The B Cell Response Flashcards
what are the four postulates of clonal selection?
- Each lymphocyte bears a single type of receptor with a unique specificity
- interaction between a foreign molecule and a lymphocyte receptor capable of binding that molecule with high affinity leads to lymphocyte activation
- The differentiated effectors derived from an activated lymphocyte will bear receptors of identical specificity to those of the parental cell
- Lymphocytes bearing receptors specific for ubiquitous self molecules are deleted at an early stage and are therefore absent form the mature repertoire.
Describe the structure of an antibody
• Constant region → one of 5 biochemically distinct forms, IgM, IgD, IgG, IgE, IgE
• Determines the effector function of the antibody
• Variable region → can be composed of a seemingly infinite variety of amino acid sequences
− The variable region determines the binding specificity
− Two identical variable regions give 2 binding sites
- The variable regions of the heavy and light chain combine to form the antigen binding site, so both chains contribute to antigen specificity
- Gene rearrangement of receptor gene segments gives an almost infinite number of specificities
- Recombinatorial assembly of a large number of gene segments generates the enormous size of the antigen receptor repertoire
- The assembly process is regulated in a manner than ensures each lymphocyte can only express one receptor specificity
- Gene segment rearrangement involves an irreversible change in a cells DNA, so all the progency of the cell will inherit the same receptor specificity
• Y shape with 4 protein subunits
• Two longer identical heavy chains
• Two shorter light chains → either kappa or lambda .
− an antibody will have two heavy chains and either two kappa or two lambda, kappa and lambda are never seen together
• The heavy chains are linked to each other and to the light chains by disulphide bonds
• Both the heavy and light chains have repeating domains
− Heavy → 4 domains, Ch3, Ch2, Ch1 and Vh
− Light → Cl and Cv
• 2 arms so two pathogens can bind
What happens upon papain cleavage of an antibody?
- Cuts the antibody at the amino terminal disulphide bonds that link the two heavy chains
- Releases the two arms of the antibody as two seperate fragments that contain the antigen-binding activity → Fab fragments
- The other fragment contains no antigen binding activity, but an crystilise readily → Fc fragment
- Corresponds to the paired Ch3 and Ch2 domains.
- Functional differences between heavy chain isotypes lie in the Fc fragment
What happens upon pepsin cleavage of an antibody?
- Cuts the carboxy terminal of the disulphide bond
- Produces a Fab fragment in which the two antigen binding arms remain linked
- Pepsin cuts the remaining part of the heavy chain into several small fragments
- This Fab fragment has the same antigen binding characteriscs as the orginal antibody, but cant interact with effector molecules → has therapeutic potential.
Describe antigen binding to antibodies
- The V regions of any given antibody differ from those of every other
- Sequence variability is not distributed evenly throughout the V region
- 3 particularly variable segments can be identified in both the Vh and Vl domains
- These are hypervariable regions
- There are 3, and the most variable part is the HV3 region
- The regions between the hypervariable regions show less variability. and are termed framework regions.
- There are 3 framework regions in each domain
- The framework regions form the beta sheets
- The hypervariable regions correspond to loops at the outer edge of the beta-barrel
- When the Vh and Vl immunoglobulin domains are paired in the antibody molecule, the hypervariable loops are brought together → forms the antigen binding site
- These 6 hypervariable loops are more commonly termed the complimentarity determining regions → there are 3 from each of the heavy and light chains
- Because CDRs from both the Vh and Vl domains contribute to the antigen binding site, it is the combination of the heavy and light chains, and not either alone, that determines final antigen specificity.
- As the amino acid sequences of the CDRs are different, so to are the shapes and properties of the surface
- antibodies bind ligands whos surfaces are complimentary to that of the antigen binding site
- A small antigen generally binds in a pocket lying between the heavy and light chain V domains
- Larger antigens may interact with all the CDRs and in some cases, part of the framework region
- The portion of antigen to which the antibody binds is the epitope → can be proteins, carbs, lipids or DNA.
