Immuno Exam 3 Flashcards
1
Q
What are 5 mechanisms of diversity in BCR?
A
- 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
2
Q
Antibody light chain genes encode how many families of DNA segments?
A
The antibody light chain gene encodes three families of DNA segments
3
Q
What are the three antibody light chain gene segments?
A
V,J,C
4
Q
How do you get different amino acids to randomly join?
A
combinations of V, J, and C allows diversity and amino acids to randomly join
5
Q
Where does the naive B or Tcell come from? What occurs to give diversity?
A
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
6
Q
How many genes are each TCR and BCR encoded by?
A
2 genes each for a total of 4
7
Q
How is the DNA rearranged?
A
through splicing and RNA tricks and with expression bits have been deleted and process differs from 1 cells to the next
8
Q
What did Tenegawa figure out about light chains when they are undifferentiated stem cells?
A
Light chains are unchanged in undifferentiated stem cells unless they become a B cell, then they have different combinations of segments
9
Q
Are there more V regions than C regions in k and lamba light chains? How does this effect the secreted forms of B cells?
A
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.
10
Q
How did they get DNA sequences?
A
Bits of DNA correspond to genes, receptors on surface and led to secreted form and purify amino acid sequence and get DNA sequences
11
Q
How to get recombination?
A
Connecting 1 V to 1 J and dropping out intermediates
12
Q
How is the k light chain formed?
A
DNA recombination between V and C region gene segments
13
Q
How many segments are light chain and heavy chain encoded be? What do they end up encoding?
A
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
14
Q
Are the lambda, kappa, and heavy chain genes on separate chromosomes?
A
Yes, each is on its own chromosome
15
Q
How many different genes does the light chain have? Which is more predominated in BCR?
A
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
16
Q
Which of the germ-line gene segments have more variability?
A
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
17
Q
What class of antibodies do naive B cells make?
A
IgM is first made for B-cell receptors
18
Q
Do the light chains have all functional products or some nonfunctional products? List specifically.
A
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
19
Q
What are the immunoglobulin variable region gene numbers for the V regions of heavy chains?
A
33-44 functional; 4 ORF; 79 pseudogenes
20
Q
Can you get nonfunctional or product that recognizes own self material?
A
False; There is a high failure rate
21
Q
Describe the two protein involved in V(D)J recombination.
A
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
22
Q
Are there base base between the V(D)J gene segments?
A
Yes
23
Q
Why are N region nucleotides important?
A
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
24
Q
What does RAG-1/2 protein recognize for recombination?
A
RAG-1/2 protein recognizes two conserved sequences in light-chain and heavy-chain in DNA that function as recombination signal sequences (RSS)
25
What is needed for recombination to occur?
RAG-1/2 protein identify 1 short RSS and 1 large
26
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
27
What are the steps of recombination of immunoglobulin V region genes?
1. binding of RAG 1/2 to V region
2. Synapsis- Matching up of RSS meaning one long V RSS to one short J RSS
3. RAG 1/2 protein cleaves and processes of signal and coding joints
4. TdT adds in N-nucleotides to heavy chain of BCR and both chains of TCR
5. Generate functional Ig variable region with a coding joint
28
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
29
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
30
What is the goal of P nucleotide addition?
To fill in overhands and get almost the same size as the strand was before
31
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
32
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
33
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.
34
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
35
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
36
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
37
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.
38
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
39
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
40
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.
41
What are three important outcomes of pre-BCR testing?
1. 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
2. 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.
3. If the first and second allele attempts don't work, then programmed cell death
42
What is the probability of creating a successful heavy chain gene?
55% There a huge failure rate
43
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
44
How does BCR get diversity?
Delete some V(D)J
45
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
46
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.
47
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
48
What receptor editing recombine with another allele?
Yes it can recombine with another allele for heavy, k, or lambda
49
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.
50
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
51
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
52
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.
53
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.
54
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.
55
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
56
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
57
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.
58
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.
59
Why are alpha and beta important to TCR?
If no alpha and beta then there is a big gap of defenses
60
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
61
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.
62
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
63
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
64
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.
65
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
66
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.
67
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
68
Do BCR and TCR both use multiple germ line V(D)J genes?
Yes, both TCR and BCR
69
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
70
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
71
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
72
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.
