Humoral Immune System

Question 1

Describe the structure of antibodies

Answer 1

Question 2

Draw a basic antibody structure

Answer 2

Question 3

What is the difference between the variable and constant regions?
What type of receptors do macrophages and eosinophils have?

Answer 3

Antigen binding Variable region=VL + VH
The tail of the Ab, the constant region (Fc region) mediates the effector function of the Ab, but is not involved in Ag binding
Macrophages and eosinophils have Fc surface receptors, therefore bind Fc and remove the microbes opsonised by the Ab

Question 4

So the antigen binding site contains the variable light and variable heavy chain (VL and VH). Describe these in more detail

Answer 4

VH and VL contain 3 unique hypervariable regions
HV regions have protruding loops which make contact w Ag
HV regions are aka complementarity-determining regions (CDR1, CDR2 and CDR3)

Question 5

HV regions are aka complementarity-determining regions. Describe these
Which one is most variable, how is the primary structure different to the tertiary etc

Answer 5

CDR3 has the highest variability due to genetic mechanisms that ensure Ab diversity
Binding of diverse Ag by Ab is mainly due to the hypervariable regions (CDRs) of the VH and VL domains
Primary structure CDRs are separated, but when folded into tertiary, CDRs form adj to each other

Question 6

What are B cells and clonal expansion?

Answer 6

B cells activated by their specific Ag differentiate-> Ab secreting plasma cell
Clonal selection and expansion occurs
Some activated cells become memory cells
Activation needs CD4+ Th via cytokine release
Natural immune responses =polyclonal: >1 B cell activated, >1 Ab synthesised
This is bc multiple Ags on organism, multiple epitopes on each antigen

Question 7

Describe the generation of Ab diversity

Answer 7

No complete Ig gene is inherited, only gene segments
Ig genes rearrange during B cell development in bone marrow
VH: encoded in 3 gene segments (V, D, J)
VL: encoded in 2 gene segments (V, J)
Random gene segment recombination=Ab diversity
This is somatic DNA recombination and occurs in both B and T cells

(VDJ rearrangement for Vβ and VJ rearrangement for Vα) THIS IS FOR TCRs

Question 8

Describe gene rearrangement during b cell development in the bone marrow.

Answer 8

Immature B cells undergo 2 rearrangements
First, successful VDJ rearrangement –> heavy chain
Then, successful VJ rearrangement–> light chain

Heavy and light chain rearrangements cannot occur side by side, they must occur sequentially
As a result, mature naïve B cells will express full IgM or Ag receptors as both heavy and light chains are present

Question 9

How is somatic DNA recombination regulated?

Answer 9

Each segment to be joined is flanked by Recombination signal sequence (RSS) - These are conserved non coding DNA sequences
RAG-1 recombinase cuts DNA at precise location
Numerous enzymes ligate the two ends to form a functional Ab chain.

Question 10

Describe the number of functional gene segments for Abs

Answer 10

Multiple copies of each region in the germline
Random gene selection enable greater diversity.
Not all genes are functional= pseudogenes- the Ab does not become functional
eg Fc region contains 9 possible gene segments; therefore 9 diff heavy chains can be produced (the type of heavy chain determines the Ab class)

Question 11

Explain what is meant by combinatorial and junctional diversity as a way to increase Ab diversity

Answer 11

Combinatorial Diversity
Somatic DNA rearrangement of genes V, J, D
Random pairing of different combinations of L and H chain regions in diff B cells

Junctional Diversity: using TdT enzyme to add nucleotides between junctions of gene segments before they’re ligated.

Question 12

What is meant by allelic exclusion?

Answer 12

B cells are diploid= 2 alleles for heavy chain so in theory could make 2 diff heavy and 4 light chains
This never happens bc of allelic exclusion:
Que one allele sea successfully rearranged and starts making heavy chain= switch off rearrangement of other. Same occurs for light chain

Question 13

What is light chain restriction?
Apply light chain restriction in the context of Polyclonal B cells

Answer 13

Each B cell either produces kappa or lambda never both= light chain restriction
Polyclonal B cells= mixture of cells making kappa or lambda

Question 14

What type of Ig do mature naive B cells have?

Answer 14

Mature naïve B cells express a membrane-bound IgM
Membrane IgM acts as B cell Ag receptor
Ag recognition by membrane IgM => activation of signalling pathways => B cell activation

Question 15

Which 2 immunoglobulins are co-expressed on B cells?

Answer 15

IgD is co-expressed with IgM on mature naïve B cells
IgM is the 1st ig to be produced
IgD is produced al mismo tiempo w IgM by differential splicing
The constant region can be μ or delta, cutting out parts of these will give rise to either IgM or IgD

Question 16

Describe the B lymphocyte subtypes

Answer 16

Follicular: spleen, lymph. Recognise Ags => Ab
MZB: spleen, lymph nodes. Recognise polysac, glycolipid, nucleic acid antigens. Produce mainly natural low-affinity IgM
B-1 cells: Found in peritoneal cavity, mucosal tissues. Recognise polysacc, glycolipid, nucleic acid antigens. Produce mainly natural low-affinity IgM

Question 17

Where is the location of the lymphocytes in the lymph node and how do they interact w each other?

