The Secondary Response: Somatic Hypermutation and Class Switching Flashcards
What is B cell activation and what are the two different types/ Stages?
B cell development does not require Ag as they rearrange their genes and express surface IgM (BCR) in the bone marrow (Antibody diversity)
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
The immune response to most antigens depends on both T and B cells recongising the antigen in a linked fashion – This is known as a T-dependent antigen (TD)
However, a small number of antigens can activate B cells with without MHC-Class 2 restricted help. These are called T-independent antigens (TI)
What is T-independent B activation?
TI antigens are antigens that don’t require T cells and are particularly resistant to degradation. Many TIs have the ability in large concentrations to activate B cell clones (polyclonal B cell activation). However in lower concentations, they can only activate B cells specific for themselves. Several signal transduction molecules are necessary for mediating these TI responses -> CD19, HS1 protein and Lyn
You can convert TI antigens into TD antigens by altering their structure
2 groups of TI Ags:
TI-1: mainly bacterial cell wall components such as Lipopolysaccharides (Gram negative bacteria cell wall). These don’t require a second signal
TI-2: predominantly large polysaccharides with repeating antigenic determinants eg dextran, polymeric bacterial flagellin, Ficoll and poliomyelitis virus. They are thought to crosslink B cells by clustering to lead to prolonged signalling but they need help from cytokines
What common charateristics can you see in many TI Ags and how are these antigens recognised by the immune system?
Many TI antigens have PAMPs which are recognised by TLRs. They therefore have the intirinsic ability to activate the immune system irrespective of their ability to bind to a specific antigen receptor on B cell clones
TI antigen-Ab responses in vitro are generally weaker than TD responses. TI antigens peak earlier and mainly maintain IgM. Their secondary response resembles the primary response compared to the far stronger secondary response of TD antigens. This means that due to lack of CD40 acitvation, TI antigens can’t induce class switiching/affinity maturation and they can produce memory cells and they tend to activate cell marker CD5+ B-cells (B-1) which can replenish themselves
What is T-dependent B activation?
T cells and B cells recognise different parts of the antigen (Mitchison 1960-70). B cells see conformational epitope (hapten), T cells see processed peptide fragment from antigen
TD B activation occurs in the lymphoid tissues (lymph nodes or spleen)
- Antigen is presented to virgin T cells by APCs e.g. dendritic cells that bind MHC class 2
a. TH cells produce stimulating signals in repsonse to processed bound antigen - B cells (BCRs) also internalise the antigen and present it to the TH cells MHC class 2
- B cells then receive signals e.g. cytokines from T cells
- They can now divide and differentiate into antibody forming cells (AFCs) and memory B cell (Bm) – Clonal Selection
Ligands and interactions
write here after completing notes
What are the two outcomes of B cell activation?
- Production of antibody forming cells (AFCs), secrete Ab to clear Ag, mostly die within 2 weeks
- Production of memory/plasma cells
• Migrate to adjacent follicles in the lymph tissue to form germinal centres before differentiating into different types of B cells (affinity maturation
What are germinal centres?
Germinal centres (GCs) are sites within secondary lymphoid organs – lymph nodes and the spleen where mature B cells proliferate, differentiate, and mutate their antibody genes (somatic hypermutation) aimed at achieving higher affinity) during a normal immune response to an infection. These develop dynamically after the activation of follicular B cells by TD antigen.
The germinal centes have two zones:
• Dark Zone – this is where affinity maturation occurs. Clonal expansion and somatic hypermutation occurs here which is where an enzyme creates mutations in the variable region
• Light zone – where the BCR is selected
This will go through several cycles until affinity is high enough and therefore achieving affinity maturation
What happens to B cells in the GCs?
- Within the lymph nodes, mature peripheral B cells are known as Follicular (Fo) B cells (Fo B)
- They acquire antigen from follicular dendritic cells (FDCs) – APC, which is presented to MHC II and T follicular helper cells (TFH)
- After clonal expansion (multiple rounds of cell division), the B cells go through somatic hypermutation (see later)
a. Involves using an enzyme to mutate antibody-encoding DNA and generate a diversity of clones in the germinal centre (dark zone) - Once affinity maturation has been achieved, maturing B cells (centroblasts) migrate to light zone and ezpress their BCR (antibody) on their cell surface
- The cells are now known as centrocytes -> those that achieved high enough affinity maturation produce Bcl-1 signal which prevents it being apoptosed and prevent autoreactive B cells produced by somatic mutations
- If they survive, with help from T cells, they will undergo class switching
- Then they will exit the germinal centre as either plasma cells (producing antibody) or B memory cells
Describe briefly the process of Affinity Maturation
The basis of affinity maturation is that the BCR that binds antigen originally has very low affinity. For a successful immunological response, we need a high affinity antibody. During this process an enzyme called AID (activation-induced cytidine deaminase)
It happens in both the light and dark zone of the germinal centre.