73
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
74
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.
75
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.
76
How do we achieve diversity in epitope presentation? 3 things!
1. Haplotypes (multiple tightly linked alleles)
2. alleles for MHC genes are all expressed: codominance (all copies present are expressed)
3. Antigen presenting groove in MCH allowing peptide diversity
77
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.
78
What are the two mechanisms for how multiple alleles are being expressed?
1. coming up on surface with little strings of amino acids on groove showing bits of self antigen
2. phagocytes engulf something puts bits of material engulfed and end up with huge number of copies on surface on either MHC II or MHC I
79
Are there a lot of different alleles expressed of MHC I and MHC II? What does this allow?
Yes, express different alleles and allows different forms of MHC I and MHC II to show different bits of peptide presenting self antigen and also alerts adaptive immune system. This how we get diversity.
80
Do class I and class II molecules look similar and similar binding grooves? What are their subunits of each? What spans the membrane and what receptors do they interact with?
Yes, class I and class II molecules similar and binding groove. Immunoglobulin folds on class I with alpha large subunit, alpha small subunit, and beta microglobulin and interacts with TCR on cytotoxic t cells. Class II molecules have two small alpha subunits and 2 small beta subunits interacting with each other and are tightly associated. both alpha and beta subunits span membrane. Interacts with helper T cell receptors to interact with appropriate peptide in binding groove.
81
How are the classes anchored at ends?
MCH I groove peptide 8-10 aa anchored at ends MHC II groove is open ended key residues hold peptide in place (hot dog shaped and aa anchored in bun)
82
What does the signaling do to MHC's from the receptors/
send both to proper T cell and antigen presenting cell too
83
Are the classes related? how?
Yes clearly related through immunoglobulin fold
84
How does the groove represent diversity? How does the aa bind to the groove of protein?
Groove in protein allows diversity by phagocytosis presenting or self antigen. Shorter peptides fit in groove. 1 aa or 2 match and MHC protein anchor in and bulge up. get one protein anchored in and allows diversity. Also get diversity through expressing multiple alleles of MHC I and MHC II and get diversity of peptides presented. MHCII is bigger and opens at ends and amino acid in bun stick in and diversity comes through variation with the rest of the amino acids. MHCI groove closed and no trough, ends are closed off and shorter peptides. anchor a peptide and bulge which is important for aa diversity
85
What is superimposed?
peptide-binding groove of MHC II superimposed over MHC I
86
What are 5 ways of peptide binding by class I MHC molecules?
1. Peptide binding domain- alpha 1/alpha 2
2. Nature of peptide-binding grooves- closed at both ends
3. general size of bound peptides- 8-10 aa
4. Peptide motifs involved in binding to MHC molecule- anchor residues at both ends of peptides
5.
nature of bound peptide- extended structure in which both ends interact with MHC groove but middle arches up away from MHC molecules
87
What are 5 ways of peptide binding by class II MHC molecules?
1. Peptide binding domain- alpha1/beta1
2. nature of peptide-binding groove- open at both ends
3. general size of bound peptides- 13-18 aa
4. peptide motif involved in binding to MHC molecules- conserved residues distributed along length of peptide
5. Nature of bound peptide-extended structure that is held at a constant elevation above the floor of the MHC groove (no bulges)
88
How do you get peptide diversity?
Put a lot of peptide diversity in MHC I cell of MHC II for helper T or cytotoxic T cells to interact with
89
How many copies of MHCI per cell? What is the frequent range?How many copies of each allele?
100,000 copies
the range is 100 to 4,000 copies of MHC and they are not on the surface unless peptide is with them.
Different alleles of MHC but get equal numbers of each allele to get two copies of each allele due to genetics=codominance. Different alleles of MHC per person
90
How many are presented in terms of peptides?
over 2,000 different peptides in MHC presented
91
Why is there a broad range of peptides presented on MHC?
sampling of cell for self. If self then no reaction of cytotoxic or virus reaction. Broad range of peptides to depend on anchors of aa and fair amount of variability in peptides that are being displayed.
92
How many hits to activate native Tc cell?
one hit on naive T cell can activate it and present a copy with Tc receptor recognizes leads to activation. interact with MHC to kill or not kill virus, cancer, etc.
