When do antigenic specificities of Ab and TCRs occur? Is it prior to or after encounter with the antigen?
Approximately how many different antibody specificities are required to recognize all antigenic structures? Why aren't more needed?
The human immune system is capable of producing antibody molecules which can recognize a virtually unlimited number of potential antigenic determinants. It has been estimated that 107-109 different antibody specificities would be sufficient to recognize all possible antigenic structures, all due to the cross-reactivity of the antibody combining sites-other Abs can also bind antigen that it was not specifically made for. binds more weakly but can still bind.
It has also been estimated that the immune system can produce at least 108 antibody molecules and even more T cell receptors.
Are the genes for the different chains of TCRs and Abs located on the same or different chromosomes? What is the consequence of this?
They are located on different chromosomes (see attached table). Because these genes are not on same chromosome, they are under independent regulation!
What are germ line genes?
During development of B and T cells, how many of these are expressed at once?
We inherit multiple V region gene segments that encode some specificities. These are called germ line genes. These are the inherited gene segments that encode for the variable domains of heavy chains, κ and λ light chains, and T cell receptor α, β, γ, and δ chains. For antibody molecules, there are about 100 of these germ line V-region gene sets for each of the heavy chains, fewer for kappa light chains and lambda light chains. During the development of a B cell, ONE of these is chosen at random from the heavy chain set, and ONE from a light chain set (either kappa OR lambda). While this mechanism generates a significant number of different combinations (and therefore a large number of different antibody specificities), it is not near enough to account for all the specificities each person can produce.
The genes that code for variable domains are split into a group of how many V gene segments?
V gene segments encode for the first 95 amino acids of the variabl domain but the domain is approximately 110 aa in length. What codes for the remaining 15 aa?
The genes that code for variable domains are SPLIT into a group of 100 V gene segments that encode the first 95 amino acids of the variable domain. However, the domain is ~110 amino acids in length. The DNA base pairs that encode the last 15 amino acids of the domain are found in a linked set of DNA segments further along the chromosome.
For light chains (and alpha chains in TCRs-equivalents) what is the variable region gene segment that can be combined with the V region to encode for the entire variable domain? How many of these are there in B cells?
How is this this segment selected to be joined with the V region of DNA?
What enzymes perform this recombination?
Recombination of variable region gene segments: This is a very important mechanism for generating diversity of human antibodies.
For the light chain, one of four possible J gene segments ("J" for joining - these DNAsequences encode for the 15 amino acids that JOIN the rest of the variable domain to the constant domain) is randomly selected to be expressed with one of the randomly selected V region sequences. Recombination is catalyzed by 2 enzymes, rag-1 and rag-2 (recombinase activating genes). The other regions of DNA not encompassing the selected coding sequences are put into a loop and are excised. Note that these enzymes are only expressed in B and T cells. This process of recombination is illustrated below:
How many gene segments are there in heavy chains that may be recombined? What are these segments called?
For the heavy chain variable domain, there are actually three gene segments that provide the DNA that encodes for the variable domain amino acids: V and J (as in the light chains) and also a D region ("D" for Diversity).
Approximately how many D regions are there? How many aa do they code for?
What enzymes catalyze the recombination?
There are ~30 of these D region gene sequences, and even though they may encode only 1-2 amino acids of the domain, each VDJ combination gives a different structure in the hypervariable regions of the heavy chain and therefore a different specificity to the antibody produced. Since there are 3 segments, these are even more variable than light chains which only have 2 segments.
Recominbination is catalyzed by rag-1 and rag-2 just like in the light and alpha chains.
Which chains (heavy or light in B cells, beta or alpha chains in T-cells) is recombined first?
Explain the order of recomibination of the segments of the heavy chain.
B-cells: heavy chain is recombined first, then light chain
T-cells: beta chain recombined first, then light chain
The D and J segments recombine first, then the V. see attached pic from slide 11 of course notes
What sequences to Rag enzymes recognize that allow them to recombine segments of genes for heavy and light chains?
Recombination Signal Sequences: highly conserved non-coding sequences next to coding sequences btwn V, D, and J genes (for heavy chain) and V and J (for light chains). RAG enzymes recognize these RSS. bind to them and form loop in DNA, excising the non-coded parts in btwn.
