基因库，可对外界的众多抗原发生应答，并能遗传。What are the problems with this theory?2 体细胞突变理论（Somatic Mutation Model）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。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,1 胚系理论1）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（Somatic Mutation Model） 1）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。2）不同类的同型的免疫球蛋白具有相同的V区。 Same variable regions on different isotypes,12,3 Dreyer Bennett 假说：,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C区基因，在胚系基因组中与V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。,两个基因 一条多肽链：,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，而另一部分相对不变。2）一个V区可以与不同类的C区结合，产生出不同类型的抗体。,13,4 The genetic foundation of antibody diversity,The Nobel Prize in Physiology 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. 1939,14,Proof of the Dreyer - Bennett hypothesis,V,V,V,V,V,V,V,V,V,Find a way to show the existence of multiple V genes and rearrangement to the C gene,15,Proof of the Dreyer - Bennett hypothesis,Size fractionate by gel electrophoresis,Cut germline DNA with restriction enzymes,A range of fragment sizes is generated,Blot with a V region probe,Blot with a C region probe,The following example describes events on only ONE of the chromosomes,16,Size fractionate by gel electrophoresis,Blot with a V region probe,Blot with a C region probe,Cut mature B cell DNA with restriction enzymes,Blot with a V region probe,Blot with a C region probe,Size fractionate by gel electrophoresis,- compare the pattern of bandswith germline DNA,V and C probes detect the same fragmentSome V regions are missingThe C fragment has got larger,Evidence for gene recombination,17,Organization of Ig and TCR Genes in the Germline,1.Organization of Ig Gene Loci,Three separate loci encode the Ig chains. 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.,18,C , 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.Organization of Ig Gene Loci,19,2. Organization of TCR Gene Loci,In 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 TCR b chain and d chain.,20,Antigen Receptor Gene Recombination,Diversity of antigen receptor genes,21,Mechanisms of Somatic Recombination of Anitgen Receptor Genes,有限的DNA是怎样产生无限的专一 性的?V区是怎样找到J区的？为什么V区不与V区相连？DNA是怎样断裂的？DNA是怎样重新组合的？,22,RSS(重排信号序列) ， recombinase(重组酶) ，12-23 rule(一个规则) 。,1、 Recombination Signal Sequences (RSS), 重排信号序列：,基因片断两端存在两个特异的保守序列：7聚体与9聚体。 7聚体与9聚体之间间隔是23bp或者是12bp。RSS: 7聚体-间隔-九聚体(The heptamer spacer nonamer),Mechanisms of Somatic Recombination of Anitgen Receptor Genes,23,RSS（重排信号序列）,24,2、重组酶：一组参与V、（D）、J基因片断重组的酶，包括以下几种：RAG-1与RAG- 2 (recombination-activating genes): 一种内切酶，只表达在T和B淋巴细胞不成熟阶段。末端脱氧核苷酸转移酶 (terminal deoxynucleotidyl transferase,TdT) ：表达于T、B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DNA的断段。其他 切开发夹结构的内切酶，参与修复DNA双链断段的DNA外切酶、DNA合成酶等，如DNA连接酶IV，DNA依赖的蛋白激酶。,25,重组酶所催化的重排步骤,RAG 蛋白复合物结合到RSS。蛋白复合物拉近将要相连的DNA片断。互补链切断，发夹结构形成。其他DNA修饰蛋白结合到发夹结构剪切RSS末端。发夹结构被随机剪切，碱基或者增加或者减少。DNA连接酶连接产生编码连接点和信号连接。,26,3、Molecular explanation of the 12-23 rule,27,Loop of interveningDNA is excised,An appropriate 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 rule,28,This rule can explain why in heavy chain, the V can not directly bind to J.,29,Recombination activating gene products, (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,互补链断裂，发夹结构形成,4. Steps of Ig gene recombination,30,A number of other proteins, (Ku70:Ku80, XRCC4 and DNA dependent protein kinases) bind to the hairpins and the heptamer ends.,4. Steps of Ig gene recombination,31,缺失性重排,5.重排方式：以重链重排为例,非缺失性重排,L,L,32,Non-deletional recombination,33,34,6.Generation of Diversity of the B and T Cell Repertoires Junctional diversity: P nucleotide additions,The recombinase complex makes single stranded nicks at random sites close to the ends of the V and D region DNA.,The 2nd strand is cleaved and hairpins form between the complementary bases at ends of the V and D region.,35,Heptamers are ligated by DNA ligase IV,V and D regions juxtaposed,36,Endonuclease cleaves single strand at random sites in V and D segment,6.1 Generation of the palindromic sequence,In 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.,The nicked strand flips out,37,6.2 Junctional DiversityN nucleotide additions,Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNA,CACTCCTTA,TTCTTGCAA,38,Junctional Diversity,NNNNNNNNNNNNNNN,Germline-encoded nucleotides,Palindromic (P) nucleotides - not in the germline,Non-template (N) encoded nucleotides - not in the germline,Creates 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.,39,Junctional Diversity,40,Maturation of B Lymphocytes,1.Pro- B cell 2.Pre- B cell3. Immature B cell4. Mature B cell,41,Maturation of B Lymphocytes,42,Stages 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 expressed most abundantly 5) Ig a and Ig b of BCR complex starts to express. 6)First recombination of Ig genes occurs in heavy chains,43,Recombination in Pro- B cell :,44,2.Pre- B cell,A 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 cell surface at low levels. Only in hematopoietic tissues;,45,46,8) 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.,47,刘老师：您好！