93
What is Human HLA complex?
MHC is referred to as HLA complex in humans. MHC is organized into MHCI, MHCII, and MHCII gene products. There are classical molecules of MHCII and MHCI. MHCII has DP, DQ, and DR alpha/beta products and MHCI is HLA-B, HLA-C, and HLA-A. MHCII is gene products include other immune function-related compounds such as the complement proteins and tumor necrosis factors (TNF alpha and Lymphotoxin alpha), and other proteins. MHCI and MHCII have multiple copies through alpha/beta subunit structure. There are multiple gene products for each class.
94
What happens on the chromosome in terms of recombination?
There is not a lot of space for recombination, 1 out of every 500 may recombine where HLA complex is split to make the MHC class. Humans tend to see certain alleles clustered together and this complex is split up. Recombination of different alleles tend to stay together. HLA has the most diversity and mutations are okay because different alleles stay together
95
What is ahaplotype?
Certain alleles clustered together
96
What is codominance?
Various recombination of MHCI/II stay linked together on same chromosome and don't get separated by recombination. They all get expressed by same of different alleles
97
What do you get for a particular haplotype?
You get a particular allele. Ex. K halotype you get k alleles
98
What percent of the time is there crossing over and recombination of a haplotype?
1/2% (1/200 meiotic events) of time crossing of alleles. Crossing over allows recombinant haplotype. 4000 kb is a small enough region for recombination to take place
99
What happens during the inheritance of MHC haplotypes in inbred mouse strains?
The result of mating of mice with two different MHC haplotypes. 2 mice are bred for a particular haplotype.This leads to 2 mice with same MHC class but different alleles. MHC genes are closely linked and inherited as a set (tightly clustered MHC), leads to the progeny (baby) when 2 are bred leads to heterozygote haplotype. 1 chromosome has 1 haplotype and the other chromosome has other haplotype b/b+ k/k =(b/k)
100
What limits transplants?
waitlist for organs, wait till die, and find a match of MHCI/II. Finding a match limits transplants
101
What happens during skin transplantation between inbred mouse strains with same or different MHC haplotypes?
b/b parent will be able to donate to only b/b parent recipciept. same for k/k haplotypes. k/k recipient parent will reject b/b donor of parent.
The progeny will be a mix of both parents of b/k. The progeny cannot donate to parents are they are k/k and b/b and will reject it. Progeny can potentially donate to a sibling if it is also b/k. Progeny can get skin graft from either parent because either haplotype is recognized as self. Overall, acceptance or rejection of skin grafts is controlled by the MHC type of inbred mice.
102
What is common in humans in terms of haplotypes?
There are enough haplotypes that most marriages between individuals where 1 parent has 2 different haplotypes compared to other parent and these two individuals do not match. There is also another situation where both parents are a/b. This leads to 4 different haplotypes being involved and a 1/4 probability that progeny serves as donor or recipient with other offspring. Probability to get 2 of the same haplotypes in 1 progeny to parent is 1/4 as well. (getting a/a of progeny when one parent is a/a)
combinations: a/a, b/b, a/b, b/a
103
What is the issue with MHC in mouse experiment? How do MHCI/II limit transplant?
Proteins predominately displayed on cells immune system is good at recognizing differences. There are 40 different blood groups in the loci (ABO and Rh) depends on how exposed and A&B antigens are mimicked by antibodies exposed to glycoproteins of bacteria. MHC limit bone marrow transplant because MHC is involved to find matching blood.
104
What are MHC used for?
1. antigen/peptide presentation
| 2. implications of transplants and antigenic determinant
105
How many genes are there for MHCI? MHCII?
6 genes for MHCI per haplotype, same alleles and all copies are expressed. 6 different MCHI's overall. Mix and match of alpha and beta subunits. MHCII has 12 or more genes
106
What are allotypes? How does this help with diversity?
Alleles with in the loci. Diversity in alleles due to multiple alleles
107
Are MHC polygenic?
