Humoral Immunity; Antibodies and the life cycle of B cells

Question 1

What are antibodies made by?

Answer 1

Antibodies are made by plasma cells to fight against pathogens and cancerous cells. They work by blocking pathogens or tagging them for removal of other cells.

Antibodies are also called IMMUNOGLOBULINS.

Question 2

What is the overall structure of antibodies?

Answer 2

Y shaped molecules made up of 2 heavy chains and 2 light chains.
The heavy chains have 4 domains. (with extra subtypes) The light chains have 2 domains.
All chains are a stream of amino acids.

Different antibodies have different variable regions for each pathogen. All antibodies of the same class have the same constant region.
 2 types of antibody: B cell receptor and secreted antibody

Question 3

What are the different classes of antibodies?

Answer 3

IgD- heavy chain delta, only antibody that is not secreted, B cell receptor, its presence indicates mature B cells

IgG- heavy chain gamma, main antibody in serum- 80%, only antibody that crosses the placenta, constant region of IgG usually binds to phagocytes, mainly used to fight off pathogens, main antibody of secondary responses, involved with neutralising toxins and opsonisation

IgA- heavy chain alpha, secreted into ,mucous, tears, saliva, colostrum

IgM-heavy chain mu, mainly involved in the primary response, best at forming immune complexes and fixing complement

IgE-heavy chain epsilon, constant region binds to mast cells and basophils, involved in allergies and getting rid of parasites

Question 4

What does heavy chain class switching affect?
What are the 2 types of class switching?

Answer 4

Heavy chain class switching affects only the constant region of the heavy chain

Class switching allows body to be more versatile with the pathogens it deals with

class switching:
1.minor class switching: differential splicing- between IgM and IgD (minor because it does not affect the DNA of the B cell itself)

2.major- DNA recombination

Question 5

How does the B cell know which class to switch?

Answer 5

by sensing chemicals around them, in which these chemicals are produced by T helper cells and indicates what type of pathogen you are dealing with.

In addition to cytokine signals, also need CD40L on T cells which interacts with CD40 on B cells

Question 6

What 3 things are needed for class switch recombination?

Answer 6

1) Cytokine signal; 2) Switch regions; 3) AID and DSB repair proteins

Question 7

How do the two types of antibodies- the secreted form and membrane form- differ/ how are they the same?

Answer 7

The secreted form is generated by plasma cells and the membrane bound B cell receptor is present on the cell since it is an immature B cell.

Both the secreted and membrane bound antibody have the same heavy and light chain variable regions and constant regions.
The difference is the secreted version has a tail piece whereas the membrane bound version has a transmembrane region and a cytoplasmic tail as an anchor.

Question 8

Summary so far

Answer 8

Antibodies – secreted by B cells to neutralize pathogens; B-cell receptor

 Structure – 2 HC, 2LC
– domain vs fragment
– membrane-bound vs secreted

 VH and VL CDRs bind to antigen

 5 classes of antibodies – different effector functions to deal with different
pathogens
– Class switching: Heavy chain constant region change, the rest stays
the same

Question 9

What are the 2 stages to B cell development?

Answer 9

The antigen independent stage and antigen dependent stage

Question 10

What does the life cycle of the B cell include?

Answer 10

The B cell starts its life in the bone marrow. It begins as a stem cell and differentiates into a pro-B cell.
The DNA of the pro-B cell undergoes V–>J and V—>DJ recombination to permanently code in the heavy chain variable region. The variable region will be expressed with the new heavy chain.
This is now known as the Pre-B cell - the cell becomes a pre-B cell when it can express a fully heavy chain with a unique variable region- which then undergoes another V—> J recombination to permanently code in the light chain variable and constant region to become immature B cells.
These immature cells express IgM and mature over time- once they can express IgM and IgD on their surface through differential splicing of their mRNA, they will become mature B cells and circulate between the bloodstream, spleen and lymph nodes.

The majority of cells further develop into professional plasma cells that secrete the antibody they code for.

After infection, some B cells remain as memory B cells.

Question 11

How can additional diversity be generated during recombination of the heavy and light chains?

Answer 11

During the VD–>J and V–> recombination of the heavy and light chains, additional diversity can be generated:

1. through junctional flexibility
2. through P and N nucleotide addition

These help form the billions of types of B cells.

