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Immunology > Humoral Immunity: Generation of Antibody Diversity > Flashcards

Humoral Immunity: Generation of Antibody Diversity Flashcards

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1
Q

Describe the structure of antibodies

A

2 heavy, 2 light chains

Heavy:

  • 4 domains of γ,𝜀,ẟ,µ,𝜶,
  • Subtypes of γ1-4 and 𝜶1-2
  • Total 9 HC regions possible

Light:
- 2 domains of kappa, lambda

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2
Q

What is the variable region of antibodies formed of?

A

Variable region formed of the first domains of light and heavy chains and bind antigens specifically, CH1 supports

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3
Q

What is the role of the constant region?

A

Constant region - same for all Ab of same class

Effector functions (activating complement, binding phagocytes) CH1,2 & 3 are the constant heavy domains

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4
Q

What is the role of disulphide bonds in antibody structure?

A

Disulphide bonds stabilize VH-CH1-CH2-CH3 heavy and light chain bonds

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5
Q

What is the role of the antibody hinge region?

A

Hinge region provides flexibility and movement to structure

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6
Q

What is the significance of carboxylic groups on the CH2 domains?

A

CH2 carboxylic groups act as anchors for immune cell interactions

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7
Q

What is the CDR?

A

Complementarity Determining Regions

The CDR is where the antibody interacts with antigens on VH and VL

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8
Q

What is the roel of the CDR?

A

CDR binds to antigen (fingers and apple)

CDR3 most variable

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9
Q

What are the 4 main functions of antibodies to combat pathogens?

A
  1. Opsonization
  2. Neutralisation
  3. Complement / MAC
  4. Apoptosis
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10
Q

What is opsonisation?

A

Tagging pathogens to make them more visible to immune cells (macrophages & NK cells)

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11
Q

Outline how antibodies cause opsonization

A
  1. Variable regions bind to pathogen
  2. Constant domains bind to FcR on macrophages

=> antibody dependent cellular phagocytosis (ADCP) of smaller pathogens

Or NK cells to produce antibody dependent cellular cytotoxicity (ADCC) by releasing chemicals to induce apoptosis (infected/cancer)

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12
Q

How do antibodies aid neutralisation of toxins?

A

Variable fragments can bind competitively to viral docking sites on cells / toxin active sites; neutralise

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13
Q

How do antibody immune complexes fight pathogens?

A

Form immune complexes composed of antibodies + pathogens that agglutinate and are removed by other immune cells

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14
Q

How do complement proteins aid immune response?

A

Can involve complement molecules (C1q,s and r) that promote inflammation, phagocytosis and MAC formation (damage membrane) to cause lysis

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15
Q

How many antibody classes are there?

A

5 different classes of antibody with different functions

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16
Q

What is the difference between each antibody class?

A

Each antibody class expresses a different heavy chain constant region

But the light and heavy chain variable regions remain the same for antibodies produced from the same B cell

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17
Q

What is the IgG antibody structure?

A

IgG has the canonical structure mentioned above with 4 domains in a gamma chain

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18
Q

Describe the IgD structure

A

The delta chain in IgD has a longer hinge region

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19
Q

What is IgE structure like?

A

The epsilon chain in IgE has 5 domains

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20
Q

What enables IgA and IgM to polymerise?

A

The alpha and meu chains in IgA and IgM are similar to IgG but they have tail pieces at the end of CH3 to facilitate polymerisation and joining to J chains

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21
Q

Describe the structure of IgA

A

Secretory IgA is 2 monomeric IgA joined by a J chain; secretory component wraps around enabling it to be secreted into the mucous → good for respiratory infections

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22
Q

Outline the structure of IgM

A

IgM is composed of 5 monomeric IgM structures joined together by a J chain

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23
Q

How is antibody class determined?

A

The heavy chain variable regions and light chain are fixed by VDJ recombination

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24
Q

Which is the heaviest Antibody?

