Lecture 3

Question 1

Describe the structure of the B-cell receptor (BCR)

Answer 1

Surface membrane-bound IgM antibody
- two heavy chains and two light chains, each with an invariable membrane-bound Fc region and variable region where antigen binding occurs

Question 2

True or false: both heavy chain and both light chain alleles are expressed in a B-cell?

Answer 2

False: only one allele of each gene is expressed (successful expression of one allele causes the silencing of the other allele)

Question 3

what are the two light chain types that could form part of the BCR?

Answer 3

Kappa or Lambda

Question 4

Why does the B-cell only express just one heavy and one light chain gene?

Answer 4

to prevent the expression of more than one type of BCR on the cell surface making it more likely that the cell will recognise self-antigens

Question 5

What is allelic exclusion?

Answer 5

the mechanism that ensures B-cells only express one heavy-chain and one light-chain allele

Question 6

What is the first stage of gene recombination in the production of a BCR?

Answer 6

V(D)J recombination of one heavy chain allele catalysed by lymphoid-specific enzymes RAG1 and RAG2 that recognise Recombination specific sequence adjacent to the coding regions.
Extra P and N- nucleotides can be added during the recombination process randomly, which increases diversity.
Exonuclease trimming can reduce the number of additional nucleotides present

Question 7

What are the two different RSS sequences that can be adjacent tot he coding regions?

Answer 7

23 base spacer (i.e. has 23 base pairs between the heptamer and nonamer)

or

12 base spacer (i.e. has 12 base pair between the heptamer and nonamer)

Question 8

How do the RSS sequences ensure correct recombination and prevent intersegment recombination?

Answer 8

23/12 Rule

Each segment will be associated with one type of RSS

Recombination can only occur between a 23 and 12 base spacer

Question 9

Following successful recombination of one heavy chain allele, what is the next step?

Answer 9

the productive heavy chain, µ, is expressed on the cell surface with a surrogate light chain in a pre-BCR

Signalling through this pre-BCR inhibits rearrangement of the second heavy chain allele (other allele is silenced)

Signalling through this pre-BCR also induces rearrangement of the kappa light chain allele

Question 10

What may cause a non-productive gene re-arrangement?

Answer 10

if P/N nucleotide addition and/or exonuclease trimming causes a frameshift mutation.

Question 11

Following signalling through pre-BCR (silencing other heavy chain allele and inducing kappa light chain re-arrangement) what is the next step?

Answer 11

Similarly to heavy chain recombination, RAG1/2 recombinases and nucleotide addition result in kappa light chain VJ recombination.

If the recombination is successful, the light chain is expressed on the cell surface with the heavy chains as a BCR.

Signalling through this BCR results in silencing of the second kappa allele and inhibits recombination of the lambda gene.

Question 12

If the first recombination of the heavy chain was non-productive, what is the next step?

Answer 12

The second allele is undergoes recombination

if this is unsuccessful, the cell dies

Question 13

If the first recombination of the light chain was non-productive, what is the next step?

Answer 13

The second kappa allele undergoes recombination

if this is unsuccessful, a lambda light chain allele undergoes recombination

If this is unsuccessful, the second lambda light chain undergoes recombination

If this is unsuccessful, the cell dies

Question 14

What is the first type of immunoglobulin expressed by immature B cells in the bone marrow?

Answer 14

IgM with a constant µ region
(µ = mu = M)

Question 15

True or false: mature B cells only express membrane bound IgM on their surface

Answer 15

False: they also express membrane bound IgD

Question 16

How does the mature B-cell also express membrane bound IgD?

Answer 16

Alternative splicing of pre-mRNA results in joining of the same variable region to the δ constant region (δ = delta = D)

Question 17

What is the name of the receptor-associated molecules required by the BCR for signal transduction?

Answer 17

Igα and Igβ

Question 18

How do BCR-associated Igα and Igβ facilitate signal-transduction through the BCR?

Answer 18

They have ITAMs (immunoreceptor tyrosine-based activation motifs)

Upon binding to antigen tot he BCR, the tyrosine residues of the ITAMs become phosphorylated and act as docking sites for SH2 and PTB domain proteins involved in the downstream signalling pathway

Question 19

What are the 5 different subclasses of antibody produced by a specific B cell and how do they differ?

Answer 19

IgM (µ constant region)
IgD (δ constant region)
IgG (γ constant region)
IgE (ε constant region)
IgA (α constant region)

Question 20

How is functional diversity of antibodies achieved?
(3 points)

Answer 20

Class switching by alternative splicing of IgM to other isotypes following B cell activation

Additional diversity by:
Somatic hypermutation
Affinity maturation

Question 21

True or false: the BCR itself does not signal?

Answer 21

True (associates with Igalpha and IgBeta for signal transduction)

Question 22

What is MHC restriction?

