1. Antibody production and B cell differentiation
Lecture 2

2. Differentiation of immune cells
All blood cells are produced in the bone marrow as haematopoietic stem cells
HSCs are both self renewing and multipotential
Large number of primed genes for several cell lineages
They will differentiate into the different cells - Haematopoiesis
and dendritic cells

3. B cells and immunoglobulins
B cells make immunoglobulins
Antibody when free in blood
B cell receptor when expressed on B cells
Antibodies are Y shaped proteins that recognise foreign particles (antigen) in the body
Antibodies are made up of a heavy chain and a light chain
Fab region (Fragment antigen binding)
Fc region

4. Antibody structure
Each immunoglobulin molecule is made up of two types of polypeptides chains
25kDa light chain (x 2)
50kDa heavy chain (x 2)
There are two classes of light chain (either  or )
Each B cell will have the genes for one or the other
There are five distinct heavy chains
 in IgG
 in IgA
 in IgM
 in IgD
 in IgE

5. Immunoglobulin synthesis
There are several genes which code for antibodies and each mature B cell carries a different combination of these genes
The three types of genes that make up an antibody light chain
Variable (V) genes (50-200)
Joining (J) genes (5-100)
Constant (C) genes (1-9)
The heavy chain also uses the Diversity (D) gene

6. V – J recombination
The V and J genes make up the variable region of the light chain of immunoglobulin
 light chain genes are on chromosome 2
40 V gene segments
5 J segments
1 C segment
 light chain genes are on chromosome 22
30 V gene segments
4 J segments
5 C segments

7. V – J recombination

8. Variation Any one B cell will express either  or  light chains
Any V region can join with any J region
Not precisely controlled so the linking can take place in any base in the region adding to more diversity
The variable region of the light chain is composed of
V region codes from approximately 1-97 residues
J region codes for approximately residues
C region codes for the constant region
Variation also occurs in the heavy chain

9. Heavy chain diversity Heavy chain genes are on chromosome 14
V region codes for 1-94 residues (51 segments)
D region codes for residues (27 segments)
J region codes for residues (6 segments)
D and J regions are joined first, then the V region
Terminal deoxyribonucleotidyl transferase can also add extra nucleotides between V and D
Recombination in both chains is executed by specific enzymes in the immune cells
RAG-1
RAG-2
Recognise recombination signal sequences (RSS) adjacent to V, D and J regions

10. Heavy chain recombination
More than 108 can be generated
Germline mutations also add to diversity

11. B cell development B cell precursors in the feotus occurs in the liver
By fourth month of pregnancy HSCs migrate to the bone marrow
Bone marrow then assumes the role generative organ for blood cells
Several cells in the bone marrow coordinate development e.g.
Osteoblasts coordinate lymphocyte differentiation
Endothelial cells regulate differentiation
Sympathetic neurones control release of from the bone marrow
Stromal cells interact with adhesion molecules on HSC to keep them in the correct bone marrow environments for appropriate differentiation signals
Maturation completes in the periphery

12. IL7 expressing cell
IL7
Pro B cell
Osteoblast
Pre B cell
Pre-pro B cell
HSC
Immature B cell
IgM
CXCL12
Stromal cell

13. 2
Stem cell
Pre-pro B cell
Pro-B cell
Pre B cell
Immature B cell
Mature B cell
Heavy chain genes
Germline
D-J rearranging
V-DJ rearranging
VDJ rearranged
Light chain genes
V-J rearranging
VJ rearranged
Surface Ig
Absent
µ chain transiently at surface as part of pre-B cell receptor
IgM expressed on cell surface
IgD and IgM made from alternatively spliced H-chain transcripts

14. B cell development The immature B cell expresses IgM on its membrane
Immunoglobulin expressed on the surface or B cells are called B cell receptors (BCR)
The immature B cells moves to the spleen to complete development
If the B cell encounters self antigens it will edit its light chain genes
If no suitable receptor (i.e. B cells that still bind to self) is made the B cell will die
Also starts to express IgD on the membrane surface
Fully developed B cell will express high levels of IgD and intermediate IgM
Only 10% of B cells made in the bone marrow enter the periphery

15. B cells activation
Each naïve B cell in the periphery expresses a different IgM (i.e. the variable regions are different)
B cells act as an antigen presenting cell and recognises soluble free antigen
Causes interaction with T cells which results in several activating signals
B cells move into the lymph nodes and spleen and differentiates into a primary foci and then plasma cells (which secretes immunoglobulin)
Plasma cells produce large quantities of IgM in early B cell response
Some plasma cells will die but others will reamin as long lived plasma cells in the bone marrow

16. B cell proliferation
Some activated B cells become germinal centres (rather than primary foci)
Germinal centres undergo hypermutation in the region coding for the immunoglobulin
Mutated cells which have higher affinity for the antigen are selected and will either
Become plasma cells with improved affinity to antigen
Re-enter another round of mutation
Become mature memory B cells
After each exposure to antigen more memory B cells and antibody to the antigen are produced

17. Naïve B cell becomes activated
Germinal centre
Primary foci
hypermutation
Plasma cell
Memory B cell

18. Immunoglobulin class switching
Ongoing T cell activation (i.e. TFH cells) also cause antibody producing B cells to start producing either IgG, IgA, IgD or IgE with same specificity as the secreted IgM
Light chain and variable region of the heavy chain remain unchanged
Only constant region of heavy chain changes (class switching)
Which class switch is made is determined by specific cytokine signals

19. Plasma cells
Are large lymphocytes with a high nucleus to cytoplasm ratio
Main function is to produce antibodies
No longer able to class switch
Cannot act as APC
Last for about 5-10 days
Some plasma cells move into bone marrow to
provide long lasting Ig memory

20. B cell expansion

21. Class switching and hypermutations
IgM
TCell
APC
IgM
Primary foci
B cell
IgM
MHC II
Plasma cell
IgM
IgM
Germinal centre
Class switching and hypermutations
IgA
IgG
Etc.
Memory B cell
IgA
IgG
Etc.
Germinal centre