Question 12

What is IgD like?

Answer 12

The IgD is like a quality control check

Question 13

How many antibody genes are inherited?

Answer 13

None! No complete genes are inherited, only gene SEGMENTS.

These gene segments are arranged in different combinations to generate many antibody( Ig) sequences.

Question 14

What is the heavy and light chain gene loci made up of?

Answer 14

different segments; V, J and C segments for the light chain and V, D , J and C (simplified) segments for the heavy chain.

There is rearrangement of these segments.

Question 15

What are the 3 genetic loci encoding Ig?

Answer 15

 Two for light chain: kappa (κ) and lambda (λ) locus
 One for heavy chain
 Located on different chromosomes

Question 16

What are recombination signal sequences (RSS)?

Answer 16

Recombination signal sequences (RSS) – conserved sequences
upstream or downstream of gene segments
made up of ‘Turns’ consisting heptamer and nonamer with a 12 or 23 bp spacer

there are 2 types of turns:
a two turn contains a 23bp spacer
a one turn contains a 12bp spacer

Question 17

Why do you need two types of turns in regards to recombination?

Answer 17

One-turn/two-turn rule (12/23 rule)

Recombination only occurs between a segment with a 12bp spacer and a 23bp spacer.

ie recombination cannot occur between a one turn and one turn or two turn and two turn ie it needs to be between a one and two turn

Question 18

What are some examples of mechanisms for generation of antibody diversity?

Answer 18

Multiple germline V, D and J gene segments
 Combination V-J and V-D-J joining
 Junctional flexibility
 P-nucleotide addition
 N-nucleotide addition
 Combinatorial association of heavy and light chains
 Somatic hypermutation during affinity maturation

Question 19

When can junctional diversity take place? what is a good thing/bad thing?

Answer 19

 Junctional flexibility during V(D)J recombination, P and N nucleotide
additions
 Good: Antibody diversity
 Bad: Non-productive rearrangements (incorrect reading frame) –wasteful process

minor hairpin formed is between 2 strands of DNA
major hairpin is whole DNA folded in half

enzymes then come to repair and process the ends of the hairpin, forming a coding joint of v17 and j3 as well as a signal joint

process from hairpin formation:

hairpin needs to be opened which is done by artemis
exonucleases/TdT then mess around with the ends of the hairpins adding/removing nucleotides
then ends are joined together again by another series of enzymes

Question 20

What does junctional flexibility involve?

Answer 20

▪ Removal of nucleotides between gene segments during V(D)J recombination
▪ Precise mechanisms unknown
▪ Involves Exonuclease - removes mismatched nucleotides

Question 21

What is allelic exclusion?

Answer 21

 Two copies of each Ig gene – one from mother and one from father
 In other cells, both genes are expressed
 Antibody genes different – Only one heavy chain allele and one light
chain allele is expressed
 Order of rearrangement: Heavy>kappa>lambda; 1st allele then 2nd
 Ensure each B cell makes one type of antibody

Question 22

Summary of antigen independent phase in the life cycle of a B cell

Answer 22

 Life cycle of B cell – antigen-independent phase (occurs in bone marrow)
 B cell receptor diversity at this stage generated by:
– Multiple germline genes from mum and dad
– Heavy and light chain combinations
– V(D)J recombination (one-turn/two-turn rule)
– Additional diversity: P and N nucleotide addition;
Junctional flexibility
 Mature, naïve B cells expresses IgM and IgD (once an immature B cell expresses both IgM and IgD, it is a mature B cell)
– Somatic recombination vs differential splicing

 Allelic exclusion

Question 23

When infection of a pathogen occurs, where do B cells go and what process occurs there?

What does that process involve?

Answer 23

T helper cells are involved in the activation of B cells when a pathogen arises/enters. The activated B cells migrate to the germinal centre, GC where it will undergo affinity maturation. This is to improve affinity for attacking pathogens.

Affinity maturation involves:
Clonal expansion
Somatic hypermutation
Selection

This process is repeated several times. Then the antibody will receive signals to identify the pathogens that entered. These antibodies will then undergo class switching to ensure they have the appropriate effective functions.

