Humoral Immunity: Generation of Antibody Diversity Flashcards

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
Q

Which is the main Ig in serum?

A

IgG is the main antibody in serum followed by IgA

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26
Q

Which antibodies are able to fix complements?

A

Only IgM and IgG fix complements

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27
Q

Which antibody is able to provide immunity to foetus?

A

Only IgG can cross the placenta to provide immunity to the foetus

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28
Q

What is the role of heavy chain class switching?

A

Provides different effector functions to deal with different pathogens

Only affects Heavy chain constant regions

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29
Q

What are the 2 types of Heavy chain class switching?

A

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
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30
Q

What causes class switching to occur?

A

Class switching occurs due to presence of chemical signalling from cytokines released by T helper cells

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31
Q

What is the main signal to initiate clas switching?

A

CD40L on T cell interacts with CD40 on B cells and cytokine signalling

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32
Q

Name the mechanism for major class switching (DNA)

A

CSR - class switch recombination

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33
Q

Outline how class switch recombination occurs

A

1) Cytokine signal
2) switch regions
3) AID and DSB ds repair proteins

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34
Q

What is the rule of Class Switching Recombination?

A

Switching only proceeds downstream

  • IgM to IgG, IgA, IgE
  • IgG to IgA, IgE
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35
Q

How do heavy chain segments recombine?

A

Heavy chain gene loci undergone VDJ recombination and affinity maturation

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36
Q

Why can’t antibodies revert back to previous IgM class after recombination?

A

Once switched to IgG all the segments before will be removed ∴can’t revert back to IgM

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37
Q

What happens to the spliced antibody regions?

A

Segments cleaved out form a switch DNA circle once spliced

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38
Q

What are the 2 types of antibodies?

A

2 types of antibodies:

  • membrane bound (BCR)
  • Secreted form
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39
Q

What is the secreted form of antibody?

A

The secreted form is the final, fully functional form of the antibody secreted by mature plasma cells

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40
Q

Why are antibodies anchored to B cells before secretion?

A

Before its secreted it’s anchored on B cells for weapon development

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41
Q

How do secreted and membrane bound antibody structure differ?

A

Secreted IgG has a tail piece while the membrane bound antibody has a transmembrane region and cytoplasmic tail anchor

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42
Q

Outline the components of the constant mu region before differential splicing occurs

A

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

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43
Q

What do M1 and M2 gene segments code for?

A

M1 and M2 code for transmembrane region and the cytoplasmic tail

44
Q

How are membrane bound antibodies converted to secreted form?

A

Whole Cμ gene region is transcribed into mRNA and introns are spliced out to form mRNA for secreted antibodies

45
Q

How are membrane bound antibodies produced?

A

Whole Cμ region (upto second polyA tail) is transcribed and 8 regions including genes encoding tailpiece and stop signal will be spliced out

46
Q

What is somatic recombination?

A

Alteration of DNA level genetic info

Once these processes have occurred, they are irreversible

47
Q

Outline the different methods of somatic recombination

A
  • V(D)J recombination
  • Tdt nucleotide addition
  • Somatic hypermutation
  • Class switching
48
Q

What is differential splicing?

A

(alternative splicing): alteration at mRNA level

2 identical mRNA copies on B cells will be altered differently to produce 2 different protein products

49
Q

What are the products of differential splicing?

A
  • IgM and IgD
  • Membrane bound
  • Secreted Ig
50
Q

What are the 2 stages of B cell development?

A

There are 2 stages to B cell development:

  • Antigen independent
  • Antigen dependent
51
Q

Where do B cells originate?

A

B cells start their life in bone marrow as stem cells that slowly differentiate into pro-B cells

52
Q

How are heavy chain variable regions coded for?

A

The Pro-B cell DNA will undergo D → J and V → DJ recombination to permanently code in heavy chain variable regions

53
Q

What is the default Heavy chain variable region?

A

The variable region will be expressed with the μ chain (VH + μ chain) - default first HC expressed by B cells

54
Q

What is a Pre-B cell?

A

B cell expressing a heavy chain

55
Q

How is the light chain variable region coded for?

A

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
Q

What are immature B cells?

A

Immature B cells express IgM and mature overtime

57
Q

When do B cells become mature?

A

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
Q

What is affinity maturation?

A

B cells hone their ability to bind to a pathogen by affinity maturation in germinal centre (GC) - only best survive

59
Q

What do majority B cells develop into?

A

Majority B cells further develop into Plasma cells that secrete the antibodies they encode for

60
Q

What is B cell first line defence?

A

As first line defence some B cells differentiate into Plasma cells expressing IgM and enter circulation

61
Q

Describe the diversity of antibodies in the body

A

Body makes 1,000,000,000 resting B cells, each contains unique ‘random’ BCR

62
Q

How do B cells differentiate to provide so much diversity?

A

To generate 1,000,000,000 resting B cells, lymphoid progenitor stem cells differentiate into Pro-B cells

63
Q

What is the first recombination Pro-B cells undergo?

A

Pro-B cells undergo D → J recombination

V segment recombines with DJ segment (V → DJ) - hard codes in variable heavy chain

64
Q

Why can B cells not class switch during development?

A

Expressed with μ (default) constant region

B cell can’t change class at this stage as pathogen type is unknown

65
Q

When does a Pro-B cell become a Pre-B cell?

