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Humoral immunity(Generation of antibody diversity) Flashcards

1
Q

What are antibodies?

A

• Y shaped molecules expressed by immune cells to target pathogens

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2
Q

How do antibodies work?

A

• Antibodies work by preventing bacteria entering by binding to the bacteria docking site or neutralizing them

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3
Q

Antibody structure?

A

2 heavy, 2 light chains

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4
Q

How many classes does the heavy chain have and what are they divided into?

A

○ Heavy chain has 5 classes: μ,δ,γ,α or ε chain
§ Divided into subclasses: γ1, γ2, γ3, γ4
§ Divided into subclasses: α1, α2

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5
Q

How many domains, constant and variable regions does the heavy chain have?

A

§ Heavy chains have 4 domains: 3 constant and 1 variable region

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6
Q

How many classes does the light chain have?

A

Variable region is very specific – binds to specific epitopes of specific antigens

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7
Q

How many variable and constant regions does the light chain have?

A

§ 1 variable region and 1 constant region

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8
Q

What is the variable region and what does it bind to?

A

Variable region is very specific – binds to specific epitopes of specific antigens

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9
Q

What is constant region very responsible for?

A

• Constant region is very responsible for biological activities

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10
Q

How many forms do antibodies have and what are they?

A

○ B cell receptor and secreted form

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11
Q

What is the final form and whats it used for?

A

The final form is the secreted form – used to fight off pathogens

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12
Q

What is the final form of antibody anchored to

A

It is anchored to plasma membrane of B cells

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13
Q

What forms can the antibody be when secreted?

A

§ When antibody is secreted it can be monomeric forms or pentameric forms

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14
Q

What is antibody initially secreted as?

A

§ Initially secreted as a monomer and then combined to produce a multimeric form

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15
Q

Where do B cells spend its life?

A

• B Cell spends its life in bone marrow, blood, spleen and secondary lymphoid organs

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16
Q

What can the life cycle of B cells be divided into?

A

• Life cycle can be divided into antigen dependent and independent stage

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17
Q

Steps involved in the lifecycle of B cells

A
  1. Start with stem cell which differentiates into pro B cell
  2. Pro B cell undergoes VDJ recombination which codes in the heavy chain constant and variable region and becomes a pre-B cell
    • Once it makes a functional heavy chain, it becomes a pre-B cell
  3. Then it undergoes another VDJ recombination to code in the light variable and heavy chain regions
  4. B cell continues to mature until it expresses IgM and IgD
  5. Becomes a mature circulating B cell and then becomes activated once it encounters a pathogen
  6. B cell migrates into germinal centre (GC) and will undergo a special selection to hone its variable region to that particular pathogen
  7. Undergoes affinity maturation and class switching
  8. Differentiates into plasma cells which secretes antibodies and memory B cells
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18
Q

What are somatic recombinations and an example?

A

• Any changes at the DNA level are called somatic recombination e.g. VDJ recombination

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19
Q

What is differential splicing?

A

• Changes at the mRNA level are called differential splicing

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20
Q

What is each chain of the antibody made of?

A

• Each chain is made of one single polypeptide

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21
Q

What is the hinge region between in antibodies?

A

Hinge region is between CH1 and CH2

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22
Q

What makes the antibody flex and so what does this expose?

A

○ These are a stretch of polypeptides that make the antibody flex so that the active site can be exposed

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23
Q

What chain is the hinge region longer in?

A

○ Hinge region is longer in heavy chain

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24
Q

What are Fv regions?