After this the B cells differentiate to plasma cells, secreting antibodies whilst still maintaining some B cell receptors on their surface.

A few B cells will become memory B cells. Both B cells and plasma cells will circulate around in the bloodstream

Question 24

In summary, what happens when they body encounters a pathogen?

Answer 24

A subset of B cells that recognise the pathogen will be activated. The rest of the B cells that are not activated continue to patrol the bloodstream.

Question 25

What occurs during B cell activation?

Answer 25

There are 2 stages to B cell activation:
The T cell independent stage
The T cell dependent stage

(separate to B cell in/dependent stages!)

When a pathogen invades, the B cells are partially activated.
The B cells will make clones of itself through clonal expansion. Some of the clones will become the first defence army, secreting IgM.
The other clones will migrate to the lymph node to wait for T cell activation.
T cell dependent, B cell activation requires a triple activation process to ensure B cells don’t get activated by mistake:

First the B cell needs to encounter the pathogen and internalise the antigens, which is presented on the surface of the B cell, via an MHC Class II receptor.
Then it has to be activated by a T cell, which in turn has been activated by the same pathogen.

So the pathogen will be engulfed by dendritic cells presented on the surface and the T helper cells would detect and be activated which then activates the B cell receptor.

The CD40/CD40L is the ‘handshake’ that confirms this cell is a T helper cell and not anything else.

The third signal it requires are cytokines produced by the T helper cells.

The fully activated B cells then undergo affinity maturation and class switching

Question 26

Short summary of the above flashcard, describing B cell activation

Answer 26

 Differentiation and clonal expansion of activated B cells
 Requires 3 signals
– Antigen binding to BCRs
– Co-stimulation by activated Th cell specific to same antigen
– Th cell-derived cytokines
 Signal transduction pathway

Question 27

What do we mean by naive B cell?

Answer 27

The B cell has not been exposed to the pathogen

Question 28

What does affinity maturation set out to do?

Answer 28

Improve the affinity of the antibody to the antigen

Question 29

Where does affinity maturation take place?

Answer 29

In the germinal centre of the lymph node- these are circular cell clusters at the periphery of the lymph node.

Apart from the B cells themselves, 2 types of cells help with the process- the T follicular helper cells and the follicular dendritic cells.

The follicular helper cells are the only T cells that can enter the germinal centre. (Other T cells stay in the T cell zone of the lymph node)

The follicular dendritic cells present antigens in the germinal centre.

Question 30

What is the germinal centre made up of?

Answer 30

The GC is made up of a dark zone and light zone.
In the GC, affinity maturation and class switching occur.

Question 31

Summary really simply of when B cell gets activated until affinity maturation and class switching.

Answer 31

B cell activated by T helper cell.
B cell migrates to GC and make clones of itself(clonal expansion)
Then B cells undergo affinity maturation- which involves somatic hypermutation and selection.
Then the B cell with the highest affinity undergoes class switching before differentiating into plasma cells or memory cells

Question 32

What happens during affinity maturation?

Answer 32

After activated B cells enter GC, they will go to the dark zone where clonal expansion takes place.
Then AID generates point mutations in the variable region gene of the B cells at random points.
So now B cells differ from each other slightly- called somatic hypermutation.
The hyper mutated B cells got to the light zone to undergo selection.
In the light zone, follicular dendritic cells (FDC)present the antigens on the surface and the B cells have to compete for the limited amount of antigens on the FDC.
After accessing antigens, they present this to the T follicular helper cells, which also give them a survival signal.
If they don’t have the survival signal, apoptosis occurs

The B cells that survive this apoptosis migrate back to the dark zone and the process begins again until antibody affinity is high enough.

While B cells undergo clonal expansion, AID makes random mutations in their variable regions. Over multiple cycles, affinity of the antigen improves and also get rid of antibodies with reduced affinity. all random process

Question 33

Summary of last bit of lecture

Answer 33

 Naïve circulating B cells that binds to invading pathogen gets
activated
 B-cell activation
– 3 step verification process
– T-cell independent vs T-cell dependent
 Affinity maturation to fine tune antibody affinity
– Occurs in germinal centre;
– Clonal expansion, somatic hypermutation followed by selection occurs- all to fine tune affinity to the pathogen.,(go on until antibody affinity to pathogen is maximised)