Describe lymph node structure
• Afferent vessels drain fluid from the tissues → free antigen diffuses through the extracelluar fluid to the lymoh node, whereas DCs actively migrate, attracted by chemokines. These chemokines also attract lymphocytes from the blood which enter via HEVs.
• Cortex may contain circular aggregates of lymphocytes
− Darker stained area = primary follicle → unstimulated B cells
− Lighter stained area = germinal center → plasma cells and memory B cells. Develop following antigen stimulation.
• Paracortex → between the follicles. Contains densely packed T cells, with some DCs and B cells
• Medulla → less densely packed, consists mainly of strands of cells containing T cells, macrophages, and large numbers of plasma cells.
• The juxtaposition of antigen, APCs, and naitve T cells in the T cell zone (paracortex) creates an ideal environment for activation of naïve T cells
• Activated T cells then move to the border of the follicle, where they can provide their helper function
Describe the structure of different antibody classes.
IgG
• Most abundant antibody in serum
• The basic 2H and 2L chain molecule
• 4 subclasses, IgG1-IgG4 → IgG1 most abundant, 70%
IgM
• Earliest to be produced
• Consists of a pentamer pf 5 Ig molecules joined by disulphide bonds and a J chain
• H chains differ from traditional IgG – have 4 constant domains, not 3
• IgM potentially has 10 binding sites, but steric hindrance means it can only use 6 at once
IgA • main class of antibody found in secretions such as mucous, saliva, sweat, breast milk and colostrum • IgA1 and IgA2 – seem to have same functions • In serum, IgA adopts the basic 2H, 2L chain conformation • IgA in secretions consists of two IgAs joined together by a J chain, and an additional protein called the secretory piece → transports it into secretions
IgE
• Lowest concentration in serum out of all antibodies
• Monomer of 2H and 2L
• Like IgM, heavy chain has 4 constant domains not 3
• Special function – asthma and allergy
Why do mature B cells express both IgM and IgD on their surface?
- The gene encoding the u-chain lies closet to the J gene segments, and therefore closest to the assembled V-region exon.
- One rearrangement is complete, a complete u-heavy chain transcript is produced, and IgM is therefore the first to be expressed.
- Immediately 3’ to the u-gene lies the d-gene, encoding the C region of the IgD heavy chain.
- IgD is co-expressed with igM on the cell surface of almost all mature B cells, but is rarely secreted.
- The function of IgD is unclear
- Has a more flexible hinge region than IgM → thought it may be an auxillary receptor that may facilitate antigen binding to IgM
- B cells expressing IgM and IgD have not undergone class switching
How are transmembrane v secreted antibodies made?
• In its membrane bound form, the Ig heavy chain has a hydrophobic TM domain with anchor residues at the C-terminus
• This domain is absent from the secreted forms
• Production of the two forms is generated by alternative splicing:
− Transmembrane → The whole transcript is created, and the secretor portion is pliced out of the middle.
− Secreted → the poly A tail is added early. The transmembrane exons are therefore not include, and the secretor portion is not spliced out.
What is the relevance of polymer formation?
- For IgA, dimerization is required for transport through the epithelia in the MALT
- IgM is found as a pentamer
- Polymerisation is thought to be important in the binding of antibody to repetitive epitopes.
- Antibodies have at least two identical binding sites
- If an antibody binds to multiple identical epitopes, it will dissociate only when all the binding sites dissociate
- The dissociation of the whole antibody will be slower than the dissociation of a single binding site → multiple binding sites therefore give greater binding strength (avidity)
Describe somatic hypermutation, and how it leads to affinity maturation
• Activation induced cysteine deaminase (AID) initiator of mutations
• Expressed only in B cells
• Requires access to the cytidine side chain of a single stranded DNA molecule → AID initiates nucleophilic attack, resulting in the deaminiation from cytidine to form uridine
• Uridine triggers mismatch repair and base-excision repair
• These generate point mutations
• Mismatch = MSH2 and MSH6 mimatch repair proteins remove the uridine along with several adjacent nucleotides, followed by fill in ‘patch repair’ → error prone and induces mutations
• Base excision = REV1 DNA repair enzyme synthesies DNA over the abasice sites, causing random nucleotide insertions.
