Humoral immunity:B cell activation, affinity maturation and class switching

Question 1

What do stem cells undergo?

Answer 1

• In the bone marrow, stem cells undergo DNA recombination to become immature B cell

Question 2

What is there in the process of joining and what contributes to the diversity of the antibody?

Answer 2

• in the process of joining there is junctional flexibility and P and N nucleotide addition to contribute to the diversity of the antibody

Question 3

What happens once the mature B cell encounters a pathogen and what is this for?

Answer 3

• Once the mature B cell encounters a pathogen, it becomes activated and undergoes affinity maturation and class switching in the germinal epithelium
  * This is to code in the heavy chain constant region of the B cell

Question 4

What is affinity maturation to further and what does it improve?

Answer 4

• Affinity maturation is to further hone the variable fragments of the antibody to that particular pathogen
-Improves affinity to the pathogen

Question 5

What does the first domain of light and heavy makeup and what does the rest make up?

Answer 5

First domain of light and heavy makes up antigen binding variable fragment and the rest of the constant regions makes up effector function

Question 6

What does the antigen binding variable fragment bind to and what does it do?

Answer 6

• In the antigen binding variable fragment, it binds to the docking site/host entry site of pathogen
○ Prevent the virus from entering cell
○ Or they bind to active site of toxins to neutralise them

Question 7

What is opsonisation mainly for and what does it do?

Answer 7

• Opsonisation: mostly for bacteria as they are larger
○ Antibody tags its surface to recruit other cells e.g. macrophages that have Fc receptors – these bind to the active site of the antibody and carries out antibody dependent cellular phagocytosis

Question 8

What region is the active site of the antibody?

Answer 8

Active site is in CH2 region of antibody ADCP

Question 9

What will antibodies do to tumour cells which are large?

Answer 9

For tumour cells which are larger, it will opsonise them and will recruit NK cells to carry out ADCC – cellular cytotoxicity

Question 10

How do antibodies form an immune complex and what does this cause?

Answer 10

• More and more antibodies can bind to the surface of the bacteria which forms an immune complex
○ This causes agglutination and precipitation as the complex precipitates out of the bloodstream, rendering the bacteria nonfunctional

Question 11

What can you form immune complexes with other than antibodies and what does this lead to?

Answer 11

• Can also form immune complexes with complement known as complement fixing
○ Can lead to phagocytosis – agglutinates and precipitates and then is cleared

Question 12

What can complement form in certain pathways and what does it do to tumour cells?

Answer 12

In certain pathways, the complement can form MAC (Membrane Attack Complex) which punches holes in the tumour cells, causing them to lyse

Question 13

How many classes of antibodies are there and what are they determined by?

Answer 13

• 5 classes, all determined by 5 classes of heavy chain

Question 14

How many heavy chain domains does IgG have and what stabilises it between the 2 chains?

Answer 14

IgG has 4 heavy chain domains – variable and constant 1, 2 3 – stabilised by two disulphide bonds between the heavy chains

Question 15

What does IgD have a long region of and what is it stabilised by?

Answer 15

IgD has a long hinge region, stabilised by one disulphide bond

Question 16

How many domains does IgE have?

Answer 16

IgE has 5 domains

Question 17

What is the modification that IgA has but IgG doesn’t and what does this allow?

Answer 17

IgA is similar to IgG but the third CH3 domain has a modification so that it allows interaction with the J chain

Question 18

What Ig is IgM similar to and what can it form and with the help of what chain?

Answer 18

• IgM is similar to IgD – 5 domains but it can form a pentamer with the help of the J chain

Question 19

Abundances of Ig in serum

Answer 19

• IgG is the most abundant in serum followed by IgA and IgM

Question 20

What is the only Ig to cross placenta?

Answer 20

• Only IgG crosses the placenta so mum can pass on antibodies to fetus

Question 21

What Ig’s fix complement?

Answer 21

• IgM and IgG fix complement

Question 22

What does IgG attract?

Answer 22

IgG attracts phagocytes

Question 23

What does IgE attract?

Answer 23

IgE attracts mast cells and basophils

Question 24

What is the first antibody to be produced after class switching?

Answer 24

• IgG is the first antibody to be produced after class switching

Question 25

Life cycle of B cells(Antigen dependent)

Answer 25

• After VDJ recombination of B cells, they become mature circulating B cells  • Lymphoid progenitor stem cells give rise to T cells  ○ T cells produced migrate to thymus where they undergo similar VDJ recombination to become TCRS  ○ T cells receive signals from APCs and become activated and in turn activate B cells when there are pathogens  • B cell migrate to GC for selection  • GC is divided into dark and light zone  ○ In dark zone: affinity maturation happens  • Honing of variable fragment to that particular pathogen  § Involves clonal expansion, somatic hypermutation (enzyme creates mutation in its DNA)  ○ In light zone: selection - selecting the BCR that binds to the pathogen the best  § Undergoes class switching because by this time you know the pathogen being dealt with so need an appropriate class • Differentiate into plasma and memory cells that will circulate in the blood and spleen 

Question 26

How many stages is B cell activation divided into and what are they?

Answer 26

• Divided into two stages  1. T cell independent  B cell activation  2. T cell dependent B cell activation

Question 27

T cell independent B cell activation

Answer 27

• When B cell encounters antigens expressed on the surface of the pathogens, it undergoes clonal expansion   ○ Some of them will differentiate into IgM secreting plasma cells  ○ This is the first line of defence 

Question 28

T cell dependent B cell activation

Answer 28

• Some of the cells will migrate into the lymph nodes  • Needs to recognize antigens, internalize them and then present it in MHC class II  • Second step: activation by TCR ○ TCR has to recognize the antigens presented on B cells and APCs • THC produce cytokines to complete the three step verification process that the b cell activation requires 

Question 29

What are the 3 signals that the B cell activation requires?

