This module will help you
Naming
and Organization
Somatic Recombination
Somatic Hypermutation and Isotype Switching
The immune system is remarkable for its ability to respond to a great many antigens, including newly synthesized compounds which did not exist during most of evolution. Unusual properties of antibody diversity include the presence of variable and constant regions on the same polypeptide chain and identical V regions used with different C regions. The somatic recombination process for generating antibody and TCR diversity is unique among mammalian systems.
Antibodies must have enough antigen-binding diversity to recognize every possible pathogen (many V regions) while maintaining the biological effectiveness of their C regions (few C regions). Ig genes are randomly spliced together from gene segments that allow many V regions to be used with a few C regions. Gene segments encoding Ig H, k, and l chains are found on three different chromosomes. During B cell development, recombinase enzymes remove introns and some exons from the DNA and splice segments into functional Ig genes.
Ig gene segments in mammals are arranged in groups of variable (V), diversity (D), joining (J), and constant (C) exons. V kappa (Vk) segments each encode the first two CDR and three framework regions (FR) of the k chain V region, plus a few residues of CDR3. J kappa (Jk) segments each encode the remainder of CDR3 and the fourth FR. C kappa (Ck) encodes the complete C region of the k light chain. DNA encoding human k chain includes approximately 40 functional Vk segments, five Jk segments, and one Ck gene segment, as well as some gene segments which contain stop codons (pseudogenes). Human l chain DNA contains approximately 30 functional V lambda( Vl) segments and four functional sets of Jl and Cl segments. A particular Jl always pairs with its corresponding Cl, unlike Jk which all pair with the same Ck.
DNA for human H chain includes approximately 50 functional VH segments, 30 DH segments, and six JH segments. The first two CDR and three FR of the heavy chain variable region are encoded by VH. CDR3 is encoded by a few nucleotides of VH, all of DH, and part of JH, while FR4 is encoded by the remainder of the JH gene segment. There are also individual gene segments in the DNA for each heavy chain domain and membrane region of each isotype, arranged in the order in which they are expressed by B cells.
Somatic recombination occurs prior to antigen contact, during B cell development in the bone marrow. One DH and one JH are randomly spliced with the removal of all intervening DNA (D-J joining). Next, a random VH segment is spliced to the rearranged DJH segment. VDJH is not spliced to the CH segments; the intervening sequences between VDJH and CH are transcribed into primary mRNA. RNA splicing of the primary mRNA in the nucleus yields mature message , which is transported to the cytoplasm and translated into H chain. A leader sequence (L) at the beginning of each VH segment encodes a signal sequence which is used to transport the newly synthesized H chains into the endoplasmic reticulum; it is not present in the final H chain.
L chain gene segment recombination occurs in a similar manner. One Vk and one Jk are spliced (V-J joining), with removal of all the DNA between them. VJk segments are transcribed with Ck and the intervening DNA. RNA splicing removes excess base pairs to allow translation of a complete kappa chain. If k chain genes are not successfully rearranged, the same process occurs with l chain genes.
V(D)J recombinase expressed in developing B cells recognizes recombination signal sequences (RSS) of nine (nonamer) and seven (heptamer) base pairs flanking each Ig gene segment. The spacers between heptamer and nonamer sequences are either 12 or 23 nucleotides long, signaling which gene segments may be linked. The DNA between the gene segments is most commonly looped out and lost from the DNA during V-JL or V-D-JH joining, but in some cases it is inverted and retained. Splicing between gene segments is imprecise, sometimes resulting in nonproductive rearrangements in which frame shift mutations yield stop codons downstream and no complete H or L chain can be produced.
Products of at least two recombination-activating genes, RAG-1 and RAG-2, plus enzymes to ligate (reattach) the DNA and terminal deoxynucleotidyl transferase (TdT) are required for somatic recombination. Each B cell makes antibody of a single antigen specificity and a single allotype. Both characteristics result from each B cell productively rearranging only one L and one H chain gene. Productive rearrangement results in the successful synthesis and membrane expression of Ig. Productive rearrangement of one allele blocks the rearrangement of the other. If a developing B cell does not successfully rearrange one H and one L chain genes, it dies.