• People with AID deficiency lack class switching and somatic hypermutation → lead to predominantly IgM and absence of affinity mutation (hyper IgM type 2 immunodeficiency)
• Somatic hypermutation operates on activated B cells
• It introduced point mutations thought the V-region at a high rate
• Detrimental mutations are selected against:
− In the germinal centre, clones are competing with each other for interaction with antigen
− Favourable mutations that increase the affinity of the receptor clones for antigen means those clones are selected for survival
− These mature into antibody secreting cells.
− This gives rise to affinity maturation → can result in an increases in affinity of 10,000 to 100,000
Describe class switching
• Enables the Vh exon to be associated with different Ch genes in the course of an immune resonse
• Involves recombination between specific switch signals
− Switch regions are repetitive DNA sequences that guide class switching, and are found upstream of the Ig C genes
− Switching is guided by the initiation of transcription by RNA polymerase though these regions
− Transcription through these regions generates R loops which serve as substrates for AID
− This introduces a high density of single strand nicks into the non-template strand
− These nicks are converted to double stranded breaks
− The two switch regions of the two isotypes are then brought together by repair proteins, and class switching is completed by excision of the intervening region of DNA and ligation of the switch regions
• Controlled by CD4+ T helper cells and cytokines
− CD40/CD40L essential, without this, cells make only IgM
− Different cytokines induce different antibody production, eg) IL-4 induces IgE
Describe B cell activation
• B cells require 2 signals to become activated
- Signal 1 → recognition by the Ig
- Signal 2 → usually from a CD4+ T cell
What happens to B cells in a LN when antigen enters?
• Draining antigen is collected by macrophages in the marginal zone
• These APCs to B cells migrating through the cortex
• You need macrophages to do this because more than one Ig on the B cell needs to be cross-linked
• The activated B cell now starts to move towards the border of the cortex and paracortex (where T cells are being activated)
How does the B cell interact with the T cell?
• When the B cell binds antigen, it becomes activated and internalizes the whole complex
• It processes the antigen and expresses it on the surface of MHC-II
• If a CD4+ Tfh cell recognizes the antigen, it delivers the second signal to the B cell
Then a series of interactions occur resulting in clonal expansion and differentiation:
• Costimulatory molecules (CD40L on the T cell binding to CD40 on the B cell)
• Cytokines from the Tfh
• ICOS on the T cell binding to B7RP on the B cell
• B cells then turn into plasma cells
What occurs in germinal centres
- After 4-7 days, some of the activated B cells and Tfh cells move to the cortex and enter primary follciles
- Primary follicles contain follicular DCs
- Form a network
- Designed to hold antigen/antibody complexes in iccosomes
- Hold it for extended periods of time to provide antigen for the next stage of the antibody response – the formation of germinal centres.
- In germinal centres, B cells undergo:
- Affinity maturation
- Class switch
- Differention into memory and plasma cells
What are antibodies functions?
- The constant region confers functional specialization
- First, the Fc portions are recognized by Fc receptors expressed by immune effector cells on phagocytic cells → facilitates the phagocytosis of pathogens
- Fc portions recognized by Fc receptors on mast cells, basophils and eosinophils → causes degranulation, eg) IgE activation of mast cells
- Fc portions can bind to C1q → activate compliment
- Fc portion can delivery antibody to places they wouldn’t reach without active transport, eg: mucous secretions, tears, mill (igA); fetal blood circulation from the pregnancy mother (IgG).
Neutralisation
• Antibody binds directly to the pathogen to prevent attachment and entry into the cell
Opsonisation
• Targets it to phagocytic cells
ADCC
• Antibody binds to an infected cell or large extracellular pathogen
• Forms briges to immune cells, eg – NK cells
• Mediates death of the cell or pathogen
Complement
• Bind to C1q