Yes, multiple gene copies all expressed (Codominance) hence they are polygenic due to a large number alleles at each locus
108
How do you mix and match subunits?
mixing subunits of different alleles allows all copies to be expressed and have different subunits of alpha and beta 2 microglobulin for MHCI and alpha/beta MHCII. Mix and match subunits for certain copy number of genes but can combine into more diversity in terms of surface of cells
109
How many allele number were found before and today?
before: 4*10^19 combinations of known class I and class I of alleles and 1.7*10^24 alleles by focusing on European populations (found more alleles). 5 additional orders of magnitude
110
How many copies and genes are there in a haplotype, explain the polymorphism?
There are 3 gene copies of MHCI in each haplotype. 1 gene-460 alleles, 2nd gene- 660 alleles, 3rd gene- 190 alleles at each lous. The alleles allow diversity. It is one of the most polymorphic genetic complexes. The number of aa differences between MHC alleles are 20 amino acid residues contribute to the structure of each allele
111
MHC and mate selection!
MHC diversity plays a role in mate selection. Aroma of person off different MHC. 1976 showed mate mice preference based off different MHC. Studies indicate that you can tell if you are interested in someone who has dissimilar MHC through odor. Rates of early pregnancy loss are lower in couples with dissimiliar MHC. A/B person likes C/D person (2 different haplotypes). Diversity plays a role in mate selection.
112
What are MHC and disease susceptibility?
Diploid where you have 2 different haplotypes leads you to be more susceptible to diseases. Particular haplotypes are better for a disease and increase levels of autoimmune disease, neurological disorders, and allergies. This is because some HLA alleles occur a a much higher frequency and diseases associated with particular MHC alleles are the ones mentioned. This can occur through the MHCI haplotype where it is a closed trough and aa bind for allelic diversity and allows things to happen.
113
MHCI, MHCII and dentritics in cells
MHCI in most cells, MHCII in rest and dentritic are MHCI and MHCII
114
What are the antigen-presenting cells?
dendritic cells, macrophages, and B cells
115
What was the swiss scientists experiment on sweet smell of diversity?
determined MHC dissimilar in humans are attracted to each other. female college students wore male shirts with out deodorants and perfumes. found percent of women preferred men with dissimilar MHC to their own. Humans and other vertebrates can tell different MHC interested in terms of odor
116
What occurs at the MHC loci?
Many haplotypes. 2 different haplotypes per person. The MHC provides more diversity and a way to fight off things
117
What is polymorphism?
Where multiple alleles concentrate in a region of a gene that encode groove in product.
118
Which subunit makes the groove in MHCI? MHCII?
The alpha subunit for MHCI. The alpha/beta make bun shape groove in MHCII
119
Codominance
same function, different structure and multiple gene copies in haplotypes, expressed as codominance with in have polymorphism
120
What are the roles of MHC?
1. peptide diversity- usually self peptides presented for self recognition. Also to demonstrate is cell is health or not through antigens
2. Less commonly foriegn peptides to show cell is infected, cancerous, old
3. MHCI- interacts with cytotocix T cells
MHCII- helper t cells
4. MHCI- sampling material with in cell usually self or non self
5. MHCII- phagocytes engulfing things are presenting those from extracellular spaces. Own cells in apoptosis or opsoninzing them.
121
What are two goals of T cell development?
1.
a. alpha/beta TCR each unique to a T cell
b. recognize appropriate MHC with "moderate" affinity
c. Does not recognize self epitopes presented with MHC
2. some gamma/delta TCR to recognize polysaccharide antigens (and others)
- want just right-> goldie locks interaction
122
What alpha/beta gene copies of T cell does it select for?
Select alpha/beta that recognize MHC with goldie locks effect, "just right"
MHC with appropriate affinity is important and those that do not recognize self epitopes
123
Describe T cell development in mouse.
hematopoietic stem cells (HSC that gives rise to all blood cells) then to DN1. DN1 T cell is not committed yet. Come become B cell, T cell of dendritic. After it is DN2 where T cell is now committed. DN3 is Beta selection, proliferation of T cell, DN4 is TCR alpha locus rearrangement. Then DP and death by neglect can occur or continue through positive/negative selection. Then DP selection makes CD 8+ for cytotoxic T and CD4+ for helper T
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DN is not..
CD4- or CD 8- expression. T cells final development is in the thymus then it migrates to blood and peripheral tissues
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What happens when TCR is committed in DN2?
1. rearrange TCR genes between beta gene and or other route genes. Can only be alpha/beta or other route
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What happens during DN3 and DN4?
beta selection, proliferation.
