(L23) Adaptive immunity, antibodies and gene rearrangement

Adaptive immunity first observed in…

jawless fish (Agnathans) that evolved ~500M years ago

Hagfish and Lamprey eels are still around today and display ___________ (VLR), a form of __________ but don’t have BCR, TCR, or MHC. Every other species from jawed vertebrates onwards has __________ meaning this was a very successful ________.

variable lymphocyte receptors, immune repertoire, adaptive immunity, adaptation

We know how this event came about because the molecular mechanisms in B and T cell rearrangement are almost identical to another mechanism called ________ that is common in species such as plants and bacteria

transposition

• shared genes with bacteria

Transposition

The changing position of a gene within a genome

Transposition requires two key elements - these two elements are highly conserved through all species with adaptive immunity

• Transposase or recombinase

• Recognition sequences

Transposase or recombinase

A specialist enzyme that cuts and repositions the gene

Recognition sequences (RS)

short conserved base pair sequences at the ends of the genes recognised by the transposase

Adaptive immunity and immune memory is

A system that changes with time to respond faster and more effectively to a pathogen. Immune memory can provide life-long protection (e.g. measles)

Adaptive immunity and immune memory have a massive…

repertoire of billions of naive B and T cells is formed randomly before birth. Each lymphocyte has a different antigen specificity

The affinity of a B/T cells antigen receptor (BCR and TCR) toward its antigen, increases over…

time and the persistence of the antigen

Diversity is caused by random __________ and __________ in genetic _______ that code for the B and T cell _____________

rearrangement, recombination, loci, antigen receptors

BCR and TCR are the only genetic region in your entire genome that can rearrange in this fashion because…

the small segments that make up your Ig and TcR loci all have the essential RS sequences at their ends

Antibodies are formed from a repeated protein unit called the…

Ig domain - similar Ig domains are found in hundreds of other proteins

The Ig protein fold is referred to as a…

B-barrel, ~110 amino acids in length

Two anti-parallel B-pleated sheets are like two cupped hands. A stabilising __________ connect the palms and the _______ join the fingers. A very stable fold

disulphides bond, loops

The loops at the ends are _________ (meaning they can bend into various shapes, are not rigid but flexible) so their ___________ sequences can _______ without disrupting the overall ________ of the domain

unconstrained, amino acid, vary, structure

The Ig-fold

• 12.5kD b-barrel ~110 amino acids

• 2 anti-parallel b pleated sheets

• Sheets joined by a central disulphides bond

• b strands joined by 3 loops

Antibody structure consists of…

4 protein chains that are constructed from repeating Ig domains

Each B cells makes two…

separate chains - Heavy (H), and Light (L)

1 H chain is disulphides linked to 1 L chain. The two H chains are also joined by disulphides bonds

L—s-s—H—s—s—H—s—sL

The Y shaped antibody has two identical…

antigen binding sites

are located at the tip of two flexible arms. This is where all amino acid diversity arises

The invariant effector region (CH2 and CH3) is where…

Fc receptors and complement component C1 bind and defines the class of antibody and its function

Function of antibodies depend on which…

H chain gene is used

Five classes of antibodies

• IgM

• IgG

• IgD

• IgE

• IgA

IgM

• Default Ig made by all naive B cells

• Comes in two forms - membrane bound (monomer) called B cell Antigen Receptor (BCR) and soluble form in blood (pentamer)

• Soluble IgM has 10 potential antigen binding sites

• Reacts to surfaces such as microbes through very high avidity binding

• Excellent at fixing complement with 5 Fc regions that bind complement components C1

Affinity

When the sum of the attractive molecular forces between two surfaces exceeds the repulsive forces, then there is an _____

The higher the _______, the fewer antibody molecules it takes per unit volume to “find and bind” the antigen

Avidity

Results from multiple contacts. Like velcro, the binding force can be orders of magnitude higher than the force associated from a single affinity interaction

Antibodies are ‘multivalent’ and allow a randomly generated naive antigen receptor e.g. IgM to distinguish a pathogen from the self

Amino acid variation is found in 3 discrete regions in the variable V domain called

Complementarity Determining Regions (CDR)

CRD1, 2 and 3 correspond to the 3 loops that connect the B-strands in the…

V domain

6 hyper variable loops from Vh and Vl come together in the folded protein to form the…

antigen binding surface

Each antibody has at least…

2 identical antigen binding sites

Germ-line Ig and TCR genes are segmented into 4 clusters called

Variable, Diversity, Joining, and Constant regions

Special recombinase enzymes (RAG1 and RAG2) which are only active in B and T lymphocytes recognise contrived base pair sequences (RS) at the..

5’ and 3’ ends of each DNA segment

As B cell develops, the following happens:

1. A D segment joins to a J

2. A V segment joins to the new DJ

3. Intervening DNA is discarded

Joining of the segments is very _______. Lots of changes where base pairs are randomly changed during DNA ligation. This creates ___________ in the central ______ loop (called the VDJ join) in the middle of the ________. C-segments code for the rest of the H and L chain

imprecise, massive sequence variation, CDR3, antigen binding site

Clonal selection and affinity maturation

Genome does not know what pathogens will be encountered before birth, so will randomly make as many receptor combinations as possible - pathogen then selects and expands just the right clone through this process

First step of Clonal selection and affinity maturation

We all start with multiple low affinity naive B cells. A single B cell clone with a BCR that reacts to a non-self antigen with sufficient avidity to trigger proliferation and expansion of that clone.

Second step of Clonal selection and affinity maturation

Over time, random mutations accumulate in the BCR gene of the selected clone, some of which increase the BCR affinity towards antigen

Third step of Clonal selection and affinity maturation

After multiple rounds of selection, a new B cell clone making high affinity, highly specificity IgG arises from the lymph node

Produces IgG secreting Plasma memory cell

Clonal selection and affinity maturation takes place in…

the lymph node follicles

Steps in affinity maturation in B cells

1. Antigen stimulates a B cell clone with a naïve BCR to grow and expand within in a germinal centre.

2. The B cell switches from using the μ (M) to the γ (G) heavy chain to make an IgG molecule.

3. Random somatic hypermutations occurs in the Ig gene of the clone as it proliferates. Some of these mutations result in new clones with improved antigen receptor affinity. These clones are more sensitive to diminishing antigen resulting in faster expansion.

4. After successive rounds the mature B cell becomes a plasma cell secreting long-lived soluble Ig designed specifically for selecting antigen

5. Some of these mature B cells take on a memory phenotype and reside in lymph nodes and tissue long-term waiting to respond to the next infection.

6. This is the primary mechanism behind acquired immunity, memory and vaccination