- Clonal Expansion occurs first – the activated B cells will first make clones of itself.
- Somatic Hypermutation -> AID (activation-induced cytidine deaminase) will induce point mutations in the variable region of the DNA of the B cells.
- After this, they are all unique B cells (made from identical clones) and will migrate to the light zone where the FDC will present the antigen on its molecular surface.
- The B cells that most closely match will bind to these antigens and present them to Tfh cells. The Tfh cells then send back a survival signal.
Since it’s a random process, the cells can end up with a reduced affinity or no change at all. Those cells will apoptose, as they do not receive a survival signal from the Tfh cell. This cycle occurs several times until the affinity is high enough, and the cells then undergo class switching.
What is Somatic Hypermutation?
Somatic hypermutation is where the AID enzyme generates point mutations in the Ig variable (V) gene segments for bith heavy and light chains, specific for a particular Ag but at random generation.
The mutations select for increased affinity and deletes low affinity Ig
• Occurs in the dark zone of the germinal centres
• Requires T cell help, CD40:CD40L (AID) and IL-4 induction
• Point mutations occur at 1000x spontaneous rate (hypermutation) and accumulate in progeny cells
• 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
• Need selection as it is random so some will get higher affinity and other won’t
How does CD40/CD40L work?
- CD40 – lies on B cells or APCs
- CD40L (CD154) – expressed on activated T cells
- When they have ligated, they can initate cell-cell communicatin, particularly on TFH cells
- Induces the expression of Activation-induced deaminase (AID)
- Involved in both class switching and somatic hypermutation > chopping up DNA
- Defect in CD40L means that
o Inability to undergo Ig class switching = Hyper IgM syndrome - Absense of CD40L =
o Stop germinal centre formation, stopping affinity maturation
What is antibody class switching?
This occurs in the heavy chain constant region in the germinal centre after the secondary response. There are two types of class switching:
- Minor – differential splicing (mRNA level) ->between IgM and IgD -> reversible as it doesn’t effect DNA
- Major – DNA recombination (irrerversible)
a. IgM -> IgG, IgA or IgE
b. IgG -> IgA or IgE
Class switching adds plasticity to the response and determines the functionality of the antibody
How does class switching work?
Class switching requires a cytokine signal, switch regions (similar to RSS), AID enzyme and a double strand break (DSB- only occurs downstream of gene sequences)
- Rearranged DNA in IgM- cells has a VDJ exon which encodes the V heavy region has a Constant u (Cu) region, Switch u (Su) region between J and C regions and upstream of this, an initiation u region (Iu)
- This sequence recombines with another constant region e.g. Ce with a Ie and Se upon cytokine and CD40L signals
- Constant regions joint to form a loop of intervening DNA which is then deleted
- Transcription of the VDJ- Ce and then RNA splicing of remaining Ce region and addition of Poly A tail
- Translation of the protein composed of original VDJ and new C
- Now get IgE produced (or equivalent)
How do cytokines determine class switching
Examples:
IgM - no signal as the Ig starts off like this
IgE - IL-4 from helminths via Th2
-> target eosinophils to helminths
IgA -> multiple factors including mucosal tissues under influence of TGFb (transforming growth factor B) or BAFF (B-cell activating factor) or APRIL (A proliferation inducing ligand)
IgG - IFNg (gamma interferon) ->Th1 cells activated by viruses and bacteria to produce IFNg
Recap: So how do we get >108 B cell specificities from 35000 genes?
Two chains per receptor - heavy and light chain which associate randomly
Multiple V (D) J segments – select from to put together a unique combination
Junctional diversity – join P and N nucleotides for more diversity
Somatic hypermutation – throw more mutations – select better antibody
C region switching – change function of Ab
