Antibody Genetics Flashcards
1
Q
The Great Dilemma
A
How can we generate 10^9 or more different antigen receptors (TCRs and BCRs) when the human has lesson than estimated 25,000 genes?
- this total variety of antigen receptors is called our antigenic repertoire
2
Q
Germline theory:
A
“One gene for one protein”
This theory suggested that the human genome contained one gene for every different BCR and every different TCR we make and display
- we now know that this can’t possibly be correct
3
Q
Somatic mutation/diversification theory
A
Tonegawa proposed the somatic mutation/diversification theory which has 2 components
- a limited number of genes can randomly combine with one another
- the limited number of genes can mutate to increase the diversity
Each of these creates greater diversity, but together they create billions of combinations
4
Q
What cells undergo somatic recombination?
A
Somatic recombination occurs in the assembly of BCR and TCR genes
5
Q
Why is it that only these types of cells undergo this type of genetic “gymnastics”?
A
??
6
Q
A person’s antigenic repertoire develops as the result of the recombining of these limited gene sets, combined with high rates of mutations within those genes.
Prior to rearrangement they are said to be in the ___________ because this is how they are present in the germ cells (eggs, sperm)
A
Germline configuration
7
Q
The process of BCR/Ig & TCR rearrangement is called ___________ because this process occurs in the somatic cells - those cells that are not germ cells.
A
Somatic recombination
8
Q
What is an exon?
A
Parts of the DNA sequence that code for the protein
9
Q
What is an intron?
A
Parts of the DNA sequence that do not code for the protein
10
Q
What is the importance of having multiple introns and exons in the genes that make up our antibodies?
A
To produce a different protein (antibodies) from the same RNA transcript
11
Q
What is alternative splicing?
A
Splicing the RNA transcript different ways
Creates different mRNAs and therefore different proteins
12
Q
Does alternative splicing occur to DNA or RNA?
A
RNA
13
Q
Each L Chain contains ___ regions namely __________
A
Two; Variable region (VL) and a constant region (CL)
14
Q
How many genes encode the CL? What are these genes called/named?
A
1 gene- C
Ckappa or Clambda
15
Q
What is CDR?
A
The complementarity determining region ie hypervariable regions
- complementary to the epitope
16
Q
How many CDRs are there in the L chain?
A
3
CDR1, CDR2, CDR3
17
Q
Where are the CDRs located within the VL (in which genes are they located)?
A
Located within the V & J genes
Variable gene- Vkappa or Vlambda
Joining gene- Jkappa or Jlambda
18
Q
How many genes encode the VH?
A
3 GENES- V,D,J
Variable gene - VH
Diversity gene- DH
Joining gene- JH
19
Q
How many genes encode the CH? What are these genes called/named?
A
1 gene- C
20
Q
Where are the CDRs located within the VH (in which genes are they located)?
A
Variable gene - VH
Diversity gene- DH
Joining gene- JH
21
Q
Rearrangement of the variable regions (for both L and H chains) is referred to as ______________.
A
V(D)J recombination
22
Q
What are the DNA sequences called that regulated the recombination of these genes in the H and L antibody chains?
A
Recombination signal sequences (RSS)
23
Q
Where are these regulatory sequences found?
A
These RSS sequences flank each V,D, and J gene in both the H & L
24
Q
Review Gene arrangement!
A
!!!!
25
Allelic exclusion
We have two copies/alleles of each gene- 1 maternal, 1 paternal
Genes from both chromosomes for the H chain (maternal allele & paternal allele) attempt simultaneous rearrangement.
- also occurs in the Kappa genes in the L chain
When one is successful, the other stops ensuring that each cell will express only one of the genes/alleles and will have a BCR of one specificity
- this is called allelic exclusion
26
Isotype exclusion
If the Kappa genes are unsuccessful, the lambda genes (both maternal and paternal alleles) attempt to rearrange
- this is called Isotypic Exclusion
- meaning each antibody molecule will have either kappa or lambda chains
- kappa goes first, inhibiting the lambda alleles
27
What happens if the H chain or the L chain do not have a successful rearrangement?
If neither allele of H chain makes a successful rearrangement, the cell dies
If neither the Kappa or lambda Cain genes make a successful L chain, the cell dies
28
Summary of rearrangement: in order for a complete Ig to be made/expressed.....
Individual genes of the H chain (V,D,J,C) must rearrange (in frame) to make a functional H chain mRNA and protein
Individual genes of the L chain (V,J,C) must rearrange (in frame) to make a functional L chain mRNA and protein
29
Explain why it is that once the H chain genes (VDJ) are rearranged, that we say antigen specificity for the H chain is now set.
Because once the H chain genes are rearranged, DNA is permanently altered
30
What is the process that makes both an IgM and IgD H chain, both with the same VDJ genes, but one with the micro C gene and the other with the delta C gene?
