Lecture 3 Flashcards
Describe the structure of the B-cell receptor (BCR)
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
True or false: both heavy chain and both light chain alleles are expressed in a B-cell?
False: only one allele of each gene is expressed (successful expression of one allele causes the silencing of the other allele)
what are the two light chain types that could form part of the BCR?
Kappa or Lambda
Why does the B-cell only express just one heavy and one light chain gene?
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
What is allelic exclusion?
the mechanism that ensures B-cells only express one heavy-chain and one light-chain allele
What is the first stage of gene recombination in the production of a BCR?
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
What are the two different RSS sequences that can be adjacent tot he coding regions?
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)
How do the RSS sequences ensure correct recombination and prevent intersegment recombination?
23/12 Rule
Each segment will be associated with one type of RSS
Recombination can only occur between a 23 and 12 base spacer
Following successful recombination of one heavy chain allele, what is the next step?
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
What may cause a non-productive gene re-arrangement?
if P/N nucleotide addition and/or exonuclease trimming causes a frameshift mutation.
Following signalling through pre-BCR (silencing other heavy chain allele and inducing kappa light chain re-arrangement) what is the next step?
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.
If the first recombination of the heavy chain was non-productive, what is the next step?
The second allele is undergoes recombination
if this is unsuccessful, the cell dies
If the first recombination of the light chain was non-productive, what is the next step?
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
What is the first type of immunoglobulin expressed by immature B cells in the bone marrow?
IgM with a constant µ region
(µ = mu = M)
True or false: mature B cells only express membrane bound IgM on their surface
False: they also express membrane bound IgD
How does the mature B-cell also express membrane bound IgD?
Alternative splicing of pre-mRNA results in joining of the same variable region to the δ constant region (δ = delta = D)
What is the name of the receptor-associated molecules required by the BCR for signal transduction?
Igα and Igβ
How do BCR-associated Igα and Igβ facilitate signal-transduction through the BCR?
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
What are the 5 different subclasses of antibody produced by a specific B cell and how do they differ?
IgM (µ constant region)
IgD (δ constant region)
IgG (γ constant region)
IgE (ε constant region)
IgA (α constant region)
How is functional diversity of antibodies achieved?
(3 points)
Class switching by alternative splicing of IgM to other isotypes following B cell activation
Additional diversity by:
Somatic hypermutation
Affinity maturation
True or false: the BCR itself does not signal?
True (associates with Igalpha and IgBeta for signal transduction)
What is MHC restriction?
Most T-cells cannot bind antigen alone- they recognise the combination of the antigen bound by a particular MHC molecule.
(TCRs are MHC restricted)
What controls TCR expression?
Allelic exclusion
In what order do the TCR genes re-arrange?
Beta gene followed by the alpha gene
What is the difference in the constant region between antibodies and TCRs?
Antibodies have different classes with different constant regions where as TCRs only have one kind of constant region
What are the coreceptors that complex with the TCR for antigen recognition?
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
Describe TCR signalling
……. do extra reading on the cellular responses of TCR signalling.
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.
What is the structure of MHC class I?
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
What is the structure of MHC class II?
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.
What is the general structure of the peptide binding grooves of the MHC molecules?
floor composed of antiparallel beta sheets with sides of alpha helices
What is the difference between the peptide binding grooves of MHC class I and MHC class II?
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
Explain how MHC molecules are inherited
The genes are tightly linked so generally inherited as a single unit from each parent as part of a haplotype block
Describe the characteristics of the MHC genes
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
Where is polymorphism mostly concentrated in the MHC molecules?
the regions that form the peptide binding groove in order to confer different specificities.
What are the two presentation pathways via the MHC molecules?
Endogenous and exogenous
Describe the endogenous processing and presentation pathway via MHC class I
- 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.
Describe the exogenous processing and presentation pathway via MHC class II
- 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
What is dendritic cell licensing how is it involved in cross-presentation?
………………….. what actually is licensing and cross-presentation - do extra reading here.
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.
How are non-peptide antigens presented and recognised by T-cells?
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.
