Chapter 13 Lymphocyte Maturation and Antigen Receptor Expression

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

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

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

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

1. Thymic microenvironment

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+ CD8+ 3) immature T cells DP CD3+ TCR+ CD4+ CD8+ the rearrangement of TCR α chain 4) mature T cells CD3+ TCR+ CD4+ or CD3+ TCR+ CD8+ SP TCR rearrangement Thymus selection

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

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

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

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

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

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

μ

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

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.

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

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

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? ---------------?

PartⅢ BCR diversity and TCR diversity

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

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)

L chain(--2 chromosome, --22 chromosome) V region encoding genes:  --V, J – 40, 5 -- V, J – 30, 4 Leader sequence—signal peptide C region encoding genes: C (1); C(4)

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.

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

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

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 

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)

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

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).

Kuby Figure 5-10 Read Kuby pages 115-117: Allelic Exclusion Ensures a Single Antigenic Specificity

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

Thymus dependent antigen APC CD4 T cells cytokines cytokines

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

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

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 

 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

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

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

(14 chromosome) (7 chromosome)

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

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

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

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

Comparison of BCR and TCR functional genes NO. of chain Chromosome V D J C ----------------------------------------------------------------------- BCR H 14 65 27 6 9  2 40 5 1  22 30 4 4 TCR  14 70-80 61 1  7 52 2 13 2  7 12 5 2  14 4 3 3 1 -------------------------------------------------------------------------