1. ANTIBODY DIVERSITY

2. V D J TCGACGTTATAT AGCTGCAATATA TTTTT Junctional Diversity
Germline-encoded nucleotides
Palindromic (P) nucleotides - not in the germline
Non-template (N) encoded nucleotides - not in the germline
Creates an essentially random sequence between the V region, D region and J region in heavy chains and the V region and J region in light chains

3. How does somatic gene rearrangement
(recombination) work?
How is an infinite diversity of specificity generated from finite amounts of DNA?
Combinatorial diversity
2. How do V region find J regions and why don’t they join to C regions? rule
How does the DNA break and rejoin?
Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity
Junctional diversity (P- and N- nucleotides, see above)

4. Gene rearrangement and the synthesis of cell-surface IgM in B cells
SUMMARY
Before immunoglobulin lightchain (center panel) and heavy-chain (right panel) genes can be expressed, rearrangements of gene segments are needed to produce exons encoding the V regions. Once this has been achieved, the genes are transcribed to give primary transcripts containing both exons and introns. The latter are spliced out to produce mRNAs that are translated to give κ or λ light chains and μ heavy chains that assemble inside the cell and are expressed as membrane-bound IgM at the cell surface. The main stages in the biosynthesis of the heavy and light chains are shown in the panel on the left.

5. Coexpression of IgD and IgM is regulated by RNA processing.
In mature B cells, transcription initiated at the VH promoter extends through both the Cμ and Cδ genes. For simplicity we have not shown all the individual C-gene exons but only those of relevance to the production of IgM and IgD. The long primary transcript is then processed by cleavage, polyadenylation, and splicing. Cleavage and polyadenylation at the μ site (pAμm; the 'm' denotes that this site produces membrane-bound IgM) and splicing between Cμ exons yields an mRNA encoding the μ heavy chain (left panel). Cleavage and polyadenylation at the δ site (pAδm) and a different pattern of splicing that removes the Cμ exons yields mRNA encoding the δ heavy chain (right panel). AAA designates the poly A tail. MC, exons that encode the transmembrane region of the heavy chain.

6. How does somatic gene rearrangement
(recombination) work?
How is an infinite diversity of specificity generated from finite amounts of DNA?
Combinatorial diversity
2. How do V region find J regions and why don’t they join to C regions? rule
How does the DNA break and rejoin?
Imprecisely, with the random addition and subsequent removal of nucleotides to generate sequence diversity
Junctional diversity

7. What mechanism is responsible for allelic exclusion?
Each B-cell produces immunoglobulin of a single specificity
Both the heavy and the light chain coding sequences are present twice in the germline.. (maternal and paternal chromosome)
Yet , the B-cell receptor (BCR) on each B-cell is mono-specific
The B-cell produces monospecific antibodies
This is important for the efficiency of clonal selection and to ensure specificity of the immune response.
The process that ensures monospecificity is called:
Allelic exclusion
What mechanism is responsible for allelic exclusion?

8. A clever genetic model provides evidence
for allelic exclusion
ALLOTYPE- a polymorphism in the Heavy chain C region of Ig
Allotypes can be identified by staining B cell surface Ig with antibodies
a/a
b/b
a/b Y B a Y B b Y B a Y B b
AND Y B a b

9. Allelic exclusion is needed for efficient clonal selection
Antibody
S. typhi
S. typhi
All daughter cells must express the same Ig specificity
otherwise the efficiency of the response would be compromised
Suppression of H chain gene rearrangement helps to prevent the emergence of
new daughter specificities during proliferation after clonal selection

10. Allelic exclusion prevents unwanted responses
One Ag receptor per cell
IF there were two Ag receptors per cell Y B Y
Self antigen B Y
S. aureus
S. aureus Y
Anti
S. aureus
Antibodies Y
Anti
self
Abs Y
Anti
S. aureus
Antibodies
Suppression of H chain gene rearrangement
ensures only one specificty of Ab expressed per cell.
Prevents induction of unwanted responses by pathogens

