Lecture 2

Question 1

Specific/adaptive immunity:

Answer 1

• is induced by exposure to a particular infection
• shows a high degree of specificity
• exhibits “memory”

Question 2

Antibodies

Answer 2

Number of different possible antibodies in humans:
>1011
< 30,000 (3 x104) genes in human genome
(approx. 170 human H/L chain genes)

Question 3

How does the number of antibosies work?

Answer 3

How can there be 3 x106 more (1011/3 x104) possible BCR/antibodies than there are genes present in the human genome?

A range of mechanisms allows the generation of an enormous antibody repertoire from relatively few (~200) genes
(and similar (but not identical) mechanisms exist for generation of equally large repertoire of different TCRs)
- very fine tuned

Question 4

Multiple genes encode a single polypeptide chain:

Answer 4

• V and C regions of antibody and TCR polypeptide chains are encoded by separate gene segments that rearrange during lymphocyte differentiation
• H chain and TCR:
V region encoded by three gene segments:
V, D and J (V is the biggest)
• L chain and TCR :
V region encoded by two gene segments: V and J

Question 5

What are the V & C regions of antibody & TCR polypeptide chains are encoded by?

Answer 5

Separate gene segments that rearrange during lymphocyte differentiation

Domains are encoded by very small mini genes

There are bits in-between V & C

Question 6

What are the bits in-between V & C?

Answer 6

D = diversity

J = joining

Question 7

What is the V region of H chains & TCR-beta encoded by?

Answer 7

V
D
J

Question 8

What is the V region of L chains & TCR-alpha encoded by?

Answer 8

V

J

Question 9

What do V, D & J encode?

Answer 9

V region of H chains & TCR-beta

Question 10

What do V & J encode?

Answer 10

V region of L chains & TCR-alpha

Question 11

Rearrangement of Ig genes in B cells

Answer 11

Genes rearrange during B cell development to form a functional gene

The genes in B cells are closer than in every other type of cell in the body

DNA is different in B cells than every other cell

Non-homologous end joining (NHEJ) recombination

B cells break their DNA in a way no other cells do (apart from T cells) & move genes so they are closer

Question 12

How do B cells produce a functional immunoglobulin?

Answer 12

Involves recombination to bring gene segments together

Question 13

Light chain of an antibody

Answer 13

After DNA breaks, a single V & a single J gene segment are joined together to encode the V region of the light chain

The 2 exons come together

DNA is broken randomly to bring 2 of each together randomly

Leads to different BCRs in every cell

B cell has to decide to break its DNA at one locus – either lambda or kappa

Question 14

What happens once V & J are next to each other for the light chain?

Answer 14

It gets transcribed into RNA like a normal gene making the light chain – can then fold up to form a unique antigen binding site

Question 15

Heavy chain of an antibody

Answer 15

Similar mechanism but extra sequence section with the D region

2 breaks needed

A single V, D & J gene fragment are joined together to encode the V region of the heavy chain

Question 16

Order of breakage in the heavy chain

Answer 16

Each B cell first breaks between D & J to bring them together

Then is sequentially broken again & the DJ joins the V

Lots of combinations – every V can go with every D & J

Question 17

Hierarchy of rearrangements in light and heavy chains

Answer 17

• greater variability in H chain as V,D and J
• first H chain genes (D-J then V-D)
• then light chain genes: κ first (V-J)
• if κ rearrangement unsuccessful then λ genes rearrange

Question 18

What forms the binding site?

Answer 18

Variable regions of H & L chain

Question 19

What chain has greater variability?

Answer 19

Greater variability in H chain as V, D & J

Question 20

What loci are the H, kappa & lambda chains encoded?

Answer 20

H chain – chromosome 14

Kappa chain – chromosome 2

Lambda chain – chromosome 22

Question 21

Recombination signal sequences (RSS):

Answer 21

• DNA rearrangement is guided by special sequences flanking the V, D and J regions = recombination signal sequences
• involves a complex of enzymes = V(D)J recombinase
• (recombination activating gene)
• RAG-1 and RAG-2 genes encode lymphoid-specific components of the recombinase
• mutations in RAG genes result in immunodeficiency

Question 22

Which gene segments encode the variable region of the Ig heavy chain?