Combinatorial diversity is generated by the random formation of many different VJL and VDJH combinations. Combinatorial diversity is increased by the ability of any VH region to pair with any VL region to bind antigen. Random pairing of 320 different VL (40 x 5 = 200 possible Vk + 30 x 4 = 120 possible Vl) with almost 11,000 different VH (65 VH x 27 DH x 6 JH) results in about 3.5 x 106 different possible antibody specificities. Actual combinatorial diversity is probably less, because some gene segments rearrange more than others and some VH-VL pairings bind antigen better than others.
Junctional diversity results from the imprecise joining of gene segments and from the addition of nucleotides to the DNA sequence at splice sites. TdT adds up to 15 nucleotides to the DNA sequence of human VH and JH regions. Junctional diversity affects predominantly CDR3; it is significant but difficult to quantify, since it also results in many nonproductive rearrangements.
B cells produce IgM and IgD receptors simultaneously by a process called alternative mRNA splicing. One mRNA is transcribes that encodes VDJH-Cm-Cd. This primary message is spliced to remove the either the Cm exons and translated into membrane IgD or the Cd exons and translated into membrane IgM. Following B cell activation, alternative splicing of mRNA allows expression of both membrane and secreted IgM by making a primary message with the carboxyl exons for both membrane and secreted m chain and then doing alternative mRNA splicing.
Somatic Hypermutation and Isotype Switching
One additional mechanism for generating Ig diversity operates after the B cell has matured and migrated to the secondary lymphoid organs. As B cells proliferate following antigen contact, mutations occur preferentially in the recombined VDJH and VJL genes. This process is called somatic hypermutation. B cells with mutations produce membrane Ig with mutated antigen-binding sites. If these altered antibodies fail to bind antigen, the B cell dies. If the altered binding sites bind antigen better than the original, the B cells receive stronger signals to proliferate and mature into plasma cells. As the immune response progresses, the average affinity of the responding B cells and the antibody produced increases (affinity maturation).
Isotype switching increases the functional diversity of antibody molecules. It occurs following antigen stimulation and Th2 cytokine production. Human gene segments for CH are arranged linearly in the order Cm, Cd, Cg3, Cg1, pseudogene Ce, Ca1, Cg2, Cg4, Ce, and Ca2. Each C gene segment, except Cd, is preceded by an intron containing a switch region sequence. Switch sequences differ from recombination signal sequences found flanking V region segments, and enzymes catalyzing isotype switching are not encoded by RAG1 and RAG2
Rearranged VDJH is always expressed first with membrane Cm in the developing B cell, with both membrane Cm and Cd in the mature B cell, and with secreted Cm as the B cell begins responding to antigen. When the B cell receives the proper signals from antigen and cytokines to switch to IgG3 production, for example, recombination occurs between the switch regions Sm and Sg3. The DNA between the two switch sequences, including the coding sequences for m and d chains, is lopped out and removed from the genome. All isotype switching events are productive, since DNA splicing occurs within introns so no nonsense codons are introduced into the transcribed DNA. Further switches to downstream isotypes (Ig A and IgE) may occur if the B cell receives the required cytokine signals.
Practice Quiz
Pick the one BEST answer for each question by clicking on the letter of the correct answer.
1. Genes for immunoglobulins are unlike other human genes in that
a. each polypeptide chain is encoded by several exons.
b. Ig genes are composed of introns and exons
c. somatic recombination occurs before mRNA is transcribed
d. there is less Ig genetic material in mature B cells than in other somatic cells
e. both c and d are true.
2. The gene segments needed to encode the variable region of a k chain are
a. one Jk plus one Dk.
b. one Jk plus one Ck.
c. one Vk plus one Dk.
d. one Vk plus one Jk.
e. one Vk plus one Jk plus one Dk.