11. Allelic exclusion is needed to prevent holes in the repertoireY B
One specificity of Ag receptor per cell Y B
IF there were two specificities of Ag receptor per cell
Anti-brain Ig
Anti-self Ig
AND
anti-S. Aureus Ig
Exclusion of anti-brain B cells i.e. self tolerance
BUT
anti S.Aureus B cells will be excluded leaving a “hole in the repertoire” B
Deletion
Anergy OR Y B
S. aureus

12. B-cell development is dependent on bone marrow stromal cells
The early stages of B-cell development are dependent on bone marrow stromal cells.
The top panels show the interactions of developing B cells with bone marrow stromal cells. Stem cells and early pro-B cells use the integrin VLA-4 to bind to the adhesion molecule VCAM-1 on stromal cells. This and interactions between other cell adhesion molecules (CAMs) promote the binding of the receptor Kit on the B cell to stem-cell factor (SCF) on the stromal cell. Activation of Kit causes the B cell to proliferate. B cells at a later stage of maturation require interleukin-7 (IL-7) to stimulate their growth and proliferation. Panel a is a light micrograph of a tissue culture showing small round B-cell progenitors in intimate contact with stromal cells, which have extended processes fastening them to the plastic dish on which they are grown. Panel b is a high-magnification electron micrograph showing two lymphoid cells (L) adhering to a flattened stromal cell. Photographs courtesy of A. Rolink (a); Paul Kincade and P.L. Witte (b).
The development of B cells in the bone marrow proceeds through stages defined by the rearrangement and expression of the immunoglobulin genes.
In the stem cell, the immunoglobulin (Ig) genes are in the germline configuration. The first rearrangements are of the heavychain (H chain) genes. Joining DH to JH defines the early pro-B cell, which becomes a late pro-B cell on joining VH to DJH. Expression of a functional μ chain defines the large pre-B cell. Large pre-B cells proliferate, producing small pre-B cells in which rearrangement of the light-chain (L chain) gene occurs. Successful light-chain gene rearrangement and expression of IgM on the cell surface define the immature B cell.

14. The pre B-cell receptor monitors the quality of heavy chain rearrangement
Mutation in λ5– arrest at
Pro-B cell stage
SEVERE IMMUNODEFICIENCY
Productive µ-chain rearrangement
---assembles pre-BCR
Switches off RAG genes, enzymes
No further µ-chain rearrangement
ALLELIC EXCLUSION
Only one specificity
Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two specificities of antibody per cell
The pre-B-cell receptor is distinguished from the B-cell receptor in that it lacks an immunoglobulin light chain, which is replaced by the surrogate light chain made up from the VpreB and λ5 polypeptides. It is thought that the pre-B-cell receptor does not appear on the cell surface but remains inside the cell in the cytoplasm, as part of membrane-enclosed vesicles.

15. Two main checkpoints during B cell development:
Rearrangment of the Ig-light chain gene leads to expression of cell surface IgM receptor (Immature B cells)
Two main checkpoints during B cell development:
1: heavy chain/ surrogate light chain (pre-BCR , pre-Bcell)
2: heavy chain/light chain (Immature B-cell)

16. Nonproductive lightchain gene rearrangements can be superseded by further gene rearrangement.
The organization of the light-chain loci allows initial nonproductive rearrangements at one locus to be followed by further rearrangements of that same locus that can lead to production of a functional light chain. This type of rescue is shown for the κ light-chain gene. After a nonproductive rearrangement of Vκ to a Jκ, in which the translational reading frame has been lost, a second rearrangement can be made by Vκ2, or any other Vκ that is on the 5' side of the first joint, with a Jκ that is on the 3' side of the first joint. When the second joint is made, the intervening DNA containing the first joint will be excised. There are five Jκ gene segments and many more Vκ gene segments, so that as many as five successive attempts at productive rearrangement can be made at each of the two κ light-chain loci.

