Antibody genetics II - The Secondary response and increasing affinity Flashcards
B cell activation
- B cell development does not require Ag
- B cells rearrange their genes and express surface IgM (BCR) in the bone
marrow - B cells then enter the circulation where they may encounter Ag in the
secondary lymph organs - If they don’t encounter Ag they die within a few weeks
Describe T-independent (TI) Ag
*particularly resistant to degradation
* 2 groups based on the manner in which they activate B cells: TI-1 & TI-2
* Generate IgM and don’t induce memory
*TI antigens don’t usually a response leading to class switching or to an increase in antibody affinity, as seen with TD antigens due to the lack of CD40 activation.
* Tend to activate CD5+ B-cells (B-1)
*predominantly activate the B-1 subset (can replenish themselves) of B cells found mainly in the peritoneum. They can be identified by their expression of CD5, which is induced upon binding of TI antigens.
Describe TI-1 antigens
*mainly bacterial cell wall components e.g. LPS. *In high concentrations they can polyclonally activate B cells.
*in lower concentrations they only activate B cells specific for themselves
*do not require a second signal
* Several signal transduction molecules are necessary for mediating TI antigen responses in B cells. These include CD19, HS1 protein and Lyn
Describe TI-2 antigens
*predominantly large polysaccharides with repeating antigenic determinants eg Ficoll, poliomyelitis virus, dextran, polymeric bacterial flagellin.
*thought to activate B-cells by clustering and cross-linking immunoglobulin molecules on the B cell surface, leading to prolonged and persistent signaling
* require residual non-cognate T-cell help, such as cytokines
Describe T-dependent (TD) response
- T cells and B cells recognise different parts of the antigen
(Mitchison 1960-70) - B cells see the epitope using antibodies, T cells see processed peptide fragment.
- T cells need peptide presented on APC e.g. Dendritic cells
- T helper cells
Therefore two processes are required to activate a B cell:
* antigen interacting with B cell immunoglobulin receptors – this involves ‘native’ antigen;
* stimulating signal(s) from TH cells that respond to processed antigen bound to MHC class II molecules.
T and B cell interaction
- Complex process between a Th cell and an Ag specific B cell, 2-way interaction
- Surface IgM binds Ag and internalizes it
- Peptide presented on MHCII
- TCR and MHC polarise on cell surfaces
- Co-stimulatory molecules bind (CD40-CD40L interaction helps to drive B cells into the cell cycle, this transduction of signals induces upregulation of CD80/86
*Signaling through CD40 is also essential for germinal center development and antibody responses to TD antigens - Cytokines produced by Th2 cells (IL-4, 5, 6, 10 & 13) promote B cell activation and production of IgG1 and IgE
- B cell proliferate and differentiate into memory cells or AFC
- T-follicular helper cells, localise to germinal centres and produce IL-21 (critical for germinal centre formation) and direct Ig class switching
Th2 cytokines in B cell response
- IL-4: Induces activation and differentiation in B cells. It also acts on T cells as a growth factor
and promotes differentiation of TH2 cells. excess IL-4 plays a part in allergic disease, causing production of IgE; - IL-5: Similar effect. A growth and activation factor for eosinophils and is responsible for the eosinophilia of parasitic disease
- IL-6: Also produced by other cell types (macrophages, B cells, fibroblasts and endothelial cells) and acts on many cell types, but induces B cells to become AFC
- IL-10: Growth and differentiation of B cells, blocks Th1 cells
- IL-13: shares a receptor component and signaling pathways with IL-4, acts on B cells to produce IgE
B cell activation - two outcomes
– Proliferation and differentiation into antibody forming cells in the lymph nodes or in the periarteriolar lymphoid sheath of the spleen. These AFCs secrete Ab to clear Ag, mostly die within 2 weeks via apoptosis)
– Production of memory B cells, long lived and responsible for long term Ab production
– Occurs in the germinal centres of the secondary lymphoid tissue, spleen (PALS- periarteriolar lymphatic sheath) and lymph nodes.
Sequence of events in B cell activation
- Antigen taken up by DCs (TLR etc)
- DCs activate Th in Lymph node (DC only cell to activate naïve T cells)
- B cells in the lymphoid tissue activated by soluble Ag
- B cells present to T cells and get some Ig production-extrafollicular activation, low level somatic mutation.