What is recombinational inaccuracy? What enzyme is involved in this?
What chain(s) does this occur in? (heavy, light or both)
Recombinational inaccuracies that occur when exons fuse further increase variability. While heavy chain variable regions are undergoing recombination (D with J first, followed by V with DJ), the enzyme terminal deoxyribonucleotidyl transferase (TdT) is active. This enzyme inserts bases randomly (without a template on the complementary chain) at the junctions of J with D and D with V. These are called N-nucleotide additions, because any base can be inserted during this process. This creates additional diversity in the messenger RNAs transcribed from this DNA. Tdt is not active when the light chains rearrange later, so this mechanism generates variabilityonly in heavy chains. Recombinational inaccuracy may also occur at the splice junctions when coding sequences are fused, with one or two base pairs beyond those that encode amino acid number 95 (for example) being utilized.
How does random assortment increase variability in Abs and TCRs? Explain this process.
Since light chains and heavy chain genes are unlinked, variable regions for each are selected at random for each cell, and since essentially any light chain can bind with any heavy chain to produce a different specificity, random assortment of heavy and light chains further increases variability. The same mechanisms apply to generation of T cell diversity.
Explain the role somatic mutations play in generation of Ig and TCR diversity.
Since the B cells divide rapidly in germinal centers of the lymph nodes, there are many opportunities for mutations. While these mutations can be detrimental to individual B cells (these cells are then eliminated), mutations which increase antibody affinity are preferentially selected, and mutations thus play a role in affinity maturation of B cell clones.
This is NOT a process involved in generation of T lymphocyte antigen receptor diversity. Mutations in TCR after antigenic stimualation invariably lead to clonal deletion. The thymus goes through a lot to ensure that TCRs have the exact right affinity for MHCs and antigens. If mutations were permitted, there would be no point of this rigorous selection process.
What is allelic exclusion? Why does it occur?
When one heavy chain gene rearranges to produce a functional gene product, it shuts off the rearrangement and expression of the other allele (on the homologous chromosome). This same phenomenon also occurs with the light chain genes. This process is called allelic exclusion, and is essential to assure that each B cell synthesizes only ONE heavy chain variable domain and only ONE light chain, and therefore makes only ONE antibody specificity. Except for rare mutations, all the progeny of this B cell will express the same VH and VL sequences, even if class switches occur during differentiation. Allelic exclusion also applies to the synthesis of the T cell receptor chains. Each T cell will express only ONE α and ONE β chain, or ONE δ and ONE γ chain.
What process determines whether an Ab will be secreted or anchored to the membrane?
Explain how/why IgD and IgM can be co-expressed on mature B-cells.
Immunoglobulin messenger RNA has information for both secreted or membrane anchored molecules, based on the amino acids encoded at the carboxy terminal end of the heavy chain.
Alternate RNA splicing also determines which heavy chain carboxyterminus will be expressed, which in turn determines whether the antibody will be secreted or membrane-anchored.
On mature B cells, IgM and IgD are coexpressed. This is possible due to alternate RNA splicing of long primary RNA transcripts that contain the coding sequences for both mu (m) and delta (d).
Both IgM and IgD on one B cell use the SAME light chain and the SAME VH sequence, so they have the SAME idiotype.
Cytokine released from what cells signal class switching of Igs produced by B-cells? What signaling molecule is required for this?
What part of the DNA is rearranged for class switching to occur? After class switching, how can B-cells revert to the original Ig isotype the expressed?
During the antibody response, the cytokine signals from helper (TH1, TH2, TFHcells (IL-4, IL-5, IL-6, IL-10), along with the stimulation from the surface CD40 molecule, signal the B cell to switch class. It does this by rearranging the DNA that encodes for the constant region of the heavy chain.
A loop is formed in the DNA between homologous switch regions in the 5' end of the non-coding DNA near each heavy chain constant region coding sequence. This is the mechanism for class switch between IgM and IgG (any subclass), IgA (either subclass) or IgE. The information in the loop is excised and degraded, so this information is lost. That is why class switching is a one-way street. Once a switch is made, the cell can never go back in the 5' direction, because the genes are gone.
NOTE that class switching does not effect the variable domain of the heavy chain, or the light chain. Thus the specificity of the antibody stays the same, even as the cell switches which isotype is expressed.