17. Allelic exclusion helps diagnose and monitor lymphoma:
Due to clonal expansion of a single cell that contains a unique
rearrangement the amount of cancer cells in blood or in bone
marrow can be determined
Can be used to monitor residual tumor cells upon treatment

18. Diagnosis and monitoring of the treatment of childhood acute lymphocytic leukemia (C-ALL)
The sequence of the framework regions are well conserved. Step 1: design primers that amplify these regions together with the hypervariable regions
Step 2: sequence the PCR fragments to obtain spec. Sequence info on the actual tumor (monoclonal, majority of amplification product is tumor-derived
Step 3: based on the sequence info, design tumor-specific PCR primers
Step 4: quantitate gene expression by Q-PCR. Follow success of therapy and detect minimal residual disease much earlier then by flow cytometry
The hypervariable regions of antibody V domains lie in discrete loops at one end of the domain structure.
The top panel shows the variability plot for the 110 positions within the amino acid sequence of a light-chain V domain. It is obtained from comparison of many light-chain sequences. Variability is the ratio of the number of different amino acids found at a position to the frequency of the most common amino acid at that position. The maximum value possible for the variability is 400, the square of 20, the number of different amino acids found in antibodies. The minimum value is 1. Three hypervariable regions (HV1, HV2, and HV3) can be discerned (red) flanked by four framework regions (FR1, FR2, FR3, and FR4) (yellow). The center panel shows the correspondence of the hypervariable regions to three loops at the end of the V domain farthest from the C region. The location of hypervariable regions in the heavy-chain V domain is similar (not shown). The hypervariable loops contribute much of the antigen specificity of the antigen-binding site located at the tip of each arm of the antibody molecule. Hypervariable regions are also known as complementarity-determining regions: CDR1, CDR2, and CDR3. The bottom panel shows the location of the light-chain V region in the Fab part of the IgG molecule.

19. II. Sequencing amplified DNA
I. Identification of monoclonal, i.e. blast-specific gene rearrangements (IGH, TCR, TCR and IGK-KDE) in the patients’ diagnostic (D1) bone marrow (BM) DNA, using multiplex PCR reactions combined with heteroduplex analysis and polyacrilamide gel electrophoresis (PAGE). Primer sequences and PCR reaction performed conform to BIOMED-1 and BIOMED-2 Concerted Actions (Leukemia 1999;13: and Leukemia 2003; 17: ).
II. Sequencing amplified DNA
ABI Prism Avant Genetic Analyzer,
ABI Big Dye Terminator 3.1 Cycle Sequencing Kit
Result of IMGT junction analysis (http//imgt.cines.fr): IGH rearrangement of patient Q015

20. Consensus TaqMan probe
Design patient-specific primers (red) and use them together with consensus primers
Consensus TaqMan probe
and reverse primer
Patient specific
forward primer
designed by us
BIOMED2 Concerted Action: IgH, TCRg, TCRd, TCRb, IgK V N1 D N2 J

24. Immature B cells with specificity for multivalent self antigens are retained in the bone marrow.
Immature B cells that are not specific for a self antigen in bone marrow mature further to express IgM and IgD and leave the bone marrow (left panels). Immature B cells specific for a self antigen on bone marrow cells are retained in the bone marrow (right panels).

25. Receptor Editing of Immature B cells with
self-reactive BCR (Bone Marrow)

26. Immature B cells specific for monovalent self antigens develop a state of anergy.
Anergic B cells have a half
life of 4-5 days
(10% that of regular B cells)

27. THE RESULT OF SOMATIC GENE REARRANGEMENTS
Combination of gene segments results in a huge number of various variable regions of the heavy and light chains expressed by different B-cells
SOMATIC GENE REARRANGEMENT
2. Successful somatic rearrangement in one chromosome inhibits gene rearrangement in the other chromosome
ALLELIC EXCLUSION
3. One B-cell produces only one type of heavy and one type of light chain
COMMITMENT TO ONE TYPE OF ANTIGEN BINDING SITE
4. The B-cell pool consist of B-cells with differently rearranged immunoglobulin genes
INDEPENDENT OF ANTIGEN
OCCURS DURING B-CELL DEVELOPMENT IN THE BONE MARROW