9: B-cell development

Question 1

Which cells induce early hematopoietic stem cells (HSC) to become committed to the lymphoid lineage?

Answer 1

Stem cell factor (SCF) and IL-7. Latter activate JAK/STAT pathway, leading to activation of TFs

Question 2

Pre-Pro-B-cells

Answer 2

B220+ (marker for B-cell lineage)

High levels of early B-cell factor 1 (EBF1): activates B-lineage genes + accessibility to D-J(H) segment + inhibit genes supporting T-cells or NK cells

Epigenetic changes for RAG1/2 => binding to RSS

Chromatin opens. No VDJ recombination

Remains in contact with CXCL12 secreting stromal cells

Question 3

Pro-B-cells (progenitor B-cell)

Answer 3

D to J(H) recombination completed

Expression of PAX5 (TF):
Igα and Igβ production start
CD19 expression
Promotes V(H)-to-DJ(H) recombination initiation

Question 4

Pre-B-cells

Answer 4

Rearrangement of heavy chain complete.

HC complexed with VpreB and λ5 => surrogate light chain (SLC)
+
Pre-B-cell receptor expressed in early pre-B stage
=>
Igα/Igβ + CD19 signaling

No rearrangement and expression of μ H chain and preBCR => apoptosis
(1st checkpoint)

Downregulation of RAG1/2, loss of TdT activity => allelic exclusion

Loss of pre-BCR expression:

• Termination of expression of SLC
• Less IL-7R signaling

Question 5

Surrogate light chain (SLC)

Answer 5

Consist of:
VpreB and λ5

VpreB homologous to light chain V region

λ5 homologous to λ light chain J+C seq

Additional aa

Cover Ag binding site

Question 6

small pre-B-cells

Answer 6

Light chain recombination
- Stimulated by: IRF4, IRF8, FOXO1,
re-expression by RAG1/2

• On K or λ loci in human

IgM BCR expressed => no more LC recombination
=> immature B-cell

If no IgM => apoptosis (2nd checkpoint)

Question 7

B-cell maturation stages (to immature B cell):

Answer 7

Pre-pro-B

Early Pro-B: DJ(H)

Late Pro-B: Some V(H)DJ(H)

1st checkpoint

Large Pre-B:

• V(H)DJ(H)
• Pre-BCR

Small Pre-B:

• V(H)DJ(H)
• V(L)J(L) begins
• Pre-BCR decreases2nd checkpoint

Immature B-cell

• V(H)DJ(H)
• V(L)J(L)
• IgM

Question 8

Fates of autoreactive B-cells:

Answer 8

Anergic
- Recognize self-Ag inside BM,
escape
- Unresponsive

Central tolerance:

Clonal deletion
- Before leaving bone marorw

Reactivation of genes to initiate light chain receptor editing.
=> change specificity of BCR

Question 9

Transitional B-cells

Answer 9

Immature B-cells:
BM -> spleen T1

T1 -> follicle: increased IgD expression => T2

B-2 cells: follicular B-cels

T3:
- Anergized
- Short half-life, need more BAFF, 
  express higher levels of Bim
- Excluded from B-cell follicles

Question 10

BAFF

Answer 10

B-cell activation factor

BAFF-R (receptor) dependent on signalling through BCR.

Necessary for the survival of transitional and mature B-cells.

Induces synthesis of anti-apoptotic factors (Bcl-2, Bcl-x_L, Mcl-1)

Interfers with the function of the pro-apoptotic molecule Bim.

Question 11

Natural Antibodies

Answer 11

Ab’s secreted in the absence of antigenic stimulation.

Secreted to self and microbial Ab’s

Question 12

Marginal zone B-cells

Answer 12

Located at the interface between the circulation and the white pulp of the spleen

Produce IgM and class-switched IgG antibodies

Low levels of IgD

Long-lived, may self-renew in periphery

Question 13

What happens after Ag binds to BCR?

Answer 13

• Phosphorylation of tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) of Igα and Igβ by Src family kinases.
• Formation of cytoplasmic signal-transducing complex (signalsome)

Outcome depends on strength and duration of binding

Question 14

Which cell receptors bind to and regulate the outcomes of Ag-BCR?

Answer 14

CD21
CD40
IL-4R
IL-21R
BAFF-R

Question 15

Interaction between T and B cells

Answer 15

T binds to processed Ag with TCR

Interactions between T cell CD28 and B cell CD80 and CD86, and between T cell CD40L and B cell CD40

Secretion of T cell cytokines necessary for complete B cell activation.

• IL-4
• IL-21

Question 16

Plasma cells

Answer 16

Secrete large quantities of nonmutated IgM and IgG antibodies.

Induce early, protective humoral immunity.
Helps to drive affinity maturation.

Question 17

Activated germinal center B cells

Answer 17

In follicles (rapid division)

B cells + associated T(FH) cells + follicular DCs

More prone to apoptosis.

Dark zones:

• Rapid division
• Somatic hypermutation.
• Centroblasts

Light zones:
- B cells interact with T(FH) and 
  FDCs.
- Selection of B cells with high- 
  affinity, mutated receptors.

Question 18

Somatic hypermutation, SHM

Answer 18

Initiated by cytidine deamination by the enzyme AID.
DNA repair mechanisms cause alteration of the sequences of the variable region of Ig genes.

Occurs only in germinal centers after T-dependent antigen stimulation

Question 19

Class-switch recombination, CSR

Answer 19

Results in the replacement of an antibody heavy chain constant region, with a different constant region, originally located 3’ to the first.

Regulated by cytokines secreted by T cells or other immune cells.

May occur in germinal centers.

May occur in presence or absence of T helper cells

Question 20

Memory B cells (plasma cells)

Answer 20

Long-lived
Generated late in a primary response
Located in bone marrow, mucosa etc.

Question 21

Negative regulation of B cells:

Answer 21

CD22 phosphorylates ITIM domain

FcγRIIb

Elimination of CD5 expression on B-1a B cells.

B-10 B cells release IL-10 => reduction of inflammatory responses.