Immuno Exam 3 Flashcards
What are 5 mechanisms of diversity in BCR?
- multiple gene segments V(D)JC
- P-nucleotide additions
- hairpin and fill in overhang on template based on sequence and allele of gene (P-nucelotide addition)
- V to J, D to J
- clipped blunt ends offset cleavage=variable amount of P nucleotide addition - exonuclease clipping- can be extensive sometimes all of D deleted more common in heavy chain joins but is seen in light chain too.
- none-templated N-nucleotide addition. This follows exonuclease clipping by TdT adding random bp through N-nucleotide which is more randomness throughout the process. It is template independent
- combination of heavy and light chains. There are 2 million variations of combinations and over a billion variants in BCR
Antibody light chain genes encode how many families of DNA segments?
The antibody light chain gene encodes three families of DNA segments
What are the three antibody light chain gene segments?
V,J,C
How do you get different amino acids to randomly join?
combinations of V, J, and C allows diversity and amino acids to randomly join
Where does the naive B or Tcell come from? What occurs to give diversity?
Hematopoetic stem cell then different combinations of regions occurs then it becomes a naive B or T cell. cut off bits from different combinations to give diversity
How many genes are each TCR and BCR encoded by?
2 genes each for a total of 4
How is the DNA rearranged?
through splicing and RNA tricks and with expression bits have been deleted and process differs from 1 cells to the next
What did Tenegawa figure out about light chains when they are undifferentiated stem cells?
Light chains are unchanged in undifferentiated stem cells unless they become a B cell, then they have different combinations of segments
Are there more V regions than C regions in k and lamba light chains? How does this effect the secreted forms of B cells?
Yes, more V regions and few C regions. When class switching from IgM exchange a C region to become IgG or IgA. Keep the V region but change C regions.
How did they get DNA sequences?
Bits of DNA correspond to genes, receptors on surface and led to secreted form and purify amino acid sequence and get DNA sequences
How to get recombination?
Connecting 1 V to 1 J and dropping out intermediates
How is the k light chain formed?
DNA recombination between V and C region gene segments
How many segments are light chain and heavy chain encoded be? What do they end up encoding?
Light chain has 2 segments V and J and heavy chain encoded by three segments V,D, J. Both have C gene segment downstream. They end up encoding an amino acid chain
Are the lambda, kappa, and heavy chain genes on separate chromosomes?
Yes, each is on its own chromosome
How many different genes does the light chain have? Which is more predominated in BCR?
2 different genes. One lambda light chain gene and one kappa light chain gene. Lambda light chain is predominated and there are some kappa light chain genes
Which of the germ-line gene segments have more variability?
lambda chain has more variability out of the light chains. There are more C regions. Kappa is easier and less variable with C region. The heavy chain has the most variation and provides additional variation to BCR and needs more C regions
What class of antibodies do naive B cells make?
IgM is first made for B-cell receptors
Do the light chains have all functional products or some nonfunctional products? List specifically.
Kappa light chains- V segment has 34-48 functional; 8 ORF; 30 pseudogenes and J segment has 5 functional; multiple alleles. The lambda light chain gene V segment has 33 functional; 6ORF; 36 pseudogenes then J segment has 5 functional; 2 ORF
What are the immunoglobulin variable region gene numbers for the V regions of heavy chains?
33-44 functional; 4 ORF; 79 pseudogenes
Can you get nonfunctional or product that recognizes own self material?
False; There is a high failure rate
Describe the two protein involved in V(D)J recombination.
1) RAG-1/2- Lymphoid-specific complex of two proteins that catalyze DNA strand breakage and rejoin to form signal and coding joints (both in TCR and BCR)- rearrangement will not occur without this protein
2) TdT-lympohid-specific protein that adds N region nucleotides to the joins between gene segments in the Ig heavy chain joints in BCR and at all joints between TCR gene segments. Add N region nucleotides
Are there base base between the V(D)J gene segments?
Yes
Why are N region nucleotides important?
TdT adds noncoding nucleotides (N-nucleotides) which are random nucleotides to add in random amino acids for more variability. May get nonfunctional products or products that recognize self molecules but need diversity of BCR or TCR to recognize different foreign material-only in heavy chain of BCR and both chains in TCR
What does RAG-1/2 protein recognize for recombination?
