1. 1 Chapter9 B Lymphocyte Xing-cheng WEI ( 韦星呈 ) Room 323, Building of Basic Medicine Department of Immunology, Tel.62215671(office)

2. 2

3. 3 Functions of Ab （ 1 ） Neutralization （ 2 ） Opsonization （ 3 ） Complement activation （ 4 ） Mediation of ADCC Binding of C1q to Ag-Ab Complex

4. 4 Patient of Bruton’s hypogammaglobulinemia (B cell deficiency) A 22 month-old male with Bruton’s hypogammaglobulinemia accompanied by vaccinia necrosum. Note the large necrotic area at the vaccination site. Progressive vaccinia occurs because of an immune defect in B cells and Ig.

5. 5 Mechanisms of Bruton’s hypogammaglobulinemia Bruton's tyrosine kinase (abbreviated Btk or BTK) also known as tyrosine-protein kinase BTK is an enzyme that isenzyme encoded by the BTK gene.gene BTK is a kinase playing a crucial role in B-cell development.kinaseB-cell

6. 6  Development  Surface Molecules  Subpopulations  Functions

7. 7 Sec.1 Maturation of B

8. 8 [Review] One HLA gene only gives one peptide chain

9. 9 [Review] Specificity of an Ab/BCR is determined by V region of the Ag-receptor

10. 10 I.Genomic Organisation of Ig Genes

11. 11 Genomic Organisation of Ig H-chain and L-chain Genes

12. 12 II. Generation of Diversity of Ig Genes II. Generation of Diversity of Ig Genes

13. 13 1. V(D)J Recombination 2. V(D)J Junction 3. Somatic Hypermutation [Three Mechanisms]

14. 14 1.V(D)J Recombination (In Bone Marrow) 1.V(D)J Recombination (In Bone Marrow)

15. 15 V(D)J Recombination

16. 16 (1) Ig H-chain Gene Rearrangement D H 1-25J H 1-6 CC V H 1-40 Recombination occurs at the level of DNA which can now be transcribed to form a H-chain peptide

17. 17 (2) Ig L-chain Gene Rearrangement Germline VkJkCk Spliced  mRNA Rearranged 1°transcript

18. 18 D V RSS and the 12-23 Rule DJ V 2312 9 7 7 9 V D

19. 19 23-mer 12-mer Loop of intervening DNA is excised Heptamers and nonamers align back-to-back The shape generated by the RSS’s acts as a target for recombinases 7 9 9 7 V1 V2 V3V4 V8 V7 V6 V5 V9 DJ V1 DJ V2 V3 V4 V8 V7 V6 V5 V9 An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the 12-23 rule) Molecular explanation of the 12-23 rule

20. 20 TCR  Organisation of TCR genes L & V x70-80 C TCR  D1D1J  1 x 6C1C1D2D2J  2 x 7C2C2 TCR genes segmented into V, (D), J & C elements (VARIABLE, DIVERSITY, JOINING & CONSTANT) Closely resemble Ig genes (  ~IgL and  ~IgH) This example shows the mouse TcR locus J x 61 L & V x52

21. 21 TCR  gene rearrangement Spliced TcR  mRNA Germline TcR  VnVn JC V2V2V1V1 Rearranged TcR  1° transcript Rearrangement very similar to the IgL chains

22. 22 Rearranged TcR  1° transcript Spliced TcR  mRNA L & V  x52 D1D1 J C1C1D2D2 J C2C2 Germline TcR  TCR  gene rearrangement D-J Joining V-DJ joining C-VDJ joining

23. 23 [Concept] (1) Antigen receptor genes consist with many V,D, and J gene segments next to RSS. (2) Recombinase (RAG1/2) recognize RSS and cooperate with other enzymes to mediate rearrangement of V(D)J segments. V(D)J recombination [Result] Multiple V, D, and J gene segments may combine randomly, so as to generate a great number of different combinations of Ig V region.

24. 24 2. V(D)J Junction (In Bone Marrow) 2. V(D)J Junction (In Bone Marrow)

25. 25 V D J TCGCATAT AGCGTATA (1) Addition of P Nucleotides at Junction TCG CATAT AGCGT ATA TCGCACATAT AGCGTGTATA *P: Palindromic (Added by Polymerase)

26. 26 V DJ TCGCCGTTATAT AGCGGCAATATA X X Germline-encoded nucleotides Palindromic (P) nucleotides - not in the germline Non-template (N) encoded nucleotides - not in the germline (2) Addition of N Nucleotides at Junction *N: Non-template encoded (Added by TdT).

27. 27 Junction Diversity [Concept] Junction Diversity [Concept] During V (D) J recombination, one to several nucleotides can be added to or removed from the DNA end generated by recombinase, which increase antigen receptor diversity greatly. During V (D) J recombination, one to several nucleotides can be added to or removed from the DNA end generated by recombinase, which increase antigen receptor diversity greatly.

28. 28 3.Somatic Hypermutation (In Germinal Center)

29. 29 Somatic Hypermutation [Concept] In proliferating germinal center B cells that finished V(D)J recombination, the Ig V genes undergo point mutation at a very high rate, which plays a very important role in affinity maturation. FR1FR2FR3FR4CDR2CDR3CDR1 Amino acid No. Variability 80 100 60 40 20 406080100120

30. 30 Estimates of Diversity VDJV(D)JJunctionRepertoireSo Hy Ig (BCR) H402566000 8.4 × 10 6 3 × 10 5 ～ 10 11 1 × 10 6  40-5200 30-4120 TCR  70-614270 5.8 × 10 6 ＞ 3 × 10 5 ～ 10 12 -  522131352  12-560 2160??-  43336

31. 31 Affecting lymphoid tissue. Predominating cells are B lymphocytes and lymphoblasts. Oral lesions include swollen and hyperplastic gingivae, ulceronecrotic lesions, and marked tendency to gingival hemorrhage. B Lymphoid leukemia maybe involved in V(D)J recombination

32. 32 III. Maturation of B Cells in Primary Lymphoid Organs

33. 33

34. 34 IV. Induction of B Cell Central Tolerance

35. 35 B-Cell Clone Deletion [Concept] In bone marrow ， immature B cells binding self-Ag with mIgM can cause die by apoptosis, so as to remove the self-reactive B cell clones. [Results] Set up B cell central tolerance to self-Ags, so that no any mature B cell clone can react to self-Ag.

