L6. Genetic Basis of Antibody Diversity

Question 1

What chain is more involved with binding to antigen and why?

Answer 1

Heavy chain, as is more variable

Question 2

What makes up the segments of heavy chain DNA?

Answer 2

DNA coding for variable regions of heavy chains are made up of 3 exons (segments of DNA) VH gene (variable) codes for most of variable region, DH gene (diversity), JH gene (joins regions) and then the constant region gene (CH), the variable exons come together during differentiation.

Question 3

What is always produced first in the early stages of an immune responseand why?

Answer 3

Early stages of immune response, or when developing, IgM is always made first, so heavy chain Cμ (IgM heavy chain) is always produced first (early in sequence)

Question 4

How many exons are present in a) Kappa chains b) Heavy chains in their germline DNA (before somatic recombination)?

Answer 4

a) Kappa chains
>Single constant region gene
>5 J segments next to this
>Downstream is around 38 V genes

b) Heavy chains
>Single constant region gene (coding for IgM)
>6 J segments next to it
>23 different D segments
>Long way downstream around 40 V segments

Question 5

What is the process that rearranges the exons in the germline DNA during B cell differentiation?

Answer 5

Somatic Recombination

Question 6

Describe a brief overview of a generic somatic recombination process in Kappa chains

Answer 6

1. A particular V gene is spliced to an aligned J gene.
2. The intervening DNA is deleted
3. Leaves mRNA with constant region joined to variable gene by a joining segment gene.
4. Once spliced, Rearranged V promotor now close to enhancer allowing transcription of this gene/ expression of this light chain for example.

Question 7

What is the generic structure of an mRNA for a kappa chain after somatic recombination?

Answer 7

mRNA with constant region connected to V gene with 10 amino acids (J segment) joining them)- for Kappa light chain.

Question 8

Describe a brief overview of somatic recombination in heavy chains.

Answer 8

1. In B cell differentiation the First event is that D segment re-arranges to join a J segment (D-J joining) then a particular V gene joins (V-D-J joining)
2. RNA processing removes unwanted DNA to produce mRNA, variable region most encoded by V segment, portion close to constant region encoded by D and J regions

Question 9

What are a) CDR1 and CDR2 b) CDR3 encoded by, how does this effect CDR3?

Answer 9

a) CDR1 and CDR2 are encoded by the V segments (i.e. germline)

b) CDR3 corresponds to the VDJ (or VJ for light chain) join
>CDR3 more variable as corresponds to VDJ not just germline.

Question 10

Why is somatic recombination called this?

Answer 10

Somatic as occurs in somatic cells rather than germ cells

Question 11

What type of somatic cell does somatic recombination occur in?

Answer 11

Only occurs in lymphocytes.

Question 12

Why does somatic recombination only occur in lymphocytes?

Answer 12

Involves lymphocyte specific recombinases and conserved recognition signal sequences (RSSs) lying adjacent of the genes needing to be joined together

Question 13

What are recognition signal sequences (RSSs)

Answer 13

> conserved heptamer (7 b.p.) + nonamer (9 b.p.) separated by 12 or 23 random nucleotides (spaces between genes). The difference between this is one half turn of the DNA double helix

> RSSs are found directly adjacent to the coding sequence of V, D or J gene segments. These guide rearrangement of the V, D and J segments.

Question 14

What is the 12-23 base pair rule of somatic recombination, what is the importance of this and why does it occur?

Answer 14

> a gene segment with a 12 bp spacer only joins with a gene segment with a 23 bp spacer.

> Ensures correct V-D-J joining (especially for heavy chain, instead of V-J-D) bylymphocyte specific recombinases.

> 12 bp is half a turn of the DNA double helix.

Question 15

What is the V(D)J recombinase?

Answer 15

Complex of several enzymes required for somatic V-region gene recombination

Question 16

What are some of the enzymes in the V(D)J recombinase usually used for?

Answer 16

Some involved in normal DNA cleavage/repair enzymes (B cells use some of these in recombination process)

Question 17

What is the most important complex in the V(D)J recombinase?

Answer 17

RAG1-RAG2 protein complex

Question 18

Why is the RAG1-RAG2 protein complex only found in developing lymphocytes?

Answer 18

RAG1-RAG2 protein complex encoded by Recombination Activation Genes* (RAG)) which is expressed only in developing lymphocytes

Question 19

What is the role of the RAG1-RAG2 protein complex in the V(D)J recombinase and an example of one of the enzymes?

Answer 19

> Specialised endonucleases for cutting DNA
terminal

> deoxynucleotide transferase (TdT) is an example

Question 20

What does mutations in RAG genes cause and why, and how can this be cured?