Answer 22

V, D, J regions

Question 23

Which gene segments encode the variable region of the light chain of an antibody?

Answer 23

V region and a J region

Question 24

How are the heavy (H) chain of an antibody and the beta (b) chain of the TCR similar?

Answer 24

Both have a V region, D region and a J region

Question 25

When does rearrangements of Ig genes occur?

Answer 25

Genes rearrange during B cell development to form a functional gene

Question 26

What are the 2 types of light chain found in antibodies?

Answer 26

Kappa and lambda

Question 27

Allelic exclusion

Answer 27

In a single B cell only one allele of H chain expressed, similarly for L chain light chain isotype exclusion a single B cell expresses either k or l ,never both These mechanisms ensure that an individual B cell produces only one specificity of antibody

Question 28

Mechanisms for generation of antibody diversity:

Answer 28

1) multiple germline genes 2) combinatorial diversity 3) junctional diversity 4) combinations of heavy and light chains 5) somatic hypermutation

Question 29

multiple germline genes

Answer 29

Multiple VH, Vk and Vl | also, multiple D and J (no D for k and l)

Question 30

combinatorial diversity

Answer 30

Different V, D and J segments recombine to produce different sequences e.g. 40V x 25D x 6J would give 6,000 combinations (note, due to D segments, H chains potentially more diverse than L chains)

Question 31

junctional diversity

Answer 31

``` Includes: imprecise joining (small differences in position of V-D and D-J join) ``` N regions (random addition of nucleotides at junctions of V-D and D-J by terminal transferase)

Question 32

combinations of heavy and light chains

Answer 32

e.g. 106 H and 104 L would give 1010 possible antibodies | (Further) mechanisms for generation of antibody diversity

Question 33

somatic hypermutation

Answer 33

mutation frequency in antibody VH genes orders of magnitude higher than normal spontaneous mutation rate occurs in germinal centres as B cells recognize Ag and proliferate/become activated

Question 34

Somatic hypermutation:

Answer 34

* Involves the enzyme, activation-induced deaminase (AID) * AID acts on DNA to de-aminate cytosine to uracil * uracil is then recognized by error-prone DNA repair pathways leading to mutations

Question 35

Membrane (BCR) vs. secreted antibody:

Answer 35

* following Ag recognition as each B cell differentiates, it will start to secrete its unique BCR as an antibody * the secreted form made by each B cell has an alternative constant region that lacks a transmembrane region. As the original re-arranged VDJ regions are not altered, the secreted antibody has the same antigen specificity as the membrane BCR. * the membrane and secreted forms are produced by alternative RNA processing

Question 36

Antibody classes:

Answer 36

``` IgM - Heavy chain μ IgD - Heavy chain δ IgG - Heavy chain γ IgA - Heavy chain α IgE - Heavy chain ε ```

Question 37

Heavy chain constant region genes and class-switching:

Answer 37

* constant region of each heavy chain is encoded by a different C region gene segment (i.e. Cμ, Cδ, Cγ, Cε, Cα) * four g chain genes correspond to the four IgG subclasses: two α chain genes * As the Cμ is physically the closest to the V, D and J genes at the heavy chain locus this is why IgM is first class/isotype BCR/antibody expressed by each developing B cell

Question 38

Summary

Answer 38

* the BCR/antibody polypeptide in each B cell is encoded by multiple genes segments * these rearrange during B cell development (randomly so each B cell will assembly a different BCR) * the random gene segments used will determined antibody specificity * the joining process introduces further variation

Question 39

After B cells have assembled a random BCR

Answer 39

After leaving bone marrow, V region genes may undergo somatic hypermutation in specialized areas of secondary lymph organs following the binding of Ag. and B cell activation this leads to further BCR specificities being generated activated B cells start to secrete their BCR (antibody) class-switch recombination may then occur resulting from the presence of cytokines