1. Immunoglobulin Gene Rearrangement
Lu Tian
Deb Walter

2. Immunoglobulin
A Y-shaped protein produced by B cells that is used by the immune system to identify and neutralize foreign objects (bacteria and viruses).
Each antibody contains a paratope that is specific for one particular epitope on an antigen, allowing these two structures to bind together.

3. Isotypes IgD IgE IgG IgM IgA

4. Structure of Immunoglobulin

5. Immunoglobulin diversity
1 Domain variability
2 V(D)J recombination
3 Somatic hypermutation and affinity maturation
4 Class switching
5 Affinity designations

6. Immunoglobulin Gene Rearrangements
The ability of cells of the immune system to make antibodies requires multiple programmed rearrangements of immunoglobulin genes.
Randomly combines Variable, Diverse, and Joining gene segments in vertebrate lymphocytes.
Because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens .

7. Rearrangement in Heavy Chain
V, D, J
First, D-J recombination
followed by the joining of V gene segment, forming a rearranged VDJ gene segment.
Any DNA between these gene segments is deleted

8. Rearrangement in Light Chain
Two types of chains:
The kappa (κ) and
lambda (λ) chains, but
Only V and J
the immunoglobulin
rearrange in a similar way

9. Germline Immunoglobulin DNA
Germline Variable, Diversity and Joining Gene segments
VDJ recombination occurs only in lymphocytes in a lineage specific manor
Our context is for B cells
Jung et al., Annual review of immunology 24 (2006)

10. V(D)J Recombination and 12/23 Rule
VDJ is site specific
Conserved Recombinational signal sequences (palindromic heptamer and an AT rich nonamer)
Tonegawa, T. Nature 302,5909 (1983)

11. RAG Mediated DNA Rearrangements
RAG introduces single strand nicks between two coding sequences and their flanking rs’s
RAG catalyzes a 3’OH transesterification Hairpin
Blunt RS ends
Remain attached to RAG
Without rag, the others happen
NHEJ has been shown in vitro but not coupled to VDJ recombination so mostly speculative
Blunt ends can be easily joined however, hair pinned coding ends must be opend before joining
Bassing et al. Cell 109 (2002)

12. Hairpin Opening and Artemis Protein
5’ overhang endonucleolytic cleavage preference is at the single to doublestrand transition
3’ overhang endonucleolytic cleavage preference is displaced 4nt into the ss region
Artemis:DNA-PK recognizes 4nt of ssDNA in an orientation dependent manner (thick arrows) and preferentially cleaves at the 3’ side of that 4nt ssDNA region
NMR suggests DNA hairpins have unpaired bases near the tip resulting in a 2-4nt single stranded loop
Endonucleolytic preference is 2 nt 3’ to the hairpin tip
Other protein binding may result in greater hairpin lengths permitting a more internal cleavage causing deletions deeper in the coding end
DNA-PK phosphorylation switches artemis from an exonuclease to endonuclease
Ma et al. Cell Press 108 (2002)

13. Murphy, Geha and Notarangelo. Immuno Biology 8th edition (2011)
V(D)J Joining
Palindromic ends
TdT (DNA pol) adds non template nucleotides (~20) which match up are trimmed and then DNA synthesis and ligation at gaps
Creates variability with each join
P- palindromic nucleotides
N- non template nucleotides
Murphy, Geha and Notarangelo. Immuno Biology 8th edition (2011)

14. Review of V(D)J Joining
PSC- post cleavage synaptic complex
These are all NHEJ proteins
Circular signal joint lost from DNA upon division
Parenthesis – different cell types may have different dependence on artemis and dna-pks
Bassing et al. Cell 109 (2002)

15. Regulation of Further Recombination
Pre B cell
Enhancers Cis elements Promoters Silencer Insulators Histones
In IgH variable region exons are assembled before IgH
DJ rearrangements occur first followed by V to DJ complex
Due to random junction difersification, only about 1/3 VDJ rearrangements are in frame and thus productive.
Arrange first allele, non productive, arrange second allele. Variability from heavy to light
CDR- complementary determining region
M and S (membrane/secreted exon)
Germline Vh and Dh gene segmens are flanked by upstream transcriptional promoters
Promoter in close proximity to strong enhancer iEu in the intron between Jh and Cu exons
Variable region diversity: variable exons encoded by the germline segment vh containing two of the three complementary determining regions (ag contact) which are different in different germline segments providing germline encoded diversity.
Recombination leads to diversity as well.
A consequence of this DNA rearrangement is that the gene becomes transcriptionally active because a promoter (P), which is associated with the V gene, is brought close to an enhancer (E), which is located in the intron between the J and C regions. As transcription initiates from the promoter a pre-mRNA is made which contains sequences from the L, V J and C regions as well as sequences for the introns between L and V and between J and C (See Figure 2). This pre-mRNA is processed (spliced) in the nucleus and the remaining introns are removed. The resulting mRNA has the L, V J and C exons contiguous. The mRNA is translated in the cytoplasm and the leader is removed as the protein is transported into the lumen of the endoplasmic reticulum. The light chain is assembled with a heavy chain in the endoplasmic reticulum and the immunoglobulin is secreted via the normal route of secretory proteins. The region V region of the mature light chain is coded for by sequences in the V gene and J region and the C region by sequences in the C gene.
The pre-mRNA is processed (spliced) in the nucleus and the remaining introns, including those between the exons in the C genes, are removed (See Figure 5). The pre-mRNA can be processed in two ways, one to bring the VDJ next to the Cmu gene and the other to bring the VDJ next to the Cdelta gene. The resulting mRNAs have the L, V, D, J and Cmu or Cdelta exons contiguous and will code for a mu and a delta chain, respectively.
The mRNAs are translated in the cytoplasm and the leader is removed as the protein is transported into the lumen of the endoplasmic reticulum. The heavy chain is assembled with a light chain in the endoplasmic reticulum and the immunoglobulin is secreted via the normal route of secretory proteins. The region V region of the mature heavy chain is coded for by sequences in the V gene, D region and J region and the C region by sequences in the C gene
Jung et al., Annual review of immunology 24 (2006)