A

IgM has highest mw; pentamer and IgA is also larger; dimer

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25
Which is the main Ig in serum?
IgG is the main antibody in serum followed by IgA
26
Which antibodies are able to fix complements?
Only IgM and IgG fix complements
27
Which antibody is able to provide immunity to foetus?
Only IgG can cross the placenta to provide immunity to the foetus
28
What is the role of heavy chain class switching?
Provides different effector functions to deal with different pathogens Only affects Heavy chain constant regions
29
What are the 2 types of Heavy chain class switching?
Minor: differential splicing (mRNA level) - IgM and IgD - Doesn’t affect B cell DNA Major: DNA recombination - IgM to IgG, IgA , IgE - IgG to IgA, IgE
30
What causes class switching to occur?
Class switching occurs due to presence of chemical signalling from cytokines released by T helper cells
31
What is the main signal to initiate clas switching?
CD40L on T cell interacts with CD40 on B cells and cytokine signalling
32
Name the mechanism for major class switching (DNA)
CSR - class switch recombination
33
Outline how class switch recombination occurs
1) Cytokine signal 2) switch regions 3) AID and DSB ds repair proteins
34
What is the rule of Class Switching Recombination?
Switching only proceeds downstream - IgM to IgG, IgA, IgE - IgG to IgA, IgE
35
How do heavy chain segments recombine?
Heavy chain gene loci undergone VDJ recombination and affinity maturation
36
Why can't antibodies revert back to previous IgM class after recombination?
Once switched to IgG all the segments before will be removed ∴can’t revert back to IgM
37
What happens to the spliced antibody regions?
Segments cleaved out form a switch DNA circle once spliced
38
What are the 2 types of antibodies?
2 types of antibodies: - membrane bound (BCR) - Secreted form
39
What is the secreted form of antibody?
The secreted form is the final, fully functional form of the antibody secreted by mature plasma cells
40
Why are antibodies anchored to B cells before secretion?
Before its secreted it's anchored on B cells for weapon development
41
How do secreted and membrane bound antibody structure differ?
Secreted IgG has a tail piece while the membrane bound antibody has a transmembrane region and cytoplasmic tail anchor
42
Outline the components of the constant mu region before differential splicing occurs
Constant region of Cμ composed of μ1-4 + tail piece + stop codon + polyA tail followed by M1 and M2 There is also a second PolyA tail and stop codon
43
What do M1 and M2 gene segments code for?
M1 and M2 code for transmembrane region and the cytoplasmic tail
44
How are membrane bound antibodies converted to secreted form?
Whole Cμ gene region is transcribed into mRNA and introns are spliced out to form mRNA for secreted antibodies
45
How are membrane bound antibodies produced?
Whole Cμ region (upto second polyA tail) is transcribed and 8 regions including genes encoding tailpiece and stop signal will be spliced out
46
What is somatic recombination?
Alteration of DNA level genetic info | Once these processes have occurred, they are irreversible
47
Outline the different methods of somatic recombination
- V(D)J recombination - Tdt nucleotide addition - Somatic hypermutation - Class switching
48
What is differential splicing?
(alternative splicing): alteration at mRNA level 2 identical mRNA copies on B cells will be altered differently to produce 2 different protein products
49
What are the products of differential splicing?
- IgM and IgD - Membrane bound - Secreted Ig
50
What are the 2 stages of B cell development?
There are 2 stages to B cell development: - Antigen independent - Antigen dependent
51
Where do B cells originate?
B cells start their life in bone marrow as stem cells that slowly differentiate into pro-B cells
52
How are heavy chain variable regions coded for?
The Pro-B cell DNA will undergo D → J and V → DJ recombination to permanently code in heavy chain variable regions
53
What is the default Heavy chain variable region?
The variable region will be expressed with the μ chain (VH + μ chain) - default first HC expressed by B cells
54
What is a Pre-B cell?
B cell expressing a heavy chain
55
How is the light chain variable region coded for?
Pre-B cells undergo another V → J recombination to permanently code in the light chain variable and constant region to become immature B cells
56
What are immature B cells?
Immature B cells express IgM and mature overtime
57
When do B cells become mature?
Once the B cells can express IgM and IgD (via differential splicing of mRNA) they become mature B cells and circulate between bloodstream, spleen and lymph nodes
58
What is affinity maturation?
B cells hone their ability to bind to a pathogen by affinity maturation in germinal centre (GC) - only best survive
59
What do majority B cells develop into?
Majority B cells further develop into Plasma cells that secrete the antibodies they encode for
60
What is B cell first line defence?
As first line defence some B cells differentiate into Plasma cells expressing IgM and enter circulation
61
Describe the diversity of antibodies in the body
Body makes 1,000,000,000 resting B cells, each contains unique ‘random’ BCR
62
How do B cells differentiate to provide so much diversity?
To generate 1,000,000,000 resting B cells, lymphoid progenitor stem cells differentiate into Pro-B cells
63
What is the first recombination Pro-B cells undergo?
Pro-B cells undergo D → J recombination | V segment recombines with DJ segment (V → DJ) - hard codes in variable heavy chain
64
Why can B cells not class switch during development?
Expressed with μ (default) constant region B cell can’t change class at this stage as pathogen type is unknown
65
When does a Pro-B cell become a Pre-B cell?
Cell becomes Pre-B cell when it can express a full heavy chain with a unique variable region
66
Describe the second recombination for the light chain region?
Pre-B cell undergoes another V→ J recombination to determine light chain variable and constant regions
67