A

Cell becomes Pre-B cell when it can express a full heavy chain with a unique variable region

66
Q

Describe the second recombination for the light chain region?

A

Pre-B cell undergoes another V→ J recombination to determine light chain variable and constant regions

67
Q

How is additional diversity created among antibodies?

A

Additional diversity is generated via Junctional Flexibility and P + N nucleotide addition
Random in nature

68
Q

Describe the structure of immature B cells

A

Immature B cell expresses full IgM and either kappa / lambda light chain

69
Q

When does an immature B cell become mature?

A

Becomes mature B cell (Naive B cells / resting B cells) when capacity to produce IgM and IgD through differential splicing - quality control

70
Q

What are the 3 genetic loci encoding for Ig?

A

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
Q

What is the role of V(D)J recombination?

A

No complete genes are inherited, only gene segments

Arranging these gene segments in different combinations generate many Ig sequences

72
Q

What gene segments are the light / heavy chains composed of?

A

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
Q

Outline the light chain segments recombine

A

Light chain V gene segment chosen at random to recombine with a J segment

74
Q

Outline how heavy chain gene segments recombine

A

Heavy chain: random VDJ segments recombine

75
Q

What do C segments encode?

A

C segments encode constant domains

76
Q

What do J or D?J segments encode for?

A

J or D/J segments code for CDR3

77
Q

What is the simplest recombination?

A

VJ recombination on kappa / lambda light chain genes (Chromosome 2/22) in humans

78
Q

What are the different possibilities of VJ recombination?

A

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
Q

What is the role of the leader sequences?

A

Leader sequence in front of each V segment : directs protein to target

80
Q

How are VJ segments recombined?

A

V + J segments randomly chosen to form L, VJ, J, C segment ⇒ transcribed into mRNA

81
Q

What happens to the extra J segment from VJ recombination

A

Extra J segment spliced out → mature mRNA (L, V, J, C)

mRNA translated into a.a sequences of light chain

82
Q

How is the final kappa light chain formed?

A

A.a. folded and leader sequence cleaved once protein reaches target ⇒ unique kappa light chain

83
Q

How many different VDJ recombination segments are there?

A
  • 51 Variable (V) segments
  • 27 Diversity (D) segments
  • 6 Joining (J) segments
  • Constant region (C) segments
84
Q

Outline the VDJ recombination that occurs on the heavy chain

A

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
Q

What happens to the VDJ recombined gene segments?

A

Recombined hard coded DNA is transcribed into mRNA transcripts

86
Q

Outline the result of differential splicing of heavy chain gene segments

A

Differential splicing of mRNA occurs resulting in expression of either:

  • LVDJCμ = IgM
  • LVDJCẟ = IgD
87
Q

What signals initiate recombination?

A

Recombination signal sequences (RSS) required – conserved sequences upstream or downstream of gene segments

88
Q

What are RSS made up of?

A

RSS made of ‘Turns’ consisting heptamer and nonamer with a 12 or 23 bp spacer

89
Q

What is the 1 turn/ 2 turn rule of recombination ?

A

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
Q

Where are two-turns located?

A

Two-turns located:

  • downstream of V segments of heavy and lambda light chains
  • upstream of heavy chain and kappa chain J segments
91
Q

Where are one-turns located?

A

One-turns located:

  • Both sides of heavy chain D segments
  • Upstream of lambda light chain J segment
  • Downstream of kappa V segment
92
Q

What are the different mechanisms of antibody diversity generation?

A
  • 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
Q

What are autoantibodies?

A

Recognise own cells and cause autoimmune diseases

- selected out by negative selection

94
Q

What is junctional diversity?

A

Removal of nucleotides between gene segments v(D)J recombination

Produces Junctional flexibility during V(D)J recombination, P and N nucleotide additions

95
Q

Evaluate junctional diversity

A

Good: Antibody diversity

Bad: Non-productive rearrangements (incorrect reading frame) – wasteful process

96
Q

Outline the process of junctional diversity

A
  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
Q

Describe the different hairpins formed from junctional diversity?

A

Minor hairpin: between 2 strands of DNA

Major hairpin: whole DNA folded in half

98
Q

How do the hairpins open and join?

A

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
Q

How does artemis enzyme open the hairpin?

A

Artemis randomly nicks one end of dsDNA

Nicked ends linearise to form overhanging ends

100
Q

When does P nucleotide addition occur?

A

Repair enzymes fill overhang gaps from Artemis enzyme → P nucleotide addition

101
Q

When are N nucleotide additions made?

A

Terminal deoxynucleotidyl Transferase (Tdt) : adds N nucleotides
Mostly in heavy chain

102
Q

What are the effects of P+N nucleotide additions?

A

P+N nucleotide addition causes frameshifts leading to new antibody formation regardless of previous V(D)J recombination selections

103
Q

How does junctional flexibility cause frameshfits?

A

There will be mismatch nucleotides that need removing via exonucleases before repair enzyme can work - can overtrim ends causing additions/deletions (frameshifts)

104
Q

How do antibody genes differ from normal?

A

Allelic exclusion

Antibody genes different – Only one heavy chain allele and one light chain allele is expressed

105
Q

What is the role of allelic exclusion?

A

Ensure each B cell makes one type of antibody