Answer 29

1. Antigen binding to BCRs 2. Co-stimulation by activated Th cell specific to same antigen 3. Th cell-derived cytokines to verify that there is an active infection going on 

Question 30

What does BCR binding cause the activation of?

Answer 30

Causes the activation of tyrosine kinase

Question 31

What does tyrosine kinase phosphorylate and activate?

Answer 31

• Tyrosine kinase phosphorylates other proteins and activates pathways involved in cell proliferation as B cell needs to make copies of itself 

Question 32

What does VDJ recombination in the body produce?

Answer 32

• VDJ recombination in the body produce 1 billion B cells with unique BCRs 

Question 33

What can BCR do when pathogen invades?

Answer 33

• When pathogen invades, the BCR that can bind to it and makes clones of itself

Question 34

Where do clones of BCR migrate to?

Answer 34

• Clones migrate to GC for affinity maturation

Question 35

Where does affinity maturation happen and what 2 cells is it helped by?

Answer 35

• Affinity maturation happens in the GC and is helped by two types of cells ○ T follicular helper cells (Tfh): subset of T cells that can enter the GC ○ Follicular dendritic cells (FDC): not your normal DCs

Question 36

Where do follicular dendritic cells stay in and present?

Answer 36

• FDC stay in GC and present antigens for selection process 

Question 37

Steps involved in affinity maturation and class switching in GC

Answer 37

• B cells will be activated by Th and migrate to GC while undergoing clonal expansion  • Enzyme causing mutation in variable region  • Migrate to light zone and with the help of FDC and TFH it will undergo selection  ○ This will repeat until affinity is high enough  • B cell will undergo class switching to select for the right antibody class before differentiating into plasma cells and memory cells 

Question 38

What zones does affinity maturation happen in?

Answer 38

• Happens in dark and light zone 

Question 39

Detailed steps involved in affinity matuation

Answer 39

• Activated T cell makes clones of itself  • Activation induced cytidine enzyme (AID) makes point mutations in the variable regions of the DNA  ○ Before this, the B cells are clones ○ After AID, they are all unique B cells with unique mutations  • These migrate to the light zone where the Tfh present antigens on its surface  • B cells with high affinity BCR on surface will try to grab the antigens on its surface and then present to Tfh  ○ Tfh will give a survival signal  • The rest also have point mutations in the DNA and these can cause improved affinity, reduced affinity or no improvement in affinity  ○ Those with reduced or no improvement will undergo apoptosis because there is no survival signal from Tfh  • Have to repeat this process multiple times until affinity is high enough 

Question 40

What happens to B cells once the affinity is high enough?

Answer 40

○ Once affinity is high enough, it progresses to the next stage – class switching 

Question 41

What does class switching only affect and not affect?

Answer 41

• Class switching only affects heavy chain constant regions ○ Doesn’t affect variable regions of the heavy and light chain  ○ Constant region of light chain stays the same 

Question 42

What are the 2 different types of heavy class switching?

Answer 42

1. Minor:Differential splicing(mRNA level) | 2. Major:DNA recombination(Irreversible)

Question 43

What is minor class switching between?

Answer 43

○ Between IgM and IgD

Question 44

Reversibility of minor class switching

Answer 44

This is reversible

Question 45

What does major class swithich occur between?

Answer 45

○ Class switching can only occur from IgM to IgG, IgA, IgE AND IgG to IgA, IgE

Question 46

Why is major class switching only between the specific Ig?

Answer 46

○ This is because of the locations of the constant regions 

Question 47

What does class switch recomination(CSR) require?

Answer 47

○ Cytokine signal ○ Switch regions – similar to RSS but is in front of heavy chain constant region gene segments  ○ AID and DSB (double stranded break) repair proteins

Question 48

What is recombination between in class switch recombination?

Answer 48

• Recombination between switch regions 

Question 49

How does switching proceed in class switch recombination?

Answer 49

• Switching only proceeds downstream ○ IgM to IgG, IgA, IgE ○ IgG to IgA, IgE ○ E..g you cannot switch from IgE to IgA 

Question 50

What does AID help in?

Answer 50

• AID helps in somatic hyper mutation

Question 51

What is the default after affinity maturation and before class switching?

Answer 51

• After affinity maturation and before class switching, B cell still makes IgM and IgD as this is the default 

Question 52

What is the final form of Ig?

Answer 52

• Secreted form is final form 

Question 53

When is Ig membrane bound?

Answer 53

• Membrane bound is during antibody development 

Question 54

What piece does secreted Ig have?

Answer 54

• Secreted has tail piece 

Question 55

What is the Ig anchor on the membrane made of?

Answer 55

• Membrane has an anchor made of hydrophobic transmembrane region and cytoplasmic tail 

Question 56

What is true differential splicing?

Answer 56

• True differential splicing is how the B cell knows how to make both of these forms of antibodies  

Question 57

What remains unchanged in DIFFERENTIAL SPLICING OF μ CONSTANT REGIONS?

Answer 57

• Leader VDJ remains unchanged 

Question 58

How does differential splicing of μ constant regions for secreted heavy chains?

Answer 58

• For secreted heavy chains, the DNA region is transcribed into MRNA and spliced at first polyA site   § This generates mRNA coding for the new secreted heavy chain

Question 59

What do you need to splice up first if you want to encode membrane bound new chain?

Answer 59

• If you want to encode membrane bound new chain, you need to splice up first polyA site 

Question 60

What does differential splicing of μ constant regions?q

Answer 60

• Prevents antibody from secreting out of B cell