Lectures 1-3: Revision of antigen recognition systems Flashcards
What are the requirements for the immune system?
To be able to recognise and respond to any invading organism
Not over react
Be able to direct different effector mechanisms against different pathogens
What are the features of specific immunity?
Mediated by B/T cells
Clonally distributed receptors
Large repertoire (low frequency of specific cells)
Response takes longer to develop memory
What are examples of large multicellular organisms that are able to infect?
Worms and protozoa
Which phase is the second response of an immune response?
Adaptive
What do TCR expressed T cells ONLY recognise?
Peptide fragments of antigen bound to MHC expressed APC
Where is the antigen binding site located on the antibody?
Towards the N-terminus
What are the structures of antibodies?
They contain: paired variable regions form 2 identical antigen-binding sites
Constant regions are responsible for structure and interacting with other molecules and cells of innate system (antibody effector functions)
What are the properties of antibody structure?
Immunoglobulin
4-chain structure
2 identical H and 2 identical L chains held by covalent and non-covalent
2 types of L chain
Variable and constant regions
Antigen binding sites Vh and Vl
Different Ch regions interact with complement and Fc regions bind to different FcRs expressed by effector cells
What are the different antibody classes?
Isotypes: IgM, D, G, E
How are the different isotypes of antibodies determined?
By the heavy chain
What are the homology domains of antibodies?
L - 2 domains
H- 4/5 domains
~110 AAs, 2 β sheets
How do antibodies and antigens interact?
Variable region with are specific for antibodies with a concentrated region of variability
Hypervariable regions HV1-3
6 hypervariable loops -> Ag binding site
Complementary determining regions (CDR)
Ag binds to amino acids in CDRs
Size/shape of Ag affects binding
How can antigens be recognised?
Epitopes are recognised by Abs that may be continuous or not
Ag be can be almost any molecule
Ab and Ag form non-covalent interactions
Ag sequence may be manipulated in vaccines
Ag may be folded
CDRs present in antibody V regions determine specificity and affinity of an Ab for an Ag
What are the 2 different chains of an antibody?
Heavy and light
Which part of an antibody interacts with the epitope of an antigen?
Variable region
Define FAB and Fc regions?
FAB - fragment antigen binding region
Fc - fragment crystallisation region (CSR)
What are the features of the TCR?
Does not bind free antigen
Binds and recognises peptides only
Presented in cleft groove of MHC class I/II
Membrane bound receptor
Smaller than antibody
What are the structural features of a TCR?
Heterodimer of α and β chain
Has a V and C region
Domains are Ig like
V domains interact with antigen = peptide bound to MHC molecule
Each chain contributes 3 CDRs to Ag binding
What are the similarities and differences between MHC molecules?
Similarities:
Related structure
Present peptides
Many alleles
Differences:
Overall different structures
Different expression patterns
What are features of MHC class I molecules?
Expressed on all nucleated cells
Heterodimer α chains & β2 microglobin
HLA-A, B and C
Encoded by separate α chain genes
α1 & α2 domains form beta-sheet - peptide binding site, DNA is very polymorphic
What are features of MHC class II molecules?
Expression limited to APC
Heterodimers, α & β chains encoded by separate genes encoded within MHC
Similar structure to class I
Polymorphic α & β domains form peptide-binding site
Which CDs are present on which MHC molecules?
CD8 = MHC class I
CD4 = MHC class II
How do B/T cells create different number of receptors?
They break their DNA into fragments
What are the different genes that encode a single polypeptide chain in antibodies?
Encoded by separate gene segments that rearrange during lymphocyte differentiation
H chain and TCRβ:
V region encoded by 3 gene segments: V,D and J
L chain and TCRα:
V region encoded by 2 segments: V and J
What do the segments V, D and J stand for?
Variable, diverse and joining
How are Ig genes rearranged?
They are found in the genome as gene segments
B cells DNA containing the Ig gene segments are deliberately broken and the gene segments are rearranged to form functional Ig genes
(non-homologous end joining (NHEJ) recombination)
Perform both breakage and rearrangement randomly (different to each B cell)
How are the Ig genes in light chains produced using gene segments?
After DNA breaks a single V and single J gene segment join together to encode a variable region of the light chain
What are the factors about the production of Ig genes on light chains?
It is a random process
There are two loci containing L chain genes λ & κ
WHat are the differences between Ig gene production in light and heavy chains?
Light: V and J only
Heavy: V, D and J
What is the precise order of the formation of the variable region on heavy and light chains of Igs?
Germline DNA
↓somatic recombination
D-J joined rearranged DNA
↓somatic recombination
V-J or V-DJ joined rearranged DNA
↓transcription
Primary transcript RNA
↓splicing
mRNA
↓translation
Polypeptide chain
What order of the Ig rearrangements occurs during B cell development?
First H chain gene segments rearrange
Light chain gene segments rearrange κ segments first
If light chain κ rearrangement is unsuccessful then λ gene segments rearrange
Where are each chain loci located?
H chain: chromosome 14
κ chain: chromosome 2
λ chain: chromosome 22
How are different isotypes encoded on the heavy chain?
The different antibody classes are encoded on the chromosome with determine the isotype of the Ig
How is the Ig gene segment rearranged?
By special sequences flanking each of the V, D an J gene segments = recombination signal sequences
What are enzymes are involved in the recombination process?
A complex of enzymes - V(D)J recombinase
What does RAG stand for?
Recombination activating gene
What do the RAG genes encode?
They encode lymphoid-specific components of the recombinase
What does mutation in RAG genes result in?