- Some T cells develop into T follicular helper cells and these cells move to follicles
- B cells move to follicles
- T and B cells cooperate to form germinal centres where extensive somatic hypermutation, affinity maturation and Ig class switching takes place
Describe Ig Class switching and what it is determined by
- Heavy chain isotype switches to IgG and IgA
- Occurs in the secondary response
- Happens in the germinal centre
- Adds plasticity to the response
- Determines the functionality of the antibody
- Immunoglobulin class expression is influenced by cytokines and type of antigenic stimulus
*It is mostly achieved by gene recombination
*It may be achieved by differential splicing of mRNA
*Class switching occurs during maturation
and proliferation
Cytokine control
- Cytokines produced in immune response direct the way in which class switch goes eg:
– Th1 cells are activated by viruses and bacteria to produce IFN-gamma. This causes switch to IgG- main complement fixing isotype.
– Helminths produce IL-4 from Th2, IL-4 directs
IgE production which target esoinophils to
helminths
– Mucosal tissue results in the switch to IgA under the influence of TGFb
Describe CD40-CD40L interactopm
- CD40, a member of the TNF receptor family, delivers a strong activating signal to B cells,
- upon activation, T cells transiently express a CD40L which interacts with CD40;
- CD40–CD40L interaction helps to drive B cells into cell cycle;
- transduction of signals through CD40 induces upregulation of CD80/CD86 and therefore helps to provide further costimulatory signals to the responding T cells.
- It also induces the expression of Activation-induced deaminase (AID)
- Involved in both class switching and somatic hypermutation
- In mice and humans with no CD40, CD40L or AID most Ig is M
Switch recombination
- Rearranged VDJ exon which encodes the heavy chain V domain recombines with a new C region
- The intervening DNA is deleted
- Switch regions in the introns between J and each CH region.
- Upstream of the switch (S) region, there is an initiation (I) sequence.
- Transcription occurs along the strand but strand breaks occur at the switch regions
- The Sm strand break then recombines with the selected downstream switch region
determined by cytokines - Translation of the protein composed of original VDJ and new C
AID: activation-induced deaminase
*AID = a DNA-editing enzyme that replaces
deoxycytidine residues with deoxyuracil in the DNA of the immunoglobulin heavy chain switch regions.
*The resulting mismatch in the DNA is recognized by the enzyme uracil N-glycosylase (UNG) that removes the deoxy-uracil residues,
leaving abasic sites that are resolved by means of DNA repair mechanisms.
*These DNA modifications trigger both CSR and somatic hypermutation.
*Dramatic expansion of germinal centers ( tonsil and lymph node enlargement), and the lack of susceptibility to opportunistic infections distinguish hyper-IgM syndrome due to AID and UNG mutations from the forms due to defects of CD40L or CD40.
What is the effect of AID and UNG mutations?
What is the treatment of AID deficiency?
*Both AID and UNG mutations cause severe deficiency of IgG, IgA, and IgE production. Furthermore, the immunoglobulins (almost entirely IgM) produced by these patients have low affinity for the antigen
*Treatment of AID and UNG deficiency is based on administration of immunoglobulins.
Somatic hypermutation and affinity maturation
*somatic hypermutation, which leads to the generation of cells with a wide range of affinities for antigen;
* Generates point mutations in the Ig V genes, specific for a particular Ag
* Selects for increased affinity and deletes low affinity Ig
* Occurs in the dark zone of the germinal centres
* Requires T cell help and CD40:CD40L (AID)
* Point mutations occur at 1000x spontaneous rate
(hypermutation)
* Point mutations can accumulate in progeny cells
* Partially understood!
* AID: C to U, then upon DNA replication this can convert to T, therefore C to T mutation, strand breaks and repair can cause errors
* Most mutations will result in lack of binding and so a reduced affinity Ab
* Some will increase affinity for Ag: resulting in high affinity Ab
* Need selection
Overview of B cell selection
FDC express C’ receptors and FcR. They ‘hold’ Ag bound to these structures and display them to B cells
TFH cells produce IL-21 which drives B cells into apoptosis unless they are ‘rescued’ by Ag recognition (Bcl-2 induction, further CD40 interaction with TFH.
As maturation continues [Ag] drops so select for higher and higher affinity B cells.