Answer 17

B and T cells are close together in the lymph para interact w each other via linked recognition.
This is done by sustained contact using the SLAM family
B cells and Helper T cells must recognise epitopes of the same molecular complex in order to interact

Question 18

Describe what happens to a naive B cell as it travels through a lymph node
What happens when B cells encounter an antigen here?

Answer 18

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Question 19

How does Ab type change during B cell responses?
What is this triggered by, how does the change occur?

Answer 19

During the B cell response, hay a switch from producing membrane-bound IgM to secreted IgM
This is triggered by Ag recognition by the membrane IgM (BCR)
The switch occurs via differential exon splicing
The Ig function changes from receptor to effector function, but Ag recognition does not change.

Question 20

Give the structural differences between membrane bound and secreted antibodies.

Answer 20

Structurally, membrane bound Ig has a hydrophobic region at its tail end, allowing it to insert in the hydrophobic lipid bilayer of the cell membrane. It also has a cytosolic tail
The secreted form of Ig has neither of these features
These structural differences are due to differential mRNA splicing

Question 21

The structural differences between membrane and secretory Ig are brought about by differential mRNA splicing. Describe this

Answer 21

Each domain (V,D,J) within the heavy chain is encoded by separate exons
IgM (μ) heavy chain has 4 exons. All other Ig heavy chains have 3

The heavy chain gene has an exon with a secretion coding sequence, and an exon with a membrane coding sequence
Differential splicing of these exons determines if the Ig produced will be secreted or membrane-bound

Question 22

Some proliferating B cells migrate to the follicle to form a germinal centre when they encounter an antigen.
What exactly is a germinal centre? What does it consist of?

Answer 22

An area of sustained B cell proliferation + differentiation, disappears when infection cleared.
3-4 weeks after first antigen presentation
More effective Abs compared to primary focus.
Somatic hypermutation and Affinity maturation.
Dark zone= B cells starting to differentiate
Light zone= more T cells for more activation

Question 23

What is affinity maturation?

Answer 23

Abs produced during primary response have low Ag affinity, later responses have high affinity
Affinity maturation aims to produce these high affinity b cells=more efficient.
Affinity maturation occurs via somatic hypermutation of Ig genes, then selecting the highest affinity B cells
This process is driven by cytokines and other T cell signals

Question 24

The main mechanism of affinity maturation is somatic hypermutation. What is somatic hypermutation? Where does it occur, what is the adv of it?

Answer 24

Occurs in the germinal centre for B cells
Introducing mutations in V region (rearranged VDJ/VJ), using the AID enzyme.
Mutations which decrease Ag affinity undergo NEGATIVE selection
Mutations which increase Ag affinity undergo POSITIVE selection
Higher affinity Abs-> stronger cell signalling; faster proliferation

Question 25

What is the importance of the AID enzyme?

Answer 25

Question 26

Describe isotope switching of antibodies

Answer 26

Diff Abs work best at certain sites/pathogens, eg IgG blood, IgA mucosa, IgE parasite
So B cell can make diff Abs sin changing specificity for Ag or the light chain!!!
Isotype switching needs signals from Th
IgM switches to IgG, IgA and IgE
IgG switches to IgA and IgE

Question 27

How do T cells help isotope antibody switching?

Answer 27

CD40L on T cell interacts w CD40 on activated B cells
The foreign Ag is presented on MHCII on the B cell
CD4R Th cell recognises this complex and activates
Cytokines produced by T cell dictate the switch:
IFNy => switch to IgG1, IgG3
IL-4 => switch to IgE
TGFBeta, IL-5 => switch to IgA

Question 28

The way that isotope switching occurs is via DNA rearrangement and is irreversible.
Explain what is meant by this

Answer 28

The Ab keeps its variable region, but exchanges constant regions for different Ig classes

In the heavy chain gene, there are switch recombination regions in between heavy chain genes
1 exception is the Cẟ region bc this is co-expressed with IgM to begin with

Question 29

Explain how cytokines help isotope switching, specifically INF-y

Answer 29

IFN-γ causes IgM to switch to IgG1 or IgG3
As a result, Sγ3 or Sγ1 gene segments become active
The DNA loop containing Cμ and Cẟ gene segments are cut out
The Cγ3 Fc region gene segment is ligated to the VDJ segment. This makes IgG3

Question 30

Explain the mechanism of isotope switching using TGF-B

Answer 30

TGF-β causes IgM or IgG to switch to IgA production
As a result, the S𝝰1 gene segment becomes active
Switching from IgG3 to IgA1:Cγ3 and Cγ1 regions are excised
Switching from IgM to IgA1:Cγ3 and Cγ1 AND Cμ, Cẟ

The end result of both is the ligation of C𝛂 with the VDJ to produce IgA1