MHC affinity. loose majority of cells that don't make it there. if all goes well then rearrange small subunits. This created diversity of beta chain rearrangement to check out if it is okay. 10 percent is good to go. If good, then alpha chain allows proliferation to go on of beta chain. each well can then DN4 and can now rearrange its alpha locus rearrangement.
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Why is beta checked for affinity?
each cell rearrange alpha chain and clone of cells with same beta/alpha but different alpha chain rearrangement. Can recognize CD8+ on MHCI or CH4+ on MHCII cells
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What is DP?
Both alpha/beta expressed and recognize either MHCI or II. If don't then death by neglect occurs.
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What are affinity issues?
need affinity for MHC
1. find cells where MHC available. If TCR is not able to recognize MHC, will not get hormonal signals to keep going ("too cold)
2. Too strong affinity- get signal to keep going and stay there and not going on to development. too high affinity means not developing further.
3. death by neglect means not interacting with epitope
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What causes final commitment?
hormonal signals they are getting
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What is needed for T cell development from HMC cells? experiment?
Need notch 1 in order to grow.
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What is notch1?
A cell membrane receptor for the notch ligand. develop as t cell and get hits on notch 1 receptor.
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What happens when no notch 1?
Artificially make notch 1, where there is no notch 1 means no receptor. no contact with ligand for hits on receptor then develop as B cell.
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What happens when Notch 1 is expressed?
Receptors get hits and the ligand getting hits on the receptor means that it is committing step to T cell development. DN2 step
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What does 4 DN mean?
earliest T cells lack CD4 and CD8 and are DN. Thymocytes pass through stages defined by changes in cell surface phenotype. DN T cells divided into DN 1-4 based on presence or absence of cell surface molecules
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What important factors occurs at DN2?
all 3 chains besides alpha rearrange because TCR alpha genes are not accessible yet. Also T cell commit to T cel lineage and reduce expression of their surface molecules
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What important factors occurs at DN3?
1 out of 3 TCR chain make decision to join to TCR alphabeta or other TCR chain gamma/delta. If T cell rearrange to beta chain then alphabeta and loss expression of surface molecule to stop prolif and enter to final phase of DN4, and mature directly to CD4+, CD8+ DP thymocytes
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What important factors occurs at DN1?
thymocytes first enter thymus and able to give rise to multiple cell type. Express surface molecule expression patterns
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TCR choices which two chains?
alpha beta or delta gamma by how fast genes that code for each four receptor chains successfully rearrange
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Which lympocyte chain appear first during mouse fetal development?
Fetal animals make more dg during DN, but this drops after birth. DG early dominance can be 4 adaptive. After birth increase in ab thymocytes
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Pre-TCR signaling?
1. Maturation to DN4 stage
2. Proliferation in cortex
3. supress TCR beta chain gene, allelic exclusion of beta chain locus
4. develop CD4 and CD8 DP
5. end proliferation
6. start TCR alpha chain rearrangement
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TEST:
| When form pre TCR what 4 things the signal forms?
1. successful beta subunit then alpha TCR rearrangement
2. stimulates expression CD4 and CD8 coreceptors and now DP
3. stimulates proliferation as appropraite. clone of cells give different clone based on alpha chain for more diversity
4. Stops additional TCR beta chain locus rearrangement (allelic exclusion)
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What is pre TCR?
(beta selection when DN rarranged TCR beta chains, uses pre-T alpha chain that acts as surrogate for TCR alpha chain which has not been rearranged. CD3 and beta chain complex is pre-TCR. Pre TCR acts as a sensor to initiate signal transduc pathway
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Allelic exclusion for beta chain gene in Pre TCR
2 alleles for beta chain gene. 1 rearrangement, 1st succeed then exclude 2nd. leads to TCR with mature ab chain. stops additional TCR beta chain locus rearrangement. immature sign path way of DP leads to positive selection (want int affinity) and neg selection (high affinity)
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TCR worries about MHC recognition or foreign epitopes or self?
foreign epitopes
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Positive selection- during immature sig path DP
death by neglect. knocks out most candidates for those that don't recognize MHC at all. low or high affinity. only want intermediate. 98% get to this receptor die by DBN. selects for thymocytes capable of binding self MHC molecules
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Negative selection
not too high affinity for self antigen. if stays any longer will kill you. can have low affinity just not too high. small percentage of cells eliminated by NS.
