1. Chapter 13 Lymphocyte Maturation and Antigen Receptor Expression

2. Chapter 13 Lymphocyte Maturation and Antigen Receptor Expression
Contents
PartⅠ Introduction of hematopoietic stem cell
PartⅡ T cell maturation and B cell maturation
PartⅢ BCR diversity and TCR diversity

3. PartⅠ Hematopoietic stem cell
Pluripotent hematopoietic stem cell--bone marrow
Marker: CD34, CD117(c-kit)
Myeloid progenitor + lymphoid progenitor
Myeloid progenitor EPO, TPO, CSF, IL
Lymphoid progenitor NK, DC, T, B
NK marker: CD56+CD16+
T cell: TCR, CD3, CD4/CD8, CD28, CD2, LFA-1
B cell: BCR, IgαIgβ, CD19/CD21/CD81,CD40
B7, MHC, CKR

6. PartⅡ T cells maturation and B cells maturation
Development and differentiation of T cells
Development and differentiation of B cells

7. SectionⅠ Differentiation of T cells in thymus
Thymic microenviroment
Differentiation course of T cells
Selection of T cells in thymus----
positive selection and negative selection

8. 1. Thymic microenvironment

10. 2. Differentiation course of T cells 1) pro T cells CD3- TCR- CD4- CD8- TCR β chain starts to rearrange DN 2) pre T cells CD3+ TCRpTα:β CD4+ CD ) immature T cells DP CD3+ TCR+ CD4+ CD8+ the rearrangement of TCR α chain 4) mature T cells CD3+ TCR+ CD4+ or CD3+ TCR+ CD SP
TCR rearrangement
Thymus selection

11. Thymus lobules Capsule Subcapsular region Cortex Medulla Bone marrow
CD3‾CD4+ CD8‾ TCR‾
CD3‾CD4‾CD8- TCR‾
CD3LOWCD4‾CD8-TCRabLOW
CD3+ CD4+ CD8+ TCRab+
CD3+ CD4+ CD8‾ TCRab+
CD3+ CD4‾ CD8+ TCRab+
CD3+ CD4+ TCRab+
CD3+ CD8+ TCRab+
CD3+ CD4+ CD8+ TCRgd+
CD3+ CD4+ CD8‾ TCRgd+
CD3+ CD4‾ CD8+ TCRgd+
CD3+ CD4‾ CD8‾ TCRgd+
CD3+ TCRgd+
CD3‾CD4‾/LOW CD8‾ TCR‾
Capsule
Subcapsular region
Cortex
Bone marrow
Medulla
Thymus lobules

12. Differentiation of T cells in thymus
Changes in thymus：
TCR rearrangement ----functional TCR
Positive selection and negative selection
T cells acquire MHC restriction and Self tolerance

14. 3. Selection of T cells in thymus
Depend on TCR , MHC and Ag peptide
TCR—MHC: positive selection
TCR---self antigen peptide : negative selection
During the course from DP(double positive) cells to SP(single positive) cells

15. Positive selection Get self MHC restriction
DP cells whose TCRs recognize and combine with MHC molecules can differentiate and develop continuously----SP
DP cells whose TCRs can’t recognize with MHC molecules or bind with high affinity go apoptosis
Get self MHC restriction
MHC molecules play an important role in positive selection:
MHC-Ⅰ------CD8+ expression
MHC-Ⅱ------CD4+ expression

17. Negative seletion Acquired self tolerance
cells whose TCRs can’t recognize with self antigen peptide develop and differentiate continuously
SP cells whose TCRs recognize and combine with self antigen peptide tightly go apoptosis or become clonal anergy
Acquired self tolerance

22. SectionⅡ Development and differentiation of B cells
Differentiation of B cells in Bone marrow
Differentiation of B cells in peripheral lymphoid tissue

23. μ

24. Differentiation of B cells in Bone marrow----Ag independent
Hematopoietic stem cells
Lymphoid progenitor
Pro-B cells( chain rearrangement)
Pre-B cell( chain + surrogate light chain )
Immature B(mIgM,  chain +κchain orλchain)
Mature B(mIgM, mIgD)
Functional B repertoire

25. Negative selection of B cells in bone marrow
Clonal deletion: Functionally immature cells of a clone encountering antigen undergo a programmed cell death. For example, auto-reactive T-cell are eliminated in the thymus following interaction with self antigen during their differentiation (negative selection). Clonal deletion has been shown to occur also in the periphery. B cells expressing only IgM (no IgD) on their surface when exposed to antigen are eliminated.