36. 36 The Fate of Immature B Cell Depends on Whether Binding with Self Antigens or Not

37. 37 Sec.2 Surface Molecules BCR Complex Co-Receptor Accessory Molecule

38. 38 [Component] -BCR: (B Cell Receptor) (mIg) -Ig  / Ig   (CD79a/CD79b) I. BCR Complex

39. 39 BCR -4 peptide chains = mIg. = mIg. -C end longer than an Ab H chain and pass through membrane. -Cytoplasmic region is short, and can not directly transfer Ag-signal into cell. -transfer Ag-signal to Ig  /Ig .

40. 40 Ig  / Ig  Heterodimer(  &  ). Extramembrane domain: belong to IgSF(CAM) Transmembrane domain: receives Ag-signal from BCR by [-][+] charge. Cytoplasmic domain ： Longer, with ITAMs, transfer Ag-signal into the B cell.

41. 41 [Functions of BCR] [Functions of BCR] ① specifically recognize Ag. ① specifically recognize Ag. ② transfer the Ag-signals to Ig  /Ig . ② transfer the Ag-signals to Ig  /Ig . [Functions of Ig  /Ig  ] [Functions of Ig  /Ig  ] Transfer Ag-signals from BCR into the B cell. Transfer Ag-signals from BCR into the B cell.

42. 42 Function of BCR Complex Ig  Ig  Intracytoplasmic signalling domains Extracellular antigen recognition domains The cytoplasmic domains of the Ig  and Ig  contain Immunoreceptor Tyrosine -based Activation Motifs (ITAMs) - 2 tyrosine residues separated by 9-12 amino acids - YXX[L/V]X 6-9 YXX[L/V]

43. 43 II.Co-receptor (B Cell) [Component] -CD19 -CD21 -CD81

44. 44 Effects of B Cell Co-receptor

45. 45 [Functions of Co-receptor] (1) enhance BCR-Ag binding. (1) enhance BCR-Ag binding. (2) help BCR transduce Ag signals. (2) help BCR transduce Ag signals. [CD21(CR2)] Bind C3b to link to Ag (other C3 splits: iC3b 、 C3d 、 C3dg can bind also). Bind C3b to link to Ag (other C3 splits: iC3b 、 C3d 、 C3dg can bind also).[CD19] Transfer Ag-signals from CD21 into the B cell to promote cell activation. Transfer Ag-signals from CD21 into the B cell to promote cell activation.

46. 46 Epstein-Barr virus (EBV) binds to CD21 to invade into a B cell

47. 47 The Epstein-Barr virus is a member of the herpes family. A person can develop chronic Epstein-Barr infection, infectious mononucleosis, Burkitt’s lymphoma, or Hodgkin’s disease. EBV infiltrates the squamous epithelial cells within the tongue.

48. 48 chronic Epstein-Barr infection Burkitt’s lymphoma Hodgkin’s disease Mononucleosis

49. 49 [Important Members] -CD40 -CD40 -B7(CD80/CD86) -B7(CD80/CD86) -LFA-1 -LFA-1 [Important Members] -CD40 -CD40 -B7(CD80/CD86) -B7(CD80/CD86) -LFA-1 -LFA-1 [Function] Transduce accessory (2nd) signals Transduce accessory (2nd) signals into a B cell for its complete activation (proliferation & differentiation). [Function] Transduce accessory (2nd) signals Transduce accessory (2nd) signals into a B cell for its complete activation (proliferation & differentiation). III. Accessory Molecules

50. 50 (B Cell) (T Cell) Effect CD40 CD40 CD40L CD40L B Act. B Act. CD80/86 CD80/86 CD28 CD28 CTLA-4 CTLA-4 T Act. T Act. T Inh. T Inh. LFA-1 LFA-1 ICAM-1 ICAM-1 B & T Act. Molecule/Ligand/Function

51. 51 B Cell Th Cell

52. 52 [ Two B Subsets] -B1 cell ， CD5 + ， innate. -B2 cell ， CD5 - ， adaptive. Sec.3 B Cell Subpopulations

53. 53 FeatureB-1B-2 SpecificityPoorStrong RenewSelf Bone marrow Th Assist (-)(+) Memory B (-)(+) Major Ig Class IgMIgG AgCarbohydrateProtein Origin time FetalNeonatal Distribution Lamina propria 2nd Lymphoid Organ Comparison of B1 & B2

54. 54 Sec.4 Functions of B Cell Sec.4 Functions of B Cell -Ab Production(Ab Functions) -Ag Presentation -Ab Production(Ab Functions) -Ag Presentation

55. 55 Double Signals & Accessory Molecules

56. 56

57. 57 The lower left a B-lymphocyte also phagotyzing particles.

58. 58 1.Concepts: V(D)J Recombination, Junction Diversity, Somatic Hypermutation, B-Cell Clone Deletion. 2.Features of B1 & B2 cells. 1.Concepts: V(D)J Recombination, Junction Diversity, Somatic Hypermutation, B-Cell Clone Deletion. 2.Features of B1 & B2 cells. [Key Points]