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Neg and positive selection
Self peptides in thymus and associate with self-MHC molecules, ensure only surviving thymocytes express TCR that have low affinity for self peptides in self MHC. 2-5% of DP thymocytes exit thymus as mature T cells
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P and N selection in thymus during MHC selection
almost all don't interact with MHC and enter apoptosis by Death by neglect. Some high affinity interact with self peptides on self MHC and die by negative selection. Positive selection allow low/inter affinity. This is all due to MHC affinity
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Become Cd8+ or cd4+
TCR recognize MHCI or MHCII with appropriate affinity. co rceptors through DP forms MHII Cd4+ and MHCI Cd8+
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Additional neg selection for CD4+ and CD8+
Too strong affinity for self epitope. sticks to AIRE+ cells too long will die. AIRE+ cells express broader range of genes to present bits of surface immune cells to interact. express tissue specific antigens and can mediate neg selection.
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autoreactive clones in neg selection with dendritic and AIRE+ (neg selection) - mech that maintain self tolerance (second round of neg seelction)
clones recog self epitopes= don't want. if avoid interacting with self epitope then survive
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If some change of recognizing self epitope..
can control later
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Mature ab TCR with mature sig pathway
Signals lead to proliferation, survival, and effector function of cytokine secretion and cytotoxicity
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Survive neg selection leads to
T reg, T helper 17, and gamma/delta cell
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T reg neg regulate immune response- mech that maintain self tolerange
stop prolif of t cells and supress auto immune responses
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some goals to decrease T reg activity
1. elimate t reg before vaccination will get better effect ( boosters used to get enough antigens to get a response to mem cells)
2. eliminate those that suppress a response to a tumor antigen and not on surface of cell
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goals to selectively increase T reg activity
1. increase T reg activity to suppress transplant rejection (low rejection rate)
2. alter activity to treat allergies and autoimmune disease
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T reg cells used to
get cells that recognize same epitope and deal with them later for cells that got through self epitope and passed thru
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4 ways T reg cells inactiviate traditional T cells with FoxP3
alt for weakly autoreactive cells. not sure how T reg cells work.
combo of 2 or 3 mechs work to catch autoreactive ce;;s
1. cytokine deprivation
2. inhibitory cytokine
3. inhibiting antigen presenting cells
4. cytotoxicity
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T reg cel can only interact with same T cell receptor as they do. True or False
True
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Apoptosis
cell break open and DAMPs exposed. cascade of caspases that lack signals of pos and neg selection. death receptors induce apoptosis. DAMPS, some sigs for phagocytosis, and not inflammation. don't kill membrane and induce neighboring cels to engluf them before release any inflamm material.
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Necrosis
inflammation= DAMPS.
| cell death form toxicity, cell burst and swells triggering damaging inflamm response.
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What two cells induce apoptosis?
NK and T cytotoxic
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Functions of complement
Act as initiators :Specific binding leads to complement activation-3 initiators c1q, mbl, ficobilins
Enzymatic mediators-cleave glycoproteins in the cascade- pro to functional forms= C3 and C5 convertase
Opsonins- tag foreign material as foreign
inflammatory mediators bind receptors on capillary endothelial cells, which is essentially signal inflammation
assemble pores in membranes- MAC
Complement receptors
Regulatory components- membrane bound or soluble-> protect host cells
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3 ways complement protects host
Inflammation
Opsonins
MAC
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2 intermediates of classical pathway
3. C3 convertase allows for C5 convertase.
Need to hydrolyze C3 convertase in order of formation of C5 convertase.
4. C3b bind C5 convertase to cleave C5
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regulation of complement
1. dissociation of C1 components
2. cleave needed C3 convertase for each pathway- wont be able to continue pathway to activate complement
3. factor 1 always on, is a protease that cleaves membrane associated C3b and C4b
4. protectin prevents C9 from coming in and inhibits lysis
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Microbial evasion
1. intereference with antibody-complement interaction
a. antibody depletion by Staphyloccal protein A
b. removal of IgG by Staphylokinase
2. Binding and inactviation of complement proteins
3. protease-mediated destruction of complement component
4. microbial mimicry of complement regulatory components
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Order of pathways found
alternative, lectin, then classical
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SLE
failure to clear immune complexes leads to tissue damage difeciencies in early complement proteins such as C3b is needed. use CR1 for both WBC and RBC for immune receptor clearance
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of layers for regulation to keep complement from being too overly aggressive
1. sialic acid protection blocks mannose residues and allows complement to not be active
2. complement instability where there is no Factor P. C3b is in an inactive forms.