上节课讲到内切酶在V和D上随机切开增加了VD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，并不位于V片段的CDR区，如何能够增加抗体的多态性？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3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U来使编码恢复正常。谢谢老师！来自崔雪晶,48,C , 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.Organization of Ig Gene Loci,49,2. Organization of TCR Gene Loci,In 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 TCR b chain and d chain.,50,8) 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.,51,上堂课回顾：1 Ig与TCR基因在染色体基因座位上的排列特点2 重排机制 1）一个信号序列 2）一个原则 3）一组酶其中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：,52,4）缺失性重排5）非缺失性重排,3、B 细胞的成熟：祖B细胞，前B细胞，非成熟B细胞，成熟B细胞,53,4、B细胞表面受体的重排：重链：等位基因排斥轻链：同种型的排斥,54,Pro-B cell,Pre-B cell,55,8.2) the pre-BCR regulates somatic recombination is by stimulating light chain gene rearrangement.,3. Immature B cell,k 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 rearrangement 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.,56,57,4. Mature B cell,Coexpressed m and 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 a few days or weeks.The majority of B cells are IgD+ IgM+.,58,59,Selection of the Mature B cell Repertoire,The 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, perhaps serve to preserve all the cells with the capacity to recognize antigens, regardless of specificity.,60,2) Negative selection: Self antigens deliver strong signals to IgM expressing immature B lymphocytes that happen to express receptors specific for these self antigens.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.,61,B-1 Cells,B cells described to-date: B-2 cellsA second population of B cells exists, called B-1 cells Account for approximately 5% total B cells in humansFound predominantly in peritoneal and pleural cavitiesRelationship to B-2 cells not understoodUse a limited set of V gene segmentsSynthesize low-affinity IgM in response to many bacteriaThought to have an important role as a first line of defense against many pathogens; maybe part of innate immunity?Express the CD5 molecule.B-1 cells spontaneously secrete IgM antibodies that often react with microbial polysaccharides and lipids.,62,B-1 Subset of B lymphocytes,63,Maturation of T lymphocytes,Pro-T cellPre-T cellDouble positiveSingle positive Nave mature T cell,64,1. Pro-T cell,Recent arrivals from the bone borrow.Contain TCR genes in their germline configuration, without expression of TCR, CD3 or z chains or CD4 or CD8RAG-1 and RAG-2 are first expressed.,4) Db-to-Jb rearrangements occur first,65,66,2. Pre-T cell stage:,Vb-to-DJb rearrangements occurPrimary RNA and mRNAThe promoter in the 5 flanking regions of Vb genes function together with a powerful enhancer that is located 3 of the Cb2 gene.The pre-T cell receptor was formed.Signals from the pre-TCR stimulate proliferation of the pre-T cells, recombination at the a chain locus.From the double-negative to the double-positive stage.,67,68,3. Double positive,Thymocytes express both CD4 and CD8 molecules. The rearrangement of the TCR a chain genes and the expression of TCR ab heterodimers occur in the double-positive population.TCR a gene recombination commences.Pre-TCR has no allelic exclusion of a chain. 30% of mature peripheral T cells do express two different TCRs, with different a chains but the same b chains.,69,70,4. Single positive,After selection, cells mature into CD4+ or CD8+ T cells.CD4+ cells acquire the ability to produce cytokines CD8+ cells become capable of producing molecules that kill other cells.,71,Role of the thymus in T cell Maturation,The thymus is the major site of maturation of T cells.2) T lymphocytes originate from precursors but seed the thymus .,Selection processes in the maturation of MHC-restricted ab T Cells,72,3) The thymic environment provides stimuli to make thymocytes proliferate and maturate.,The stimuli come from cells including:Thymic epithelial cells ;Bone marrow derived macrophages;Dendritic cells;ii) Two types of molecules produced by the nonlymphoidMHC molecules: epithelial cells, dendritic cells and macrophages,Cytokines: thymic stromal cells including epithelial cells, Stimulate the proliferation of immature T cells, especially IL-7.,73,Positive Selection of Thymocytes: Development of the Self MHC-Restricted T cell Repertoire,1. Positive selection,Double positive thymocytes are produced spontaneouslyIn the thymic cortex, immature cells encounter MHC TCR recognize the MHC I-peptides complex, the CD8 interacts with the molecules, the T cell will develop to CD8+ T cell.Thymocytes can not recognize self MHC-restricted will die by apoptosis.,74,In the positive selection, three molecules are essential in maturation of T cells: self MHC molecules; TCR; peptides bound to MHC,Table 7-2. Development of MHC Restriction in Bone Marrow plus Thymus Chimeras,1.