B cells I. Differentiation of B cells in Bone marrow II B cells I. Differentiation of B cells in Bone marrow II. BCR and B cell accessory molecules III. The subsets of B cells

I. Differentiation of B cells in Bone marrow 1 I. Differentiation of B cells in Bone marrow 1. process of B cell maturation 2. events in the differentiation of B cells 3. mechanisms of Ig diversity

I. Differentiation of B cells in Bone marrow 1 I. Differentiation of B cells in Bone marrow 1.Process pro-B cell  chain pre-B cell surrogate light chain +  chain immature B cell κ chain or λ chain +  chain (membrane IgM,mIgM) mature B cell mIgM, mIgD

2. Events in the differentiation of B cells 1) Negative selection immature B cells : mIgM--self antigen mIgM self antigen apoptosis or anergy surviving to develop mature B cells 2) gene rearrangement

It is estimated that in the mouse the bone marrow produces about 5x107 B cells/day but only 5x106 (or about 10%) are actually recruited into the recirculating B-cell pool. This means that 90% of the B cells produced each day die without ever leaving the bone marrow. negative selection Immature B cells that express auto-antibodies against self-antigens are eliminated in the bone marrow.

2) gene rearrangement (1) germline gene structure of Ig (2) rearrangement of Ig genes (3) characteristics of Ig gene rearrangement

(1)   Germ-line gene structure of BCR H chain: chromosome 14 V region encoding genes: VH (variable gene segments), DH (diversity gene segments), JH (joining gene segments) C region encoding genes: CH (constant gene segments): Cμ , Cδ, Cγ et al. (9)

L chain(-- chromosome 2, -- chromosome 22) V region encoding genes:  --V, J -- V, J 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.

(2) Gene rearrangement of BCR VDJ rearrangement of H chain pro-B cells: D-J V-DJ VDJ DNA transcription pre-B cells: VDJCμ VDJ- Cμ RNA mRNA splicing V-J rearrangement of L chain pre-B cells: V-J VJ DNA immature B cells: VJC VJ-C RNA mRNA

BCR(membrane type) and secretory type Ig

(3) Characteristics of BCR gene rearrangement Allelic exclusion: only one of the two alleles in homologous chromosomes can be expressed. isotypic exclusion: only one of the two types of light chain genes can be expressed.

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

3. mechanisms of diversity of Ig (BCR or Ab) 1) 3. mechanisms of diversity of Ig (BCR or Ab) 1). Mutiple germline gene segments 2). Combinatorial V(D)J joining 3). Junctional flexibility 4). combinatorial assocination of heavy and light chain 5). somatic hypermutation

1). Mutiple germline gene segments There Are Numerous Germ-Line V, D, and J Gene Segments.

2). combinatorial V(D)J joining The multiple germ-line gene segments are combined randomly during the rearrangement of BCR genes. human Ig: 51VH×27DH ×6JH= 8262 possible combinations

3). Junctional flexibility imprecise joining In the junction of V-J, V-DJ or D-J, several nucleotides are lost to increase the diversity of the V region encoding gene of L chain or H chain. N-nucleotides addition During the D-J and V to D-J joining process, several nucleotides are inserted to increase the diversity of V region encoding gene of H chain. N-nucleotides insert by TdT(terminal deoxynucleotidyl transferase) without template There is no N-nucleotides insert in L chain.

4). Combinatorial association of heavy and light chains 5) 4). Combinatorial association of heavy and light chains 5).Somatic hypermutation Somatic hypermutation occurs at a frequency approaching 10-3 per base pair per generation. This rate is at least a hundred thousand-fold higher (hence the name hypermutation) than the spontaneous mutation rate, about 10-8 /bp/generation, in other genes. Somatic hypermutation adds diversity in already-rearranged gene segments

Somatic hypermutation Ag activated B cell proliferate gene mutation in V region encoding genes affinity maturation

II. BCR and accessory molecules of B lymphocytes B cell receptor complex B cell accessory molecules

1.BCR complex a group of membrane molecules on B cells that can specifically bind to the antigen and pass an activation signal into B cells, consisting of BCR and Ig-Ig  heterodimer BCR membrane immunoglobulin on B cell, mIg: IgM, IgD Ig-Ig , (CD79) ITAM transduce an activation signal

ITAM An immunoreceptor tyrosine-based activation motif (ITAM) is a conserved sequence of amino acids (YXX(L/V)X7-11YXX(L/V)) in the cytoplasmic tails of certain cell surface proteins of the immune system. The tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands and transduce an activation signal. ITIM An immunoreceptor tyrosine-based inhibition motif (ITIM), is a conserved sequence of amino acids (S/I/V/LxYxxI/V/L) in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-possessing inhibitory receptors interact with their ligand, their ITIM motif becomes phosphorylated and tranduce an inhibitory signal.

2. Co-receptors complex 1) CD19, CD21, CD81, CD19: CD21(CR2): receptor of iC3b and C3d

3.CD40 ---co-stimulatory receptor CD40 on B cell binds to CD40L on activated T cell---pass a costimulatory signal into B cells 4. B7 5. CD45 6. MHC molecules 7. Mitogen receptors 8. Cytokine receptors

B7(CD80,CD86) ------co-stimulator to T cells ligand of CD28

3.CD40 ---co-stimulatory receptor CD40 on B cell binds to CD40L on activated T cell---pass a costimulatory signal into B cells 4. B7 5. CD45 The cytoplasmic domain of CD45 has an intrinsic phosphatase activity that removes an inhibitory phosphate group on a tyrosine kinase called Lck (in T cells) or Lyn/Fyn/Lck (in B cells) and activates it. 6. MHC molecules 7. Mitogen receptors 8. Cytokine receptors

6. MHC molecules MHC-I and MHC-Ⅱ 7 6. MHC molecules MHC-I and MHC-Ⅱ 7. Mitogen receptors Receptors of mitogen: SPA,PWM (pokeweed), LPS 8. Cytokine receptors

III. Subset of B cell --------------------------------------------------------------------- Comparison of B1 and B2 cells ------------------------------------------------------------------------------------ B1 B2 CD5 + - location thorax, abdominal cavity lymph organs lamina propria of intestine Recognized Ag TI Ag and auto-Ag TD Ag --------------------------------------------------------------------------------