3. Proteases inactivate complement and are often enhanced by sialic acid, which is found on our own normal cel membranes. Proteases clear anaphylaxatoxins
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Opsinization of MBL, C3b, antigen-antibody complex
1. promotes receptor mediated phag
2. promotes immune complex clearance for phags and RCB via CR1
3. virus cannot be in a sutiable reaction for infection so coat complement opsonins on virus particle.
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What are receptors for opsonins?
PRR and DAMP.
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example of clearing immune complexes
Soluable C3b binds CR1 of erothrocyes and picks up immune complex and hanging it to phag to engluf
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C3D helps with activation of
CD21. C3d is a coreceptor protein that binds to antigen to help with B cell activation enhancement via IgM
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C3a and C5a most potent in anaphylaxatoxin and leads to
1. baso, neut, ecino, mast degran
| 2. chemotaxis. If C3a and/or C5a binds too robust then response is anaphylaxatoxin
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CR1 and CR2
CR1 bind immune complexes for clearance through liver and release complexes of antibody-antigen. RBCS in erythrocytes clear complexes phags go through immune complexes and deal with them.
CR2 enhancement of B cell activation where native B cells with B cell receptors with IgM dont want to be too active. Complement hits CR2 and complement is actiavted
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C3 or C5 convertase Reg proteins
prevent making holes in our own membranes. limits pores from own thing or normal cell. appropriate levels of anaphylaxis and opsonins
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C8 and C9
C8 makes small pores leads to influx of Ca ions which leads to apoptosis
C9 most effective forms larger pore
and most effective level of dealing with foreign invader
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Enveloped virus
MAC pores forms in envelope and renders them to no longer infect host, however they have strategies to evade compleemtn
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comp
shut down or help kill off immune response
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C5- MAC cascade
defects later are forgiving if have C5 convertse sequence assembly of proteins from membrane attack complex.
C5 is activated by fragments with initial glycoproteins that are active when cleaved. Products that are active are C5b that interacts with C6 and allows C7 to interact then change conformation. Then C8 and then C9. This is a cascade.
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worst case of anaphylaxatoxin
overreaction. induce too much degranulation and too much inflammation. but can also fight off an infection with them
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complement clears what to damp down immune response- complement in contraction phase of immne response
contraction phase means no overreaction of immune response. shut down immune response and clear immune complexes. antibody-antigen, induce Treg cells- help shut down immune response, and apoptosis of cells.
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Why do antigen antibody complexes need to be cleared
Act as a red flag for inflammation, needs to be cleared out
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comp altert T reg
this is interface between innate and adaptive. comp alter adaptive to shut down immune system.
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Tickover pathway
activation of key components but unstable at normal cell surface. regulatory proteins inactivate it
189
complement cascade is activated by three pathways. true or false
true
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instability
helps complement cascade keep in check of some key components and by regulatory proteins on normal cell surfaces
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Three main classes of complement activity in the service of host defense
1. innate defense against infection
- lysis, opsonization, inflam/chemotaxis by anaphylatoxin
2. interface between innate and adaptive
- augmentation of antibody responses, ehancement of immunologic memory, enhancement of antigen presentation
3. complement in the contraction phase of immune response
- clearance of contraction phase, clear complexes from tisues, clear apoptotic cells, induce T reg cells
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polygeny
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multiple genes in the genome w the same function
ensures that even MHC homozygous ind express 3 diff class I and II proteins, each w diff binding profile to expand types of antigens presented
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polymorphism
presence of mutliple alleles for a given gene locus within the population. MHC polymorphism and codominant expression facilitate inheritnce and expression of alleles at each locus, increase number of antigens individuals can present
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codominant
ability of an individuals to express maternal and paternal alleles in genes in same cell.