A

○ Variable regions of light and heavy working together

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25
What are Fab regions?
○ Variable fragment + first constant domains 
26
What are Fc regions?
○ Heavy chain CH2 and CH3 working together 
27
What does glycan form a part . of and what does it attract?
• Glycan (sugar group) forms part of the active site  | § Forms a pocket where the receptors of the other immune cells will be attracted to 
28
What stabilises the antibody structure?
• Disulphide bonds stabilise the structure 
29
What are CDRs?
Protrusions that interact with the antigens
30
Where are CDRs located in the antibody?
○ Are located in the variable light and variable heavy chains 
31
Lifecycle of B cells(Antigen independent stage)
• Stem cell differentiates into pro-B cell   • Pro-B cell undergoes DNA recombination ○ First DJ • Then V region combines with the DJ (VDJ recombination) ○ This codes for the heavy chain variable region  § Heavy chain variable region is co-expressed with μ constant region – constant region for IgM antibody class (this is the default for all B cells before they encounter pathogens)  • Pre B cell expresses valid functional chains  • This pre B cell expresses placeholder light chain to hold it in place as light chain is not generated yet  • Undergoes third recombination on the light chain – VJ recombination  ○ Codes in the variable and constant regions on light chain  • There are also additional processes such as junctional flexibility and P and N nucleotide addition ○ Contributes to the diversity of the antibody   • Once B cell can express IgM and IgD (has capacity to alternatively splice MRNA), it becomes a mature/resting/naïve B cell 
32
How many loci are there that encode Ig?
There are three genetic loci encoding Ig • Two for the light chain: kappa (κ) and lambda (λ) locus • One for heavy chain
33
What chromosome is the λ chain found on?
○ In humans, the λ chain is found on Chr22, κ on Chr2
34
What chromosome is the heavy chain found on?
○ Heavy chain: Chr14
35
What genes regarding antibodies are inherited and not inherited?
• No antibody genes are inherited | § Only gene segments are inherited 
36
What does arranging gene segments in different combinations generate?
○ Arranging these gene segments in different combinations generate many Ig sequences causing the B cell to be unique 
37
What segments are there in the light chain?
• In the light chain there is variable and J gene segments 
38
What regions are there in heavy chain?
• In the heavy there is V, D and J regions 
39
What do segments code for once they've been rearranged?
• Once they are rearranged at DNA level, they will code for the CDRs 
40
What do the J or DJ regions code for?
The J or DJ region codes for CDR3 (3RD CDR) region
41
What is the most variable region?
CDR3 region
42
What gene are the kappa light chain genes on?
• On chromosome 2 
43
How do the segments allign in chromosome 2 for the kappa loci?
• In the kappa loci in chromosome 2, there is V1-V40, J1-5 and then constant region segments 
44
What is there on front of each V segment?
• On front of each V segment there is a leader sequence 
45
Steps in VJ recombination
* Within V1-40, one segment will be selected at random  * At DNA level, it will be recombined together  * This is transcribed to MRNA  * In the primary RNA transcript, there will be extra segments and these are spliced out (this change does not affect the DNA) * Mature RNA has a stop codon and polyA tail  * After translation, leader sequence is cleaved off and therefore forms the light chain of BCR or secreted antibody 
46
What chromosome is gamma heavy chain genes on?
On chromosome 14 
47
Steps in VDJ recombination
* D7 and J3 to join at random  * This is transcribed into mRNA transcript * Only the first two constant segments are transcribed  * B cells can express IGM and IgD when they are mature  * By alternative splicing, the B cell can make two different classes of cell receptor
48
How is antibody diversity generated?
* In the body there are two copies from each parent and each allele has different polymorphisms to contribute to the diversity  * 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 
49
What is the recombination signal sequence?
conserved sequences upstream or downstream of gene segments
50
What does the RSS consist of?
• ‘Turns’ consisting heptamer and nonamer with a 12 or 23 bp spacer
51
What is the one turn/two turn rule(12/23 rule)?
• Recombination only occurs between a segment with a 12bp spacer and a 23bp spacer
52
Turns in the heavy chain?
○ Two turn is located downstream of every V segment and upstream of D segment  ○ One turn is located upstream and downstream of D segment 
53
What is the mechanism if we want to recombine V and J with their RSS?
• Rag 1 and Rag 2 would have to clamp down on the RSS to form a major hairpin • Create nicks in DNA forming minor hairpin  • Two hairpin structures; ○ Major: between upper and lower DNA strands • Results in V and J joining and a circular signal joint 
54
What does artemis do?
Comes in and opens the hairpin by generating a nick 
55
Steps involved in antibody diversity?
• Exonucleases and tdt perform end processing – adding, deleting BPs before joining  • P and N nucleotides form in between due to exonucleases  • Results in a frame shift due to additions -This generates antibody diversity as well due to different AAs being produced 
56
Steps involved in junctional diversity
• Artemis randomly nicks between the BPs • Because it is nicked it will uncoil • Results in overhang ○ Repair enzymes add nucleotides to repair this  ○ P nucleotides  • Tdt enzyme adds end nucleotides (tdt is only present in the heavy chain) ○ This adds more DNA before the V and J segments join
57
When is there junctional flexibility during?
• Junctional flexibility during V(D)J recombination, P and N nucleotide additions
58
Steps in junctional flexibility
* Involves exonucleases  | * Remove mismatched nucleotides but sometimes can remove dna of gene segment 
59
What does junctional flexibility mean?
Junctional flexibility means the coding regions would lose BPs whereas the signal joint is always precise
60
How many copies of Ig genes do we have?
• Two copies of each Ig gene – one from mother and one from father
61
What mechanism is in place for the heavy and light strand?
Mechanism in place to ensure that there is one heavy chain allele and one light chain allele is expressed
62
What is the order of rearrangement?
• Order of rearrangement: Heavy>kappa>lambda; 1st allele then 2nd 

Immunology (8 decks)

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