Answer 20

> Mutations in these genes* (RAG) result in severe combined immunodeficiency (scid).

> Mutations in RAG1 or RAG2 genes so can’t produce antibody or T cell receptors, so no adaptive immunity

> Can be cured by bone marrow transplant.

Question 21

Describe the role of the RAG1-RAG2 complex (specialised endonuclease) in somatic recombination?

Answer 21

> The RAG1-RAG2 complex recognises RSS and aligns them, has endonuclease activity and cleaves the DNA adjacent to RSS sequences

> The cleaved DNA is repaired to form the coding joint (V and J segments now next to one another) and the signal joint (intervening DNA excised)

(During recombination, the RAG1-RAG2 complex recognises and aligns the RSSs adjacent to the gene segments to be joined)

Question 22

What are the 4 mechanisms of diversity in lymphocytes, describe each one and which ones are antigen dependent?

Answer 22

1. Multiple copies of each V region gene segments
   >[Vn x Jn or Vn x Dn x Jn]
2. Heavy x light chain combination
   >[Vκ x Jκ] + [Vλ x Jλ] x [VH x DH x J]
3. Recombination is imprecise –> junctional diversity.
   >Nucleotides may lost or added: variable addition of nucleotides at junctions contributes to diversity of CDR3
   >Terminal deoxynucleotide transferase (TdT) randomly adds up to 12 nucleotides to V-D-J joins (heavy chain)

These 3 are antigen independent (occurring during B cell development) giving 3*10^7 diversity

4. Somatic mutation of V regions following antigen activation
   >Point mutations
   >Base changes tend to be clustered in CDRs (loops/ parts which recognise antigen)
   >Somatic mutation is antigen dependent,giving 3*10^14 diversity

Question 23

Describe somatic recombination in a heavy chain in 8 steps

Answer 23

1. RAG-1-RAG-2 complex recognises and aligns the RSSs adjacent to the gene segments to be joined (germline DNA folded)
2. Single stranded break by RAG-1-RAG-2
3. Two ssDNA breaks are made close to the RSSs.
   >Free 3’-OH attacks phosphodiester bond on other strand of DNA to create a hairpin at the segments to be joined and a flush ds break at RSS boundary.
4. 4 – 7.Repair of DNA. Other proteins bind to repair the joints, but this process is imprecise, with nucleotides added or subtracted.
   >DNA hairpins are cleaved at random, symmetrically

(4). or asymmetrically
(5). For V-D-J joining of the H chain, nucleotides can be added randomly by terminal deoxynucleotide transferase (TdT)
(6). Unpaired overhangs are filled in by DNA polymerase
(7). or may be excised by an exonuclease.

8) DNA ligase joins the nicked and repaired hairpins to form the “coding joint” (part that codes for heavy or light chain). (The blunt ends formed at Stage 3 are ligated to form the “signal joint” and this DNA is typically excised).

Question 24

Describe in 3 steps an overview of immunoglobulin gene expression and B cell differentiation after infection

Answer 24

1. Antigen independent changes occur in bone marrow
   a. Heavy chain gene rearrangement (D-J then V-DJ) occurs first, TdT adds up to 12 nucleotides and is then switched off
   >μ heavy chain produced
   b. Light chain gene rearrangement (V-J) –> Expresses membrane IgM on cell surface as receptor.
2. Selection against self-recognising B cells to be deleted.
3. Secondary lymphoid tissue
   a. “Naïve” B cell expresses membrane IgM or IgM + IgD

Question 25

Why is somatic hypermutation called this?

Answer 25

hyper as mutation rate is much higher than in normal cell (while in a somatic normal cell = 1bp per 10^10bp is the normal mutation rate))

Question 26

What is a point mutation?

Answer 26

A single base pair changes

Question 27

Where does somatic hypermutation occur on the gene (where are the point mutations introduced)?

Answer 27

Point mutations introduced in rearranged V regions (not in germline/ after somatic recombination as is antigen dependent.

Question 28

Where in the body does somatic hypermutation occur and what has to happen for it to take place?

Answer 28

Secondary lymphoid tissue, when a naïve B cell recognising antigen/ dividing in germinal centers

Question 29

Why is a B cell responding to antigen necessary for somatic hypermutation to occur?

Answer 29

> Activation-induced cytidine deaminase (AID), an enzyme expressed by B cells in lymphoid tissue responding to antigen.

> AID is only expressed in B cells responding to antigen, as it induces mutations don’t want it acting anywhere else.

Question 30

Where do the point mutations particularly get introduced during somatic hypermutation in mature B cells?