3. Pseudogenes are DNA sequences which look very similar to functional genes except for the presence of a(n)
a. intron.
b. leader sequence.
c. promoter codon.
d. signal sequence.
e. stop codon.
4. Combinatorial diversity says that by random combination of 40 functional Vk segments with five Jk segments, the number of possible different k chains that could be made are
5. Which does NOT contribute to Ig antigen-binding diversity
a. Any L chain can combine with any H chain to form a functional antibody.
b. Any Vk can be joined to any Jk to encode the light chain V region.
c. Many CH genes are present in the germline DNA.
d. Random numbers of N nucleotides can be added during somatic recombination.
e. VJL and VDJH joining is imprecise.
6. The proper joining of one VL to one JL is regulated by
a. heptamer and nonamer sequences.
b. leader sequences.
c. P-nucleotide addition sites.
d. 12 and 23 nucleotide spacers between heptamer and nonamer sequences.
e. TdT binding site for DNA.
7. Since each B cell productively rearranges a single H and L chain allele, it exhibits
a. affinity.
b. allelic exclusion
c. antibody restriction.
d. antigen-binding diversity.
e. cross-reactivity
8. Primary mRNA for H chain encodes
a. one VH, one DH, and one JH segment.
b. one VH, one DH, and multiple JH segments.
c. multiple VH, one DH, and one JH segments.
d. multiple VH, one DH, and multiple JH segments.
e. multiple VH, DH, and JH segments.
9. Somatic recombination occurs
a. in the bone marrow stem cell.
b. in the progenitor cell as it is becoming a B cell.
c. in the mature B cell following antigen contact.
d. in the plasma cell after antigen contact.
e. in the plasma cell after antibody secretion.
10. Junctional diversity affects primarily the amino acid sequence in
11. Isotype switching
a. changes the leader sequence exon so the antibody is secreted.
b. improves the antigen binding specificity of an Ig molecule.
c. increases the affinity of antibodies in a process called affinity maturation.
d. increases the functional diversity of Ig molecules.
e. occurs randomly between switch regions.
12. Isotype switching resembles somatic recombination because both processes
a. are catalyzed by the products of RAG1 and RAG2
b. are regulated by helper T cell cytokines.
c. can result in stop codons in coding sequences.
d. occur in developing B cells in the bone marrow.
e. result in the irreversible loss of DNA from the B cell.
13. Alternative mRNA splicing
a. allows the B cell to improve its antigen-binding fit after antigen contact.
b. allows the B cell to make membrane IgM from the mature mRNA for secreted IgD.
c. can be used for the simultaneous production of any two Ig isotypes.
d. is a process by which a B cell can simultaneously synthesize m and d chains.
e. occurs in response to T cell cytokines.
14. Because of the order of the CH gene segments (Cm, Cd, Cg3, Cg1, pseudogene Ce, Ca1, Cg2, Cg4, Ce, and Ca2), a human B cell which undergoes isotype switching from IgM to IgG1 can never in the future secrete
15. Isotype switching is always productive because
a. B cells produce all isotypes simultaneously.
b. isotype switching does not involve recombination of DNA gene segments.
c. no DNA is deleted from the chromosome in isotype switching.
d. no effector diversity results from isotype switching.
e. recombination between switch sites occurs in introns so it cannot introduce stop codons into coding regions.
16. Somatic hypermutation does NOT
a. occur by somatic recombination.
b. occur during B cell proliferation.
c. occur in the B cell following antigen stimulation.
d. result in increased affinity of antibodies secreted later in immune responses.
e. result in the death of some B cells which no longer bind antigen.
Problem
1. Make a line drawing of germline DNA encoding an Ig H chain, with several VH, DH, and JH segments and with Cm. Now make a second drawing showing the same DNA following somatic recombination of one of your D and one of your J segments. What DNA did you eliminate? Do the same for the H chain DNA after V-DJ joining.
2. Draw the primary mRNA for H chain in a mature resting B cell that is expressing both IgM and IgD on its membrane. Explain how this message can be used to produce m and d chains with identical variable regions but different C regions.