27. 2. Differentiation of B cells in peripheral lymphoid tissue----Ag dependant
Virgin B/naïve B cell most die
Plasma cell Ab
Memory B cell secondary immune response

29. 3. Events in the differentiation of B cells: Gene rearrangement of Ig Negative selection Immature B cells : mIgM--self antigen mIgM -- self antigen apoptosis or anergy surviving to develop mature B cells

30. Questions? Why can TCR or BCR recognize so many Ag in nature?
Why does IgM produce earlier than others?
How does Ig produce BCR and Ab?
How can B produce different type of Igs? ?

31. PartⅢ BCR diversity and TCR diversity

32. SectionⅠ BCR diversity
Gene structure of Ig
Gene rearrangement of Ig
Characteristics of Ig gene expression
Mechanism of Ig diversity

33. 1. Gene structure of Ig (human)
H chain:14 chromosome V region encoding genes: VH (variable gene segments) – 65
DH (diversity gene segments) – 27
JH (joining gene segments) – 6
Leader sequence—signal peptide
C region encoding genes: CH (constant gene segments): Cμ, Cδ, Cγ et al. (11)

34. L chain(--2 chromosome, --22 chromosome) V region encoding genes:  --V, J – 40, -- V, J – 30, Leader sequence—signal peptide C region encoding genes: C (1); C(4)

35. In heavy chains, the V, D and J segments encode the variable domain while the C segment encodes the constant domain.
In light chains, the V and J segments encode the variable domain while
the C segment encodes the constant domain.

37. (a)  Chain (22 chromosome))V
JC JC JC JC
(2 chromosome)

38. 2. Gene rearrangement of Ig V-D-J rearrangement of H chain pro-B cells: D-J V-DJ VDJ DNA pre-B cells: VDJCμ VDJ- Cμ RNA mRNA V-J rearrangement of L chain pre-B cells: V  -J  V  J  DNA immature B cells: V  J  C V  J  -C RNA mRNA
transcription
splicing

39. C C C3 C 1 C1 C2 C4 C C2
C C C3 C1 C1 C2 C4 C C2
C C C3 C1 C1 C2 C4 C C2 C C C C 

41. The expression of BCR Intranuclear: DNA rearrangement V region encoding gene (VDJ or VJ) Transcription and splicing leader sequence + V region encoding gene + C region encoding gene (L gene-V gene –C gene) Extranuclear: translation nascent peptide L-V-C Endoplasmic reticulum: assembly H chain and L chain (IgM or IgD) transportation------BCR (membrane Ig, mIg)

42. 3. Characteristics of Ig gene expression
① recombination enzyme:
RAG(recombination activating gene)
TdT(terminal deoxynucleotidyl transferase)
other DNA enzymes

43. ② Allelic exclusion and isotype exclusion Allelic exclusion: only one of the two alleles in homologous chromosomes can be expressed Isotype exclusion: only one of the two types of light chain genes can be expressed(:=65:35).

44. Kuby Figure 5-10
Read Kuby pages : Allelic Exclusion Ensures a Single Antigenic Specificity

45. ③ Isotype switching ( class switching ) Ag activated B cells proliferate VDJ is switched to recombine with another C region encoding gene IgM IgD, IgG, IgA, IgE Switching region

47. Thymus dependent antigen
APC
CD4 T cells
cytokines
cytokines

48. ④ Membrane type (BCR) and Secretory type Ig (Ab)

50. 4. Mechanism of Ig diversity
① Combinatorial diversity
human Ig:
65VH×27DH ×6JH=10530V
40V ×5J =200V
30V ×4J  =120V

51. C C C3 C 1 C1 C2 C4 C C2
C C C3 C1 C1 C2 C4 C C2
C C C3 C1 C1 C2 C4 C C2 C C C C 

52.  ② Junctional diversity
CDR3 lie in V-DJ or D-J junctions
Lose or insert of several nucleotides will increase the diversity of CDR3.
N-nucleotides insert by TdT without template
There is no N-nucleotides insert in L chain

53. ③ Somatic hypermutation Ag activated B cells proliferate gene mutation in V region encoding genes affinity maturation mature B cells which finished V gene rearrangement

54. SectionⅡ Gene structure and rearrangement of TCR
Gene structure of TCRαβ
αchain(14 chromosome): V, J, C
βchain(7 chromosome): V, D, J, C

55. (14 chromosome)
(7 chromosome)

56. 2. Gene rearrangement of TCRαβ
TCR β chain rearrange first
inactivate δ gene within αgene

58. 3. Gene structure of TCRγδ
γchain(7 chromosome): V, J, C
δchain(14 chromosome): V, D, J, C

59. 4. Gene rearrangement of TCR γδ No junctional diversity in TCR γδ
(7 chromosome)
(14 chromosome)

61. 5. Characteristics of TCR gene expression
Without somatic hypermutation
More N- nucleotides insert than BCR
More valid rearrangement in V region of TCR
BCR: 1014
TCR: 1016

62. Comparison of BCR and TCR functional genes
NO. of chain Chromosome V D J C
BCR H  
TCR    