The Generation of Lymphocyte Antigen Receptors

Transcription

1 The Generation of Lymphocyte Antigen Receptors Questions (outlines): 1. Do you really understand V(D)J recombination? Functions of RAG? 2. What are the differences between the Ig and TCR gene recombination? 3. What is class switch? Functions of AID? 不提 : Evolution of the adaptive immune response (page 186 最後一段 - 195)

2 The generation of diversity in immunoglobulins Repertoire: a group of diverse molecules, segments, or motifs (page 158)

3 10 11 Ab in humans Dryer and Bennett 2-gene model, 1965 Tonegawa gene recombination model, 1976

4 Susumu Tonegawa ( 利根川進, 1939-) The Nobel Prize in Physiology or Medicine 1987, for his discovery of the genetic mechanism that produces antibody. Although he won the Nobel Prize for his work in Immunology, he has turned his attention to the molecular and cellular basis of memory formation In his later years. David Baltimore (1938-) The Nobel Prize in Physiology or Medicine in 1975 (shared with Howard Temin and Tenato Dulbecco), for the discovery of reverse transcriptase. In 1981, working with Vincent Racaniello, Baltimore generated a plasmid encoding the polio virus genome, using recombinant DNA technology. Other important breakthroughs from Baltimore's lab include the discovery the transcription factor NF- B and the recombination activating genes RAG-1 and RAG-2. 曾擔任校長 : Rockefeller University California Institute of Technology

5 Ig genes are rearranged in B cells A population of normal B cells has many different rearranged genes, so they yield a smear of DNA fragment sizes.

6 Probe: mrna encoding light chain ( )

7 The germline organization of the Ig heavyand light-chain loci in the human genome (Ch #22) (8 families or clans) (Ch #2) (7 families or clans) (Ch #14) (3 families or clans)

8 V-region genes are constructed from gene segments ( aa) (< 13 aa) (90-95 aa) (2-3 aa) (10-15 aa) CDR 3

9 V-region sequences are constructed from gene segments V l aa aa V aa 2-3 aa aa H ( 原始體細胞狀態 )

10 老鼠胚胎發芽母細胞基因發生重組連接 所有可能產生之不同抗體數目 輕鏈 V 區 = 3 * J 區 = 3 組合數 = 3X3 = 9 k 輕鏈 V k 區 = 250 J l 區 = 4 組合數 = 250X4 = 1,000 重鏈 V H 區 = 250 D H 區 = 10 J H 區 = 4 組合數 = 250X4 X10 = 10,000 抗體多樣性含 k 抗體 = 10,000 X1,000 = 10 7 種含 l 抗體 = 9 X10,000 = 90,000 種 * 大約數

11

12 but naive

13 The numbers of functional gene segments for the V regions of human heavy and light chains usually with pseudogenes (page 160)

14 Conserved heptamer and nonamer sequences (recombination signal sequences, RSSs) flank the gene segments encoding the V regions of heavy (H) and light ( and ) chains

15

16 (recombination signal sequences)

17 The V-region gene segments are joined by recombination Deletional recombination Inversional recombination (more for V -J )

18 The reaction that recombines V, D, and J gene segments involves both lymphocyte-specific and ubiquitous DNA-modifying enzymes RAG: recombination-activating genes (no introns) Ku70:Ku80 + DNA-PKcs to form DNA-PK Artemis (with nuclease activity) TdT: terminal deoxynucleotidyl transferase DNA ligase IV (form complex with XRCC4) DNA polymerase and λ SCID: severe combined immune deficiency -Lack of DNA-PKcs, Ku, Artemis, RAG-1/2 Human diseases: Omenn syndrome : mutations of RAG1/RAG2 RS-SCID: sensitive to radiation, Artemis defect Ataxia telangiectasia: ATM (protein kinase, 似 DNA-PK C ) defect (low # T and B cells/ 少 class switch, 但機制尙不明確 ) film 課本 Fig 5-6

19 The introduction of P-and N-nucleotides at the joints between gene segments during Ig gene rearrangement

20 RAG: recombinationactivating gene 1/2 P-nucleotide addition (palindrome) N-nucleotide addition TdT: terminal deoxynucleotidyl transferase 3 OH 5 P DSBR: double-strand break repair

21

22 allelic exclusion

23

24 T-cell receptor gene rearrangement The germline organization of the human T-cell receptor and loci C1 and C2 are highly homologous.

25 TCR - and -chain gene rearrangement and expression CDR1 CDR2 CDR3

26 The organization of the human T-cell receptor and loci page 172 -chain locus is located within the -chain locus

27

28 TCR Ig (all TCR molecules) -gene is within -gene fewer V fragments, more Js of and chain use RAG1/2 enzymes follow the one-turn/two turn joining rule allelic exclusion is less stringent

29 The numbers of human TCR gene segments and the sources for TCR diversity compared with those of Igs

30 Structural variation in immunoglobulin constant regions

31 The properties of the human Ig isotypes

32 The Ig isotypes are encoded by a cluster of Ig heavy-chain C-region genes : pseudogene

33 The IgM and IgA molecules can form multimers

34 2 forms of Igs

35 Transmembrane and secreted forms of Ig are derived from the same heavy-chain sequence by alternative RNA processing

36 Coexpression of IgD and IgM is regulated by RNA processing

37 Synthesis, assembly, and secretion of the Igs IgM: H+L HL (HL) 2 IgG: 2H H 2 +L H 2 L+L H 2 L 2 (rough endoplasmic reticulum)

38 Immunoglobulin isotype (class) switch IgM 多 IgG 多

39 Isotype switching involves DNA recombination between specific switch signals 150 X (GAGCT)n(GGGGGT), n=3-7 Enzyme: unknown AID (activation-induced cytidine deaminase) UNG, APE1, Ku70:Ku80

40 AID (activation-induced cytidine deaminase) in class switching and somatic hypermutation Normal IgM, little IgG in AID -/- mice. AID 基因缺損相關疾病 ;hyper-igm syndrome (type II) AID 基因發現 : by Dr. Honjo in 2002

41 AID (activation-induced cytidine deaminase) in class switching and somatic hypermutation No somatic hypermutation in AID -/- mice.

42 Activation-induced cytidine deaminase (AID) is the initiator of mutations in somatic hypermutation, gene conversion, and class switching The sequence of AID is related to that of a protein known as APOBEC1 (apolipoprotein B mrna editing catalytic polypeptide 1), which convert cytosine to uracil by deamination in apolipoprotein B mrna.

43 Generation of single strand nicks in DAN by sequential action of AID, UNG, and APE1 UNG: uracil-dna-glycosylase (to form abasic site in the DNA) APE1: apurinic/apyrimidinic endonuclease 1 (to generate a single-strand nick) C U

44 Somatic hypermutation introduces variation into the rearranged Ig variable region that is subject to negative and positive selection to yield improved Ag binding (affinity maturation)

45 AID initiates processes that lead to somatic hypermutation, gene conversion and class switch recombination (synthesize DNA over the abasic sites and have a random nucleotide insertion ) (through homologous recombination with pseudogenes)

46 The primary antibody repertoire is diversified by three processes that modify the rearranged Ig gene

47 Class switching involves recombination between specific switch signals S : 150 repeats of (GAGCT) 3-7 (GGGGGT) - form R-loop 與 V(D)J recombination 不同之處? (page 186)

48 Changes in Ig and TCR genes that occur during B-cell and T-cell development and differentiation

49 Transcription regulation promoter enhancer NF- B 許多與癌症或細胞凋亡相關的重要轉錄因子

50

51 translocation bcl-i bcl-ii