Answer 22

Most T-cells cannot bind antigen alone- they recognise the combination of the antigen bound by a particular MHC molecule.
(TCRs are MHC restricted)

Question 23

What controls TCR expression?

Answer 23

Allelic exclusion

Question 24

In what order do the TCR genes re-arrange?

Answer 24

Beta gene followed by the alpha gene

Question 25

What is the difference in the constant region between antibodies and TCRs?

Answer 25

Antibodies have different classes with different constant regions where as TCRs only have one kind of constant region

Question 26

What are the coreceptors that complex with the TCR for antigen recognition?

Answer 26

CD3 (contain ITAMS that can be phosphorylated to transmit signal) CD4 and CD8 (increase avidity of peptide binding by TCR) CD28 (engages CD80/86 on APC to fully activate a naive T-cell

Question 27

Describe TCR signalling ....... do extra reading on the cellular responses of TCR signalling.

Answer 27

APC presents peptide antigen on MHCII. TCR recognises antigen, causing clustering of TCR with coreceptors within lipid rafts. This causes activation of CD4/CD8-associated Lck that phosphorylates tyrosines in ITAMs of CD3 and Zeta-chains. ZAP-70 SH2 domains bind to the intracellular phosphorylated ITAMs of the zeta chains and is activated and phosphorylates adaptor proteins including LAT. The phosphorylated adaptors become docking sites for cellular enzymes such as PLCγ1 and GDP-GTP exchange factors that activate Ras and other small G proteins upstream of MAP kinases which all activate various cellular responses.

Question 28

What is the structure of MHC class I?

Answer 28

Three extracellular alpha domains (alpha 1-3) that form one single polypeptide chain with peptide binding groove between alpha1 and alpha2. Transmembrane segment and cytoplasmic C-terminal tail Beta2-microglobulin associates with the alpha domains

Question 29

What is the structure of MHC class II?

Answer 29

Formed of two extracellular beta domains (beta1-2) and two extracellular alpha domains (alpha1-2) (the alpha domains form one polypeptide chain and the beta domains are a separate polypeptide chain, which associated via a peptide binding groove between alpha1 and beta1 domains). Each polypeptide chain has a transmembrane spanning domain and a cytosolic tail.

Question 30

What is the general structure of the peptide binding grooves of the MHC molecules?

Answer 30

floor composed of antiparallel beta sheets with sides of alpha helices

Question 31

What is the difference between the peptide binding grooves of MHC class I and MHC class II?

Answer 31

MHC class I: - peptide binding groove between alpha1 and alpha2 domains - each nucleated cell can express several unique class I molecules that each have slightly different peptide binding specificity - the groove can only accommodate a smaller peptide of around 10 amino acids MHC class II: - binding groove between alpha1 and beta1 domains - expression is only restricted to antigen presenting cells - the groove can accommodate much longer peptides

Question 32

Explain how MHC molecules are inherited

Answer 32

The genes are tightly linked so generally inherited as a single unit from each parent as part of a haplotype block

Question 33

Describe the characteristics of the MHC genes

Answer 33

Highly polymorphic (many alleles exist for each gene within a population) Polygenic (several different MHC genes exist within an individual) Co-dominantly expressed (both maternal and paternal copies expressed) Do not undergo gene rearrangement

Question 34

Where is polymorphism mostly concentrated in the MHC molecules?

Answer 34

the regions that form the peptide binding groove in order to confer different specificities.

Question 35

What are the two presentation pathways via the MHC molecules?

Answer 35

Endogenous and exogenous

Question 36

Describe the endogenous processing and presentation pathway via MHC class I

Answer 36

- endogenous antigens are degraded into peptides within the cytosol by the proteasome - these peptides are assembles with MHC class I molecules in the rough endoplasmic reticulum - assembled complexes are presented to CD8+ cytotoxic T cells.

Question 37

Describe the exogenous processing and presentation pathway via MHC class II

Answer 37

- Exogenous antigens internalised and degraded within acidic endocytic compartments - the peptides are complexed with MHC class II molecules in the rough endoplasmic reticulum and presented to CD4+ T-helper cells

Question 38

What is dendritic cell licensing how is it involved in cross-presentation? ....................... what actually is licensing and cross-presentation - do extra reading here.

Answer 38

Dendritic cells are licensed by T-helper cells via CD40 on DC/CD40L on T cell in addition to TCR binding to peptide on MHC class II. This can allow a dendritic cell to gain access to the class I presentation pathway and stimulate CD8+ T-cells against intracellular microbes by capturing antigen from dying cells or the extracellular environment.

Question 39

How are non-peptide antigens presented and recognised by T-cells?

Answer 39

Presented on CD1 family of non-classical class I molecules (structural homology to MHC class I) - glycolipids or lipoproteins are bound within the deep lipid binding groove (deep to accommodate the hydrophobic lipid chain) - the hydrophilic part is exposed for binding by the TCR.