16. Somatic Hypermutation
After maturation, ag stimulates proliferation and somatic hypermutation occurs
SHM diversifies B cell receptors used to recognize foreign elements (antigens) and allows the immune system to adapt its response to new threats during the lifetime of an organism.[1] Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. Unlike germline mutation, SHM affects only individual immune cells, and the mutations are not transmitted to offspring.[2]
When a B cell recognizes an antigen, it is stimulated to divide (or proliferate). During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least fold greater than the normal rate of mutation across the genome.[2] Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin.[4] The exact nature of this targeting is poorly understood, although is thought to be controlled by a balance of error-prone and high fidelity repair.[5] This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.[1]
Following activation with antigen, B cells begin to proliferate rapidly. In these rapidly dividing cells, the genes encoding the variable domains of the heavy and light chains undergo a high rate of point mutation, by a process called somatic hypermutation (SHM). SHM results in approximately one nucleotide change per variable gene, per cell division.[8] As a consequence, any daughter B cells will acquire slight amino acid differences in the variable domains of their antibody chains.
This serves to increase the diversity of the antibody pool and impacts the antibody's antigen-binding affinity.[35] Some point mutations will result in the production of antibodies that have a weaker interaction (low affinity) with their antigen than the original antibody, and some mutations will generate antibodies with a stronger interaction (high affinity).[36] B cells that express high affinity antibodies on their surface will receive a strong survival signal during interactions with other cells, whereas those with low affinity antibodies will not, and will die by apoptosis.[36] Thus, B cells expressing antibodies with a higher affinity for the antigen will outcompete those with weaker affinities for function and survival. The process of generating antibodies with increased binding affinities is called affinity maturation. Affinity maturation occurs in mature B cells after V(D)J recombination, and is dependent on help from helper T cells.[37]
Jacobs and Bross. Curent opinion in immunology. 13 (2001)

17. Class Switch Recombination
Upon activation they juxtapose their VDJ exon to a downstream C by csr allowing different ab classes and effector functions
Teal – repetitive switch region where csr takes place
Switch region is an intron
Isotype or class switching is a biological process occurring after activation of the B cell, which allows the cell to produce different classes of antibody (IgA, IgE, or IgG).[7] The different classes of antibody, and thus effector functions, are defined by the constant (C) regions of the immunoglobulin heavy chain. Initially, naïve B cells express only cell-surface IgM and IgD with identical antigen binding regions. Each isotype is adapted for a distinct function, therefore, after activation, an antibody with an IgG, IgA, or IgE effector function might be required to effectively eliminate an antigen. Class switching allows different daughter cells from the same activated B cell to produce antibodies of different isotypes. Only the constant region of the antibody heavy chain changes during class switching; the variable regions, and therefore antigen specificity, remain unchanged. Thus the progeny of a single B cell can produce antibodies, all specific for the same antigen, but with the ability to produce the effector function appropriate for each antigenic challenge. Class switching is triggered by cytokines; the isotype generated depends on which cytokines are present in the B cell environment.[38]
Class switching occurs in the heavy chain gene locus by a mechanism called class switch recombination (CSR). This mechanism relies on conserved nucleotide motifs, called switch (S) regions, found in DNA upstream of each constant region gene (except in the δ-chain). The DNA strand is broken by the activity of a series of enzymes at two selected S-regions.[39][40] The variable domain exon is rejoined through a process called non-homologous end joining (NHEJ) to the desired constant region (γ, α or ε). This process results in an immunoglobulin gene that encodes an antibody of a different isotype.[41]
Manis et al. Trends in immunology. 23, 1 (2002)

18. Severe Combined Immunodeficiency (SCID) Mice
Immunodeficiency, autoimmunity, cancer
Scid
Omenn syndrome- effects circulating levels of t and b cells
Types of SCID
Bubble boy
Omenn syndrome
SCID- Severe combined immunodeficiency
disease, vaccine, and transplant research
Deficient protein kinase involved in DNA repair
Because V(D)J recombination does not occur, the humoral and cellular immune systems fail to mature. SCID mice, therefore, present with impaired ability to make T or B lymphocytes, or activate some components of the complement system, and cannot efficiently fight infections, nor reject tumors and transplants.
Jackson Laboratories <jax.org>