RAG-1/2 protein recognizes two conserved sequences in light-chain and heavy-chain in DNA that function as recombination signal sequences (RSS)
What is needed for recombination to occur?
RAG-1/2 protein identify 1 short RSS and 1 large
What is needed to generate a complete light chain gene? What are the summary of events?
All events need to be done to get a complete light chain gene. Recombination between gene segments is required to generate a complete light chain gene.
1) germline where it chooses which V and which J out of cluster of gene families
2) DNA recombination and RNA splicing
3) Some V and J segments that are left behind and not chosen are not expressed and spliced out
What are the steps of recombination of immunoglobulin V region genes?
- binding of RAG 1/2 to V region
- Synapsis- Matching up of RSS meaning one long V RSS to one short J RSS
- RAG 1/2 protein cleaves and processes of signal and coding joints
- TdT adds in N-nucleotides to heavy chain of BCR and both chains of TCR
- Generate functional Ig variable region with a coding joint
What is the mechanism of V(D)J recombination?
1) RAG 1/2 recognizes RSS
2) Clips DNA and then ligates them together (joining) but not this simple (goal)
3) Hairpin is open and results in overhangs or blunt ends
4) for variability, P nucleotides are added which creates sloppyness. Enzyme that adds P nucleotides fill in gaps from blunt ends and ligate them together
4) segments join together and get additional nucleotides due to P nucleotide
5) Variability for a good adaptive immune system in BCR and TCR genes is through exonuclease where there is a loss of nucleotides on either or both sides of joints (only heavy chain joints of BCR or both of TCR)
6) TdT adds in nontemplated nucleotides
What is the overarching big picture of V(D)J recombination?
1) RAG 1/2 recognize RSS
2) Protein clips DNA and intervening DNA is lost and V(D)J is chosen
3) TDT comes in and adds noncoding nucleotides depending if TCR or heavy chain BCR
What is the goal of P nucleotide addition?
To fill in overhands and get almost the same size as the strand was before
Can recombination occur between DNA segments that are flipped (opposite) or the same transcriptional direction on chromosome?
Yes, recombination can occur between DNA segments aligned in the same or opposite transcriptional direction on the chromosome. Some regions can get flipped. Any V to J region can join together as long as it is a short RSS with long RSS and it can even be flipped
List increasing to decreasing combinational antibody diversity of gene segments for heavy and two light chain genes? Which of the genes have the most combinations?What are the possible number of heavy-light chain gene combinations?
V has that most combinational diversity in all three genes (41,41, 33), then D (23), and then J(6,5,5). Heavy chain gene has the most combinations, then kappa, then lambda. Any heavy with a light chain has over 2 million combinations in humans
What does the addition of P-nucleotides and N-nucleotides do in respect to functional products? What is the trade-off?
WIth addition of P-nucleotides in all chains and N nucleotides (heavy BCR and both TCR), you get random nucleotides and get out of frame shifts, leading to nonfunctional product. This is a trade of where the benefit is more variability/diversity and the cost is when you add 3 bp at a time and add random nucleotides, throws off the gene. Allows 3 orders more magnitude of diversity.
What was first appeared in jawed vertebrates?
emergence of RSS-dependent recombination-based adaptive immune system coincides with the first appearance of the jawed vertebrates
How do B-cell receptor get its final expression of its genes?
1) Gene in hematopoetic stem cells
2) Clip off bits
3) May get nonfunctional product through frame shift
4) If there is a functional product, test to see how well it works and if it recognizes self antigens
How does pre-BCR receptor testing work of heavy chain work?
With two components of the heavy chain gene it puts out testing components called VpreB and lambda5 signals. These two components substitute for light chains
What is allelic exclusion?
Progenetor B cell progression tries heavy chain gene first and works with surrogate light chain with 1 allele at a time. There is a vast majority that gives us something that does not work. The success rate is low. Start with heavy chain allele, the pre BCR excludes the second chain allele of heavy. If the first allele works through allelic exclusion, then it rearranging light chain gene.
What are the steps of pre-BCR testing?
If the first attempt at the heavy-chain gene first is unproductive due to P and N nucleotides or exolnuclease trimming at joints causing unproductive product, then rearrangement of nucleotides occur at second heavy chain allele. If the second copy of heavy chain is also unsuccessful, the B cell will die
What happens with testing of pre-BCR if it works?