Immunodeficiency
What are the 2 types of light chain found in antibodies?
Lambda and Kappa
What is allelic exclusion?
In individual B cells only one H chain gene rearranged from one chromosome is expressed
Light chain - similar thing happens
What is light chain isotype exclusion in allelic exclusion?
Each B cell expresses either a rearranged Kappa or Lambda (NEVER BOTH)
Why is allelic exclusion used?
To ensure an individual B cell produces just one randomly generated BCR/antibody
(similar thing in T cells)
What are the mechanisms for generating antibody diversity?
Multiple gene segments for each chain (VH, Vκ and Vλ) also multiple J and only DH
Combinatorial diversity - different V,D and J segments recombine to produce different sequences
Combinations of heavy and light chains
Junctional diversity - imprecise joining, N regions
Somatic hypermutation - mutation frequency in antibody V genes have a magnitude higher than other areas of the genome
Where does somatic hypermutation occur?
In germinal centres as B cells (ONLY) recognise Ag and proliferate/become activated
What are the mechanisms that somatic hypermutations occur via?
Performed by the enzyme activated-induced deaminase (AID)
AID acts on DNA to de-aminate cytosine to uracil
Uracil is recognised by error-prone DNA repair pathways leading to mutations
What are the similarities and differences between membrane and secreted antibody?
Following antigen recognition, B cell differentiates secreting unique antibody
Secreted form has an alternative constant region (lacks transmembrane)
Secreted antibody has same antigen specificity as membrane
Both produced by alternative RNA processing
What are the different antibody classes?
IgM - µ
IgD - δ
IgG - γ
IgA - α
IgE - ε
What is the first class of antibody expressed by developing B cells?
IgM on the C region gene on the heavy chain
How can classes be coexpressed?
By differential processing of the RNA from the 2 C region genes
What is the mechanism of isotype switching?
Looping out - cuts sections of the class not wanted so the correct isotype is expressed
How are antibody classes co-expressed?
Using differential processing of the RNA from the two C region genes
How does class switching take place?
It uses further DNA recombination using the enzyme AID in a process of loping out and switch-region recombination
What are the factors of TCR genes?
They are also encoded by rearranging gene segments
Variable regions encoded by V+J on light chain and V+D+J on heavy
Segments rearrange in the thymus
Similar to Ig genes
What are the similarities in the diversity of TCR and BCR?
Multiple V+D+J segments
Combinatorial diversity
Junctional diversity
What are the differences in diversity generation between TCR and BCR?
TCR is never secreted
No somatic hypermutation occurs in TCR genes
What are the different polypeptides TCRs have?
TCRα - V+J+C - Ch 14
TCRβ - V+D+J+C - Ch 7
TCRγ - V+J+C - Ch 7
TCRδ - V+D+J+C - Ch 14
How is MHC diversity developed?
No gene rearrangement
Co-dominantly expressed
Class I - all nucleated cells
Class II - on particular cell types (APCs)
Induced and up-regulated by interferon
What is an example of a non-nucleated cell that cannot express MHC?
Red blood cells
What is the difference between a polymorphism and an allele?
A polymorphism is the variation of the gene but the allele is the gene variant at a specific locus
What is an example of genes that are vey polymorphic?
MHC molecules which have many different alleles
How can MHC be co-expressed?
3 Class I molecules if heterozygous the classes can be expressed 6 different times, similarly with MHC class II
How are the polymorphic residues structured on the MHC?
Non-randomly which impacts peptide binding
Why are MHC molecules so polymorphic?
To allow binding of a vast range of peptides presented to T cells, evolutionary advantages the population (respond to many pathogens)
What are the risks of MHC polymorphism?
Immune-mediated disease (likelihood of presenting self antigens)
Reduces pool of available donor organs for transplantation
How are peptides found on the surface of cells bound to MHC?
Synthesised inside a cell (endogenous Ag)
Synthesised outside a cell (exogenous Ag)
How are antigens processed and presented by MHC class I?
Antigen synthesised into cytoplasm
Protein cleaved to peptides by proteasome
Peptides transported to endoplasmic reticulum by TAP transporter
Peptides bind to MHC class I
MHC-I/peptide complex transported to cell surface
What are examples of intracellular antigens?
Viral antigens and self antigens
What is the function of the proteasome in antigen processing?
Cytoplasmic protein turnover
They receive inflammatory cytokine signals that are modified to produce altered peptides
How does the TAP peptide loading complex work?
It uses tapasin and calreticulin to deliver the peptide that binds to the MHC class I molecule to complete folding which is then released from the TAP complex and exported
How are antigens processed and presented by MHC class II?
Antigen endocytose into intracellular vesicles inside the cell
Protein cleaved to peptides by acid proteases in vesicles
Vesicles fuse with vesicles containing MHC class II
Peptides bind MHC class II
MHC-II/peptide complex then transported inside vesicles to cell surface
How is the invariant chain used in antigen processing of MHC class II?
It binds to the invariant chain in the ER which prevents binding in the groove
How is the CLIP peptide used in antigen processing of MHC class II?
Lysosomal enzymes are used to degrade the invariant chain which leaves CLIP associated with the binding groove
Peptides from the antigen displace CLIP
Which MHC associated molecule is required for loading peptides into the groove?
HLA-DM (class II)
What do MHC molecules bind and present in healthy uninfected cells?
Peptides from self proteins
Where are the different proteins required for antigen processing encoded?
Within the MHC (class I - TAP, LMP and class II - HLA-DM)
Which MHC molecules process extracellular antigens?
MHC class II