Answer 30

> Mutations can be introduced throughout V regions ,BUT in mature B cells mutations appear to be clustered in CDRs (CDR1,CDR2, CDR3)

Question 31

What genes encode for a)CDR3 b)CDR1-2

Answer 31

a) CDR3 is the V(D)J joined together, more varied as different segments can be joined differently.

b) CDR1-2 are also made of V region, less variability as is just the V region which codes for them.

Question 32

What is the role of somatic hypermutation in the immune response?

Answer 32

> Somatic hypermutation can add diversity

> But main function:
AFFINITY maturation - higher affinity receptors selected as immune response proceeds - “survival of the fittest”

Question 33

Describe how affinity of antibodies increases over time of the infection (affinity maturation induced by somatic hypermutation).

Answer 33

> At start of immune response antibodies don’t bind very well but over time of infection become higher affinity.

> Early in immune response, as are many antigen around, even a B cell with weakly binding receptors can bind and hold on (antigen binding determines B cell survival), when antigen concentration decreases, only B cells tightly binding to antigen (through mutations) will survive and produce high affinity antibodies (mutations are found in CDR regions as this is the region that binds to antigen)

Question 34

Does the memory response antibodies have high affinity? Why?

Answer 34

Memory response has higher affinity than at the start of an immune response, as the B cells which survive affinity maturation have receptors of those that can form tight interactions with the antigen.

Question 35

Where does class switching occur?

Answer 35

In secondary lymphoid tissue

Question 36

What is class switching?

Answer 36

Antibodies always make IgM first (in primary response) but can switch to make other heavy chain classes. (Class switching: IgM –> IgG, IgA etc.)

Question 37

What is a very brief overview of how class switching occurs?

Answer 37

The same recombined V region associates with different C region genes (so the V region with high affinity for an antigen swaps to another constant region/ heavy chain)

Question 38

Why is class switching important and give an example?

Answer 38

> Why do this: antigen specificity retained, different localisation/effector functions induced - flexible response to pathogens

> E.g. IgM good for early stages, then if a parasitic infection IgE is better.

Question 39

What help does both somatic hypermutation and class switching require?

Answer 39

Somatic hypermutation and class switching require T cell help and AID (Activation-induced cytidine deaminase)

Question 40

Why is IgM always produced first?

Answer 40

Mature B cell heavy chain genes: V region encoded by recombined VDJ, and IgM is always expressed first in an immune response as is Cμ is closestdownstream to the VDJ region.

Question 41

What is a switch region and how does it work in class switching?

Answer 41

Adjacent to the heavy chain genes is a switch sequence, this allows recombination of VDJ genes so can recombine with an IgA (Cα) or IgE (Cε) constant region gene instead of Cμ

Question 42

Why is class switching irreversible?

Answer 42

As intervening DNA is lost, it is irreversible as DNA is lost, so can’t go back to making IgM as Cμ gene is lost

Question 43

Does class switching use different mechanisms to V-(D)-J (somatic recombination) joining?

Answer 43

Yes it uses different machinery

Question 44

How does AID know where to initiate class switching?

Answer 44

Initiated by AID acting at switch regions (not conserved regions but rich tandem repeated DNA sequences which AID loves to bind to found close to C region genes), so as it deaminates lots of C to U then a double stranded nick must occur removing the upstream heavy chain constant region which is replaced by the downstream one

Question 45

What is the mechanism of action of activation-induced cytidine deaminase (AID)?

Answer 45

AID deaminates cytidine to form uracil (Uracil normally found in RNA, so as is in DNA cell tries to repair.)

Question 46

Describe how AID causes mutations in B cells?

Answer 46

> AID is expressed in activated B lymphocytes, only active on ssDNA

> Activity triggers DNA repair pathways due to adding Uracil.

(The ssDNA on which AID acts is generated transiently during transcription of the immunoglobulin genes. The transcription process creates regions of ssDNA as the RNA polymerase moves along the DNA template. AID targets these single-stranded regions, initiating a cascade of events leading to mutation or recombination.)

Question 47

What are the mutations AID can cause and why do these occur, and what do they lead to?

Answer 47

> Repair pathways initiated in B cells are error-prone, leading to different mutational outcomes:

1. Mismatch repair, base excision repair (single base pair changes)= SOMATIC HYPERMUTATION
2. Nucleotide-excision repair: Single strand nicks → double strand nicks caused by more uracil added (common in G-rich tandem repeat switch regions)= CLASS SWITCHING (V region lies next to whole new constant region).

Question 48

What can a mutation in AID cause?

Answer 48

> AID mutation causes immunodeficiency: Hyper-IgM Syndrome Type 2- can only make IgM, make lots of it but don’t have effective memory responses.

> If overactive can cause cancer through inducing more mutations.