If the heavy chain gene that is tested with surrogate light chain works, then test to see if it recognizes self antigens or not. If the 1st or 2nd copy works, then pre-BCR receptor is placed there and will progress to maturity
What occurs to light chain locus of K or lambda if testing is successful?
Once a complete heavy chain is expressed, light chain rearrangement begins at second heavy chain allele. In humans light chain rearrangement may begin at either kappa or lambda light chain locus. After light chain gene rearrangement is complete, it results in expression of BCR on cell surface.
What are three important outcomes of pre-BCR testing?
- Prevents rearranging 2nd heavy chain allele if the 1st heavy chain allele works. This is important because you only want one functional naive B cell and don’t want different receptors recognizing different antigens (can be messy). The prevention of rearranging the second allele is important because if the first allele works, then there is no recombination of the second heavy chain allele
- If the first heavy chain allele doesn’t work then use the second allele. If that second allele works then you can express light chain gene. Allelic exclusion is out the window at this point.
- If the first and second allele attempts don’t work, then programmed cell death
What is the probability of creating a successful heavy chain gene?
55% There a huge failure rate
What is the end goal of BCR after pre-BCR testing?
Want one heavy chain allele and one light chain allele. The other copies are excluded through allele exclusion
How does BCR get diversity?
Delete some V(D)J
How is the success rate of generation of functional immunoglobulin receptors determined for productive arrangement at heavy and light chain alleles?
Success rate is low and screen for self antigens. Appropriate number of functional BCR depends on genetics and how good of an immune system you have
What is receptor editing? What gene is often edited?
The immune system can make changes after completed receptor is on cell surface. If the receptor is found to be auto-reactive (recognizes self antigens), the cell can swap it out for a different one in the receptor editing process. Receptor’s binding site has both heavy and light chain, just swapping just of one of these us good enough to change specificity. Kappa light chain receptor editing is show frequently.
What is a specific example of kappa light chain receptor editing in respect to how it functions?
1) first arrangement between V3 and J3 is productive, but combination of the resultant light chain with Ab heavy chain results in an autoimmune antibody
2) Second rearrangement betweenV2 and J4 is productive, and combination with the Ab heavy chain results in an non autoimmune antibody
What receptor editing recombine with another allele?
Yes it can recombine with another allele for heavy, k, or lambda
What is the importance of poly A sites?
Proteins select particular choices of Poly A sites and RNA splicing sites. This makes a primary transcript of RNA after mRNA is spliced. It allows more diversity as a result of membrane bound forms of IgM that B cells express.
What is differential expression of membrane forms of u and delta chains and how is it done? Which chain is membrane form?
Differential expression of secreted and membrane forms of u and gamma for heavy chain is done by alternative RNA processing. u chain is membrane form, IgM
What is the C region for and what is the poly A sites for?
C region is for M or D form. Poly A stie is for IgM membrane bound form or soluble form
Describe the process of an antibody being membrane bound to secreted.
Have a naive B cell and have poly A sites for IgM which is membrane bound form of antibody. Want B-cell receptor on surface of naive B cell and scan for antigen recognized. If activated, it gets assistance from helper T-cells and helps get it differentiated into a plasma cell and now secretes antibodies. Poly A sites are now at a different location from the IgM membrane bound form to secreted form where there is no membrane bound domain.
Why is splicing important?
Splicing is important because going from primary transcript to RNA copy of gene sequence and need to splice out bits we don’t want to express and get to mRNA and have recombined bits that we want to express. extra copies not being expressed are clipped out.
What are the importance of splicing and poly A? What are the steps for alternative RNA processing?
poly A gets us from membrane form or secreted form and splicing is to get just gene segments you want for particular naive B cell. For alternative RNA processing need poly A and then RNA splicing.
How do you get from membrane found form to secreted?
clip off a poly A site through mRNA splicing and get secreted form by using different poly A site. Then with that poly A site, differentiate into a plasma cell and get secreted forms of antibodies
What is the final composition of a B-cell receptor from a naive B cell? How many genes total are there?
1 allele for light chain and 1 allele for heavy chain. Recognize one epitope per foreign material. 2 genes total
What are T-cell receptors made of? How many genes total?
alpha and beta subunits are always in T-cells and small subset of gamma delta. There are four TCR genes in total.
Why is TCR harder puzzle to figure out than BCR? What did they do to fix this?
There are no secreted forms of TCR to assist tracking down. TCR is always on surface of cell. They came up with a method to trick subtractive RNA hybridization to narrow search of B cells vs. T cell RNA.