Alternative splicing
VhDhJhCmicroCdelta—>
VhDhJhCmicro or VhDhJhCdelta
31
Since both the IgM and IgD transcripts (and therefore proteins) have the same VDJ genes, they will have identical ___________
Antigen specificity
32
Why is it important for naive cells to have IgD on the surface?
Variable gene - VH
Diversity gene- DH
Joining gene- JH
33
Why is it important for naive cells to have IgD on the surface?
IgD is essential to active B cells
34
Are the IgM and IgD BCRs on this naive follicular B cell identical? How are they same? How do they differ?
No.
Contain the same antigen specificity, but different Constant Heavy chain regions.
IgM- Cmicro
IgD- Cdelta
35
What happens when we need a different isotype of antibody? What is the process called?
Isotype switching - type of DNA Rearrangement
36
When the isotype is changed, what remained the same in the antibody and what gets changed?
The heavy chain is changed - altering isotype
Variable region unchanged- therefore, maintaining antigen specificity
37
Explain the difference between alternative splicing and isotype switching?
Alternative splicing- splicing of the primary RNA transcript
Isotype switching- germline DNA rearrangement
38
Which L chain genes are rearranged first in humans?
Kappa
39
How is lambda gene rearrangement different from kappa gene rearrangement?
Kappa first then lambda
40
Is the fact that kappa gene rearrangement occurs prior to lambda rearrangement allelic or Isotypic exclusion?
Isotypic exclusion- kappa before lambda so each antibody molecule will have either kappa or lambda chains
Allelic exclusion- maternal vs paternal alleles
41
What is the function of the RSS in Ig gene rearrangement?
Conserved DNA sequences that regulate recombination events
| - these RSS sequences flank each V,D,and J gene in both the H&L chains
42
What are the parts of each RSS?
3 components
- Hepatmer (7 bp): always next to the gene
- Spacer
- Nonamer (9 bp): always away from the gene
Nonamer:spacer:heptamer-gene-heptamer:spacer:nonamer
43
What are the 2 types of RSSs?
One-turn RSS has a 12 bp spacer
Two-turn RSS has a 23 bp spacer
44
Explain the 12/23 (1 turn/2 turn) rule.
A 12 bp spacer RSS is always moved next to a 23 bp spacer RSS - called the 12/23 rule or 1 turn/2 turn rule
45
What is the name of the enzymes that carry out the recombination events?
Endonuclease RAG-1: DNA is nicked
46
What prevents 2 D genes from being rearranged next to each other or from skipping the D genes and rearranging a V gene with a J gene in an H chain?
RSS sequences
47
What are the four ways of generating diversity in BCRs/antibodies?
1. Combinatorial diversity
2. Combinatorial association
3. Junctional diversity
4. Somatic hypermutation (Antigen dependent)
* 1-3: Antigen Independent
48
Combinatorial Diversity
Random joining of VDJ genes of the H chain
Random joining of the VJ genes of the L chains
- ie V(D)J recombination occurs at the DNA (gene) level
49
Combinatorial Association
Any L chain (kappa or lambda), with any combination of VJ genes, can associate with any H chain, with any combination of VDJ genes, to form a functional antibody
- in 2 different cells, there could be the same H chain combining with different L chains (or vice versa)
Occurs at the PROTEIN (chain) level
50
Junctional Diversity
Imprecise joining of the VDJ and VJ genes
- P nucleotide addition
- Junctional flexibility
- N nucleotide addition
Involves the addition or subtraction of nt’s within the DNA
51
Somatic Hypermutation
The ONLY mechanism of generating diversity that occurs AFTER antigen exposure
- involves point mutations within the V regions of both the H & L chains
- Does NOT change the number of nt’s
52
Somatic hypermutation is the mechanism behind the phenomenon called _______________.
Affinity maturation
- results in the generation of antibodies with increased affinity for their cognate Ag
- the reason that this type of immunity is adaptive
53
Which of these four mechanisms occur during B cell development (ie in the bone marrow) and which of these mechanisms occur after B cell development (ie after activation, in the secondary lymphoid organ)?
Combinatorial diversity, combinatorial association, junctional diversity- occur during B cell development in the bone marrow. Prior to, and independent of, antigen exposure
Somatic Hypermutation- after antigen exposure
54
Which enzyme involved in these processes requires a template to add to the DNA strand?
DNA polymerase during Junctional Diversity - P nucleotide addition
Adds nt’s to the overhang (caused by endonuclease RAG-1 nicks the DNA leaving a hairpin loop which is then cleaved by endonuclease Artemis leaving an overhang)
55
Which of these enzymes involved in these processes does not require a DNA template to add to the DNA strand?
TdT - terminal deoxynucleotidyl transferase adds nucleotides
- during Junctional Diversity: N-nucleotide addition
56
Why is only CDR3 affected by P-nucleotide addition, Junctional flexibility, and N-nucleotide addition?
Because its at the end of the V region
???
57
Which CDRs can be affected by somatic hypermutation? Why?
All of them because somatic hypermutation is a point mutation that can occur anywhere within the V region.
