User:Kinkreet/Immunology/Generation of diversity in B cells

Antibodies produced by B cells contain huge diversities. Some antibodies are non-specific and can bind any antigens, others are specific to one antigen; different antibodies also have different effector functions.

This diversity is generated both in the germline and somatic level.

Somatic recombination (VDJ)
The genes for both the variable and constant regions of immunoglobulins are grouped into segments, and are located on the same chromosome, but separated by a large distance. The genes that make up the heavy chain is located on chromosome 14, those that make up the λ light chain is located on chromosome 22, and those that make up the κ light chain is on chromosome 2. The heavy chain contains a V segment containing ~40 different genes (95-100 residue), a D segment containing 25 genes, a J segment containing 6 genes (<13 residue), and finally a constant segment. The light chains contain a V segment with 30-40 different genes, and multiple C segments separated by a J segment. All the segments apart from the C segment forms the variable region.

In B cells, the variable and constant region genes are closer than in other cells, because of somatic recombination of pre-B cells in the bone marrow. First, if a D (Diverse) segment is present, unwanted D segment genes on the 3' end of the segment are removed along with the unwanted J (Joining) segment genes. Then, unwanted V segment genes and unwanted D segment genes on the 5' end are removed. This recombined gene is then transcribed to produce a primary transcript containing the segments: V-D-J-Cμ-Cδ. The primary transcript is first polyadenylated at the 3' end and then alternate spliced to remove the translated to give the respective chains.

The flanking regions of the V, D and J segments are required for recombination. V(D)J recombination is mediated by recombination signal sequences (RSSs), containing conserved heptamer (CACAGTC) and nonamer (ACAAAAACC) signal sequences, separated by a non-conserved spacer sequences of 12 or 23 bp. Lymphocyte-specific recombination activating proteins RAG1 and RAG2 cleave DNA at the border of the RSS (at the 3' end of the V segment, and 5' end of the J segment, now found to be small regions of active chromatin encompassing J (and where present J‑proximal D) segments of Igκ, Igh, Tcrα and Tcrβ, which they have termed recombination centres. ) by recombining dissimilar spacers (12-spacer with 23-spacer). Mice without the RAG proteins do not produce any lymphocytes.

The light chain only begins its rearrangement once the heavy chain has completely rearranged.

Mutation at junction
This cleavage is done by the Artemis complex. The Artemis protein has single-strand-specific 5' to 3' exonuclease activity, but it can also complex with the 469 kDa DNA-dependent protein kinase (DNA-PKcs) to gain endonuclease activity on hairpins and the 5' and 3' overhangs; the DNA-PKcs phosphorylates Artemis to give it this new function. During V(D)J recombination, the RAG complex (made up of RAG-1 (RAG1 is homologous totransposase) and RAG-2 complexed with HMG1 or HMG2) binds to two recombination signal sequences (RSSs); the complex associates with each other, bringing the strands together, creating a loop which contains all the DNA between the two RSSs. Some of this DNA is then deleted, and the RAG complex then induce a nick precisely at the 5' end of the heptamer. This creates a 3' OH group which acts as a nucleophile in a transesterification attack on the antiparallel strand, yielding a DNA hairpin (two hairpins, as the RAG complex dimer binds to two strands). In lymphoid cells, recombination can only occur between a 12-RSS and a 23-RSS; this is known as the 12/23 rule. The four ends of DNA (two hairpinned coding ends and the two signal ends) are held together in a postcleavage complex by the RAG complex. DNA-PKcs binds to each end of the broken DNA and recruits the Artemis nuclease, Ku and DNA ligase IV/XRCC4 (X-ray repair cross-complementing factor 4) dimer, Cernunnos (also called XLF or XRCC4-like factor), and any of several DNA polymerases, can then close up the signal ends into a 'signal joint'. It also opens the hairpins of the coding ends, and this process is thought to be mediated by the RAG complex (the RAG complex can open free hairpins by itself, but this is only observed in manganese-containing buffers, and not in magnesium-containing buffers). XRCC4 and Cernunnos aligns the two DNA strands together, and recruits terminal deoxynucleotidyl transferase (TdT), which add nucleotides at the open ends. This occurs until there are complimentary sequences at which point the opposite strands will pair up. Exonucleases then remove the unpaired nucleotides, and Ligase IV fill in the gaps. This creates a junction between each joined segment, containing an unspecified number of nucleotide additions, flanked by a 2-residue palindromic sequence.