Why are alpha and beta important to TCR?
If no alpha and beta then there is a big gap of defenses
What was the hybridization method used to sequence TCR?
TCR genes are different than the two genes of BCR but they come from the same lineage. The genes expressed in T cells are not expressed in B cells. This is called substrate RNA hybridization
How does substrate RNA hybridization work?
Use closely rated cell so most RNA expressed match up and subtract out genes that are not TCR. Genes we want is in one cell type and not the other. Get some genes that overlap, but get rid of genes that are not TCR.
How did scientists produce and ID cDNA clone encoding the T-cell receptor beta gene?
take RNA from 1 cell or complementary RNA from other so RNA sequence from one can hybridize with the other. Use reverse transcriptase to to complementary base pair through making a cDNA. They will hybridize if genes are expressed in both T and C cells. Enrich for SScDNA genes only expressed in T cells and discard cDNAs that hybridized with B cell mRNA. Recover T cell specific cDNAs and use them to ID hybridizing DNA clones from a T-cell specific library
What is the key to the puzzle of TCR in terms of gene rearrangement?
If rearrangement is different in each cell then compare the controls of different T cell clones which gives different recombination. This means the controls are B cell and liver cells which have no rearrangement but different T cell clones have different rearrangement patterns. Genes look different in T cell clones because gene fragments are rearranged and restriction sites are close together
What gene did they find through this method?
The major Beta gene of TCR was cloned and the size was right for the alpha subunit and both subunits glycosylated.
How did they find out that there are other subunits?
Saw that alpha and beta are glycosylated and saw there is a different subunit by looking at the amino acid sequence and saw that one is not glycosylated and found the other two gene segments
What is different of TCR from BCR?
for TCR you can either express the alpha/beta route or gamma/delta route. The segments are interspersed. Recombine to get one and get get the other. For BCR try heavy chain first then try different light chain genes.BCR can only go with kappa or lambda light chain gene.
Why are pseudogenes important?
Better for the immune system. Gives variation and diversity of epitope recognition of antigens. They are random events by duplication and random mutation for diversity and leads to a better immune system
Do BCR and TCR both use multiple germ line V(D)J genes?
Yes, both TCR and BCR
Do BCR and TCR both use light chain segment use?
No, B cells use kappa and lambda variable regions encoded by V and J segments and T cells use alpha and gamma variable regions encoded by V and J segments
Do BCR and TCR both use heavy chain segment use?
BCR uses V heavy regions encoded by V,D, and J segments and TCR uses beta and delta variable regions encoded by V,D, and J segments
Do BCR and TCR both use RAG 1/2 for expression? How about junctional diversity of P and N nucleotide addition?
Yes to both for TCR and BCR
Does TCR have secreted form on activation and secrets product with the same binding site as the receptor?
BCR has secreted form when activated by antigen and TCR is not found in secreted form. C region of TCR does not chain. BCR can class switch from membrane bound IgM through changing C region for class switching.
Does the receptor genes of both BCR and TCR undergo somatic hypermutation following antigen stimulation?
Additional diversity only in B cells and not in T cells
What is SCID? Why is it bad?
Disease when mutation in either RAG 1 or RAG 2. It is bad because no B or T cells will be made. Adaptive immunity is knocked back. There is no diversity of BCR and TCR for adaptive leads to risk of infection.
Developing fetus with SCID after birth rate have..? How do you resolve this, what difficulties may one have when trying to resolve?
IgG crosses placenta. Antibodies from mother so most commonly see effects of no B or T cell susceptible. More susceptible to fungal infections or viral infections. Developing infant cannot develop own T-cells. Individual with SCID will not last long. Resolve it with bone marrow transplant from hematopoietic stem cells (reproductive ability) will help with this issue. Issue with bone marrow is getting a match.
How do we achieve diversity in epitope presentation? 3 things!
- Haplotypes (multiple tightly linked alleles)
- alleles for MHC genes are all expressed: codominance (all copies present are expressed)
- Antigen presenting groove in MCH allowing peptide diversity
What does the MCH II interact with? How about MHC I? Which one is more abundant?
MHC II interacts through the helper T (TH) cells through CD4+ proteins on surface. MHC I groove interacts with Tc cells that are CD8+. Most cells have MHCI and the rest have MHC II.