How is additional diversity created among antibodies?
Additional diversity is generated via Junctional Flexibility and P + N nucleotide addition Random in nature
68
Describe the structure of immature B cells
Immature B cell expresses full IgM and either kappa / lambda light chain
69
When does an immature B cell become mature?
Becomes mature B cell (Naive B cells / resting B cells) when capacity to produce IgM and IgD through differential splicing - quality control
70
What are the 3 genetic loci encoding for Ig?
Two for light chain: kappa (κ) chr.2 and lambda (λ) chr.22 locus One for heavy chain chr.14 Located on different chromosomes
71
What is the role of V(D)J recombination?
No complete genes are inherited, only gene segments Arranging these gene segments in different combinations generate many Ig sequences
72
What gene segments are the light / heavy chains composed of?
The light and heavy chain gene loci are made up of different gene segments; - Light: V, J and C - Heavy: V, D, J and Cμ
73
Outline the light chain segments recombine
Light chain V gene segment chosen at random to recombine with a J segment
74
Outline how heavy chain gene segments recombine
Heavy chain: random VDJ segments recombine
75
What do C segments encode?
C segments encode constant domains
76
What do J or D?J segments encode for?
J or D/J segments code for CDR3
77
What is the simplest recombination?
VJ recombination on kappa / lambda light chain genes (Chromosome 2/22) in humans
78
What are the different possibilities of VJ recombination?
V segments far from J segments, but J are relatively close to C segments - 40 Variable (V) segments - 5 Joining (J) segments - Constant region (C) segment
79
What is the role of the leader sequences?
Leader sequence in front of each V segment : directs protein to target
80
How are VJ segments recombined?
V + J segments randomly chosen to form L, VJ, J, C segment ⇒ transcribed into mRNA
81
What happens to the extra J segment from VJ recombination
Extra J segment spliced out → mature mRNA (L, V, J, C) | mRNA translated into a.a sequences of light chain
82
How is the final kappa light chain formed?
A.a. folded and leader sequence cleaved once protein reaches target ⇒ unique kappa light chain
83
How many different VDJ recombination segments are there?
- 51 Variable (V) segments - 27 Diversity (D) segments - 6 Joining (J) segments - Constant region (C) segments
84
Outline the VDJ recombination that occurs on the heavy chain
First C regions are Cμ and Cẟ coding for IgM and IgD ∴ first recombination is D → J joining (randomly selected) Random V segment also joined
85
What happens to the VDJ recombined gene segments?
Recombined hard coded DNA is transcribed into mRNA transcripts
86
Outline the result of differential splicing of heavy chain gene segments
Differential splicing of mRNA occurs resulting in expression of either: - LVDJCμ = IgM - LVDJCẟ = IgD
87
What signals initiate recombination?
Recombination signal sequences (RSS) required – conserved sequences upstream or downstream of gene segments
88
What are RSS made up of?
RSS made of ‘Turns’ consisting heptamer and nonamer with a 12 or 23 bp spacer
89
What is the 1 turn/ 2 turn rule of recombination ?
Recombination only occurs between a segment with a 12bp spacer and a 23bp spacer One-turn can recombine with two-turn (not one-turn and one-turn or two-turn and two-turn)
90
Where are two-turns located?
Two-turns located: - downstream of V segments of heavy and lambda light chains - upstream of heavy chain and kappa chain J segments
91
Where are one-turns located?
One-turns located: - Both sides of heavy chain D segments - Upstream of lambda light chain J segment - Downstream of kappa V segment
92
What are the different mechanisms of antibody diversity generation?
- 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
93
What are autoantibodies?
Recognise own cells and cause autoimmune diseases | - selected out by negative selection
94
What is junctional diversity?
Removal of nucleotides between gene segments v(D)J recombination Produces Junctional flexibility during V(D)J recombination, P and N nucleotide additions
95
Evaluate junctional diversity
Good: Antibody diversity Bad: Non-productive rearrangements (incorrect reading frame) – wasteful process
96
Outline the process of junctional diversity
1. RAG1 / RAG2 enzymes bind to V/J 1/2-turns 2. Pulls segments together to form (major) hairpin 3. DNA nicked to form (minor) hairpin at end of gene segments 4. Enzymes repair and process segment ends 5. Results in: - Coding joint of V and J segment - Signal joint of turns and excess DNA between segments
97
Describe the different hairpins formed from junctional diversity?
Minor hairpin: between 2 strands of DNA Major hairpin: whole DNA folded in half
98
How do the hairpins open and join?
RAG1/2 processing forms hairpins Hairpin opened via Artemis Exonucleases, TdT mess around with free DNA ends (add nucleotides) Ends joined by another series of enzymes
99
How does artemis enzyme open the hairpin?
Artemis randomly nicks one end of dsDNA Nicked ends linearise to form overhanging ends
100
When does P nucleotide addition occur?
Repair enzymes fill overhang gaps from Artemis enzyme → P nucleotide addition
101
When are N nucleotide additions made?
Terminal deoxynucleotidyl Transferase (Tdt) : adds N nucleotides Mostly in heavy chain
102
What are the effects of P+N nucleotide additions?
P+N nucleotide addition causes frameshifts leading to new antibody formation regardless of previous V(D)J recombination selections
103
How does junctional flexibility cause frameshfits?
There will be mismatch nucleotides that need removing via exonucleases before repair enzyme can work - can overtrim ends causing additions/deletions (frameshifts)
104
How do antibody genes differ from normal?
Allelic exclusion | Antibody genes different – Only one heavy chain allele and one light chain allele is expressed
105
What is the role of allelic exclusion?
Ensure each B cell makes one type of antibody

Immunology (14 decks)

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