58
Why does N-nucleotide addition only occur during H chain gene recombination?
Due to temporal expression of TdT enzyme
| - ie TdT enzyme is not present during the pre-B cell step of development when the L chain rearrangement occurs
59
What is meant by antigen-independent vs antigen-dependent changes?
Antigen-independent: occur during B cell development in the bone marrow- prior to, and independent of antigen exposure.
Antigen-dependent: after antigen exposure
60
Which of these mechanisms that we discussed are antigen-independent and antigen-dependent?
Antigen Independent: combinatorial diversity, combinatorial association, junctional diversity
Antigen dependent: somatic hypermutation
61
What is the role of follicular dendritic cells during affinity maturation?
Presentation of antigen to B cells thereby driving their affinity maturation (generation of ab’s with increased affinity for their cognate Ag- adaptive immunity!)
B cells interact with Ag on the FDCs to see if mutations have made their ab’s better, worse, or unchanged
- B cells only with the better ab’s receive survival signals from the FDCs
62
Clinical Correlation: Mutation in either RAG-1 or RAG-2 can result in ________________.
A lack of mature B cells
63
Clinical Correlation: Mutation in the endonuclease Artemis can cause ___________________.
A lack of mature B cells
64
Heavy chains are composed of how many genes? What are they?
4 chains
| Vh, Dh, Jh, and Ch
65
Light chains are composed of how many genes? What are they?
3 genes
| VL, Jl, and CL
66
What are the two types of L chains?
Kappa and lambda
67
Explain how the 12/23 rule shapes the specificity of an antibody molecule for an antigen.
• Because a 12 base pair spacer must be paired with a 23 base pair spacer therefore controlling shape.
68
Which mechanism(s) of generating antibody diversity occur during B cell development – i.e. are antigen-independent?
* All except somatic hypermutation
* Combinatorial diversity (random joining of VDJ in H chain and VJ in L chain), Combinatorial association (random association of H and L chains), Junctional diversity - P nucleotide addition, Junctional flexibility, N nucleotide addition
69
Which mechanism(s) of generating antibody diversity can change the number of nucleotides?
• Junctional diversity- involves the addition or subtraction of nucleotides within the DNA
◦ P nucleotide addition- RAG-1 (endonuclease), Artemis, DNA polymerase
◦ Junctional flexibility- Exonuclease (removes up to 10 nt’s)
◦ N nucleotide addition- TdT (adds up to 10 nt’s), DNA polymerase, ligase
70
Which genes are affected by SHM?
• Point mutations within the V region of both H and L chains
71
Which genes are affected by isotype switching?
• Ch genes
72
Explain the difference between combinatorial diversity & combinatorial association.
* Combinatorial diversity: Random association of VDJ genes in H chain and VJ genes in L chain
* Combinatorial association: Random association between H and L chains
73
Explain the difference between allelic exclusion & isotypic exclusion
• Allelic exclusion: We have two copies/alleles of each gene (maternal and paternal). Both attempt simultaneous rearrangement. When one is successful, the other stops ensuring that each cell will express only one of the genes/alleles and will have a BCR of one specificity
◦ Applies to both H and L (kappa) chains
• Isotypic exclusion: If kappa genes are unsuccessful, the lambda genes (both maternal and paternal alleles) attempt to rearrange ie each antibody molecule will have either kappa or lambda chains
74
How can a single B cell have both lgM & lgD with the same antigenic specificity but be expressed simultaneously?
• Antigen specificity determined by variable region. Isotype determined by constant region. Alternative splicing of the primary RNA transcript allows both IgM and IgD to be expressed simultaneously
75
What types of B cells express both IgM & IgD simultaneously?
• Mature, naive follicular B-2 B cells
76
Explain what affinity maturation means.
Generation of antibodies with increased affinity for their cognate antigen
77
Explain how affinity maturation occurs.
• Somatic hypermutation- point mutations within the V regions of both the H and L chains.
◦ SHM occurs in the follicles (of spleen or LN’s) after B cell activation
◦ Progeny B cells use the same VJ and VDJ genes, but point mutations are introduced during clonal proliferation
◦ Can occur at the end of a long primary response or during secondary IR’s (ie any subsequent challenge by the same Ag)
◦ Activated B cell proliferates, creating many clones of itself, introducing mutations in the VDJ of the H and L chains as the cells copy their DNA
◦ Antibodies (with mutations) are made and expressed on the surface of the B cells
◦ B cells interact with Ag on the FDCs to see if mutations have made their ab’s better, worse or unchanged
◦ Only B cells with better ab’s receive survival signals from FDCs
78
What is the difference between isotype switching and alternative splicing?
* Isotype switching - DNA rearrangement. The same VDJ are moved next to a new Ch gene. Any upstream Ch genes are deleted ie permanently removed from the DNA sequence.
* Alternative splicing- splices out of the primary RNA transcript generates to mature mRNA transcripts for the IgM and IgD heavy chains.