This recombination is nearly-random, meaning, through chance, the body is able to produce a diverse range of antibodies against any possible antigen the body may encounter. The κ or λ light chains and the μ heavy chain come together to form IgM, which is displayed on the cell surface of B cells. Other proteins involved are Ku70:Ku80, DNA-dependent protein kinases (Artemis), TdT (terminal deoxynucleotidyl transferase), exonucleases, DNA ligase IV and XRCC4.

Combination of light and heavy chain
Immunoglobulins can exist bound to the membrane, or it can exist freely; and alternative splicing of the same primary transcript determines the type of C-terminus it will have, which is what determines whether it will have a transmembrane domain or not.

Affinity Maturation
Affinity maturation is the process by which the population of antibodies expressed on different B cells, over time, will bind to the antigen with higher affinity over the course of the immune response. Therefore, the affinity of antibodies found in the secondary response may be several logfold higher than in the primary response. The process of affinity maturation consists of repeating cycles of mutations and selection.

Somatic hypermutation (SHM)
Mutation rate at the antigen-binding sequences of the variable region (complementary-determining regions, CDRs) are up to 106 times higher than in cell lines not in the lymphoid system. This results in a mutation rate of 1-2 point mutations per CDR per division. Note that the constant segment is not affected.

Clonal selection
The mutation induced may have increased or decreased the binding affinity of the antibody. But when follicular dendritic cells of the germinal centers present antigens to B cells, only the B cells with the highest affinities are able to bind effectively and survive, those with low affinities will not be able to compete with those of higher affinity, and thus will not survive.

Allelic exclusion
As B cells only express one type of antibody on one cell, the other rearrangement on the second chromosome must be suppressed. This is done only after the antibody is expressed on the cell surface.

Selection
After the receptor is displayed on the surface of the B cells, it is technically an immature B cell; it will detach from stromal cells but remain in the bone marrow where it will be negatively selected - any B cells with receptors that binds to self molecules are removed. When the B cell encounters antigens, it is activated and undergo class switching

Class Switching
The CH genes of every class available are all located 3' of the J segment, the class which gets are incorporated into the immunoglobulin varies at different stages of an immune response, as different classes of immunoglobulins perform different effector functions; for example, IgG and IgM are involved in complement activation, whereas IgE are involved in binding to mast cells and basophils. Alternative splicing occurs at switch regions, which are located 5’ of the C region, where the different switch regions undergo recombination to incorporate the desired C gene into the immunoglobulin; the mRNA then undergo polyadenylation 3' of the C gene, which prevents its degradation in the cytoplasm as well as aiding in transcription termination.

Class switching is signalled by cytokines; IL-4 induces the class switch to IgG1 and IgE (in mice); TGFβ induces the class switch to IgG2b and IgA; IFNγ induces the class switch to IgG2a and IgG3. These cytokines causes changes in chromatin conformation (probably by acetylation) at specific sites which makes the switch site available to recombinases.

Without a signal, B cells will simply produce IgM. For it to produce anything else, you need CD40-gp39 interaction.

Flagellin of bacteria in the small intestine are recognised by lamina propria dendritic cells, which expresses a high level of TLR5; these lamina propria DC help naive B cells to differentiate into IgA-secreting plasma cells.

Plasma cells and memory cells
Plasma cells will secrete antibodies, whereas memory cells will retain the high affinity antibodies, and return to the bone marrow and lymphoid tissues.