Humoral Immunity - Generation of B-cells and Antibody Diversity Flashcards Preview

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Flashcards in Humoral Immunity - Generation of B-cells and Antibody Diversity Deck (23)
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1

What is the difference between somatic recombination and differential splicing? 

Use examples from the B-cell life cycle 

Somatic recombination = Alters genetic information at the DNA level 

  • V(D)J recombination 
  • Tdt nucleotide addition 
  • Somatic hypermutation 
  • Class Switching 

Differential Splicing = Changes made at the mRNA level 

(Two identical copies of mRNA will be altered differently to produce two different protein products) 

  • IgM and IgD 
  • Membrane bound and secreted Ig 

2

What are the two stages of B-cell development? 

  • Antigen dependant 
    • D→J (Heavy chain variable)
    • V→DJ (Heavy Chain variable) 
    • V→J (light chain variable and constant) 
  • Antigen independant 
    • Affinity maturation + Class Switching 

3

Describe the Life Cycle of B cells 

  1. The B cell will start in bone marrow 
    • It starts as a stem cell and differentiate to a pro-B cell 
    • The DNA of the pro B cell undergoes 
      1. D→ J recombination 
      2. And V→DJ recombination 
        • This permanently codes in the heavy chain variable region 
  2. The variable region will be expressed with the μ heavy constant chain
    • This is the first default heavy chain expressed by the B-cell
  3. The pro-B cell will become a pre-B cell when it expresses a heavy chain with a light chain placeholder 
  4. The pre B cell will undergo 
    1. V→J recombination of either kappa or lambda to permanently code the light chain variable + constant region to become immature B-cell  
  5. The pre-B cell will become an immature B-cell once it can express both heavy and light chain IgM 
    1. Note during V(D)J or VJ recombination we have junctional flexibillity + P/N nucleotide addition to generate diversity between B-cells with same comb of gene segments 
  6. Once they express both IgM and IgD on their surface (via alternative splicing of mRNA) = mature B-cells + circulate between blood stream, lymph nodes + spleen 
  7. They will patrol resting until they encounter a pathogen 
  8. The B-cell is activated once it encounters a pathogen and undergoes affinity maturation in the germinal centre (GC) 
  9. B-cells will recieve information on the type of pathogen and undergo major class switching for the appropriate effector function 
    1. Note: B cell receptor can become IgG or IgA 
  10. Majority of B-cells will develop into professional plasma cells to secrete the antibody that they code for + some B-cells coding for IgM will differentiate into plasma cells secreting first defence IgM
  11. After infection some B-cells will remain as memory B-cells 

 

4

Further describe the antigen independant phase 

  • This stage occurs in our bone marrow, makes 1, 000, 000, 000 resting B cells which each contain a unique random BCR
    • Lymphoid progenitor will differentiate into pro-B-cells 
      • Pro-B undergo D to J recombination in its DNA 
      • V segment will recombine with DJ segment to hard code the variable region of the heavy chain 
    • This will be expressed with the μ constant region
    • The cell will become a pre-B cell when it can express a full heavy chain with unique variable region 
      • Pre-B cells will express a light chain placeholder which forms a pseudoantibody with the heavy chain 
    • The pre-B cell will undergo V – J recombination on the light chain regions, this will determine the light chain variable and constant region

    • The immature B-cell will express a full IgM (μ heavy chain, κ/λ light chain)
    • It becomes a mature B cell (also known as resting B cell/ naïve B cells) when it has the capacity to produce both IgM and IgD through differential splicing of the mRNA

5

What processes occur during V→ DJ and V→J recombination which allow for additonal diversity? 

  1. Junctional flexibillity 
  2. P + N nucleotide addition 

6

How do we know that the immature B cell has become a mature B-cell? 

What acts like the quality control? 

  • We know the immature B-cell has become mature when it has the capacity to produce both IgM and IgD through differential splicing 
    • IgD = like quality control 

7

How are antibody genes inherited? 

NONE are inherited 

  • No complete genes are inherited, only gene segments
  • These gene segments are arranged in different combinations to generate many Ig sequences

8

What different gene segments is the light chain and heavy chain loci made up of? 

  • Light chain is made up of V, J and C gene segments
  • Heavy chain is made up of V, D, J and Cμ
    • In the light chain one V segment will be selected at random to recombine with one J segment 
    • In the heavy chain random V(D)J segments will recombine  
      • The DNA will now encode a complete antibody with the VJ of VDJ segments encoding for the light or heavy chain variable fragments and the Cμ segment coding for the constant domains

9

What does the J or the J/D portion code for? 

  • The J or the D/J portion will code for the CDR3 (complementarity determining regions)
    • These are the finger like protrusions interacting with the antigen

10

How many genetic loci are there coding Ig chains? 

3 Genetic Loci 

  • 2 for light chain = Kappa and lambda locus
  • 1 for heavy chain

 

11

Explain the example of VJ recombination of kappa light chain genes on chromosome 2

 

In humans the Kappa chain contains: 40 (V) variable segments, 5 (J) segments and a constant region (C) segment 

  • In front of each segement there is a leader sequence which is like a postcode so the cells know where the protein will end up 
  • The V and J segments are randomly chosen 
  • This is then transcribed in to mRNA 
  • The extra J segment will be spliced out to form the leader with V, J and C segments 
  • The mRNA will be translated into amino acid sequences of the light chain
  • The amino acid will be folded, and the leader sequence will be cleaved off when the protein reaches where it needs to go
    • This will result in a unique kappa light chain

12

Describe VDJ recombination of heavy chain genes on chromosome 16

In humans the H chain will contain: 51 (V) variable segments, 27 (D) Diversity segments, 6 (J) joining segments, Constant region (C) segments 

  • The VDJ segments are rearranged on the chromosome below 
    • The Cμ and Cδ code for the IgM and IgD heavy chain respectively
  • First recombination which will take place is the is the D →J joining
    • In this D7/J3
  • Then there will be V→ DJ joining
    • In this case it will be the V20 joining to D7 and J3
  • The recombined hard coded DNA will be transcribed into mRNA transcripts
  • The mRNA transcript will then undergo differential splicing
  • In a population of mRNA transcripts some will express the leader sequence followed by VDJ and finally Cμ sequence
    • This will be translated into the IgM heavy chain
  • The other mRNA transcript will express the leader, VDJ and Cδ
    • This will be translated later IgD heavy chain to signal maturity of B-cells

13

How can V(D)J recombination take place? 

It involves the use of recombination signal sequences (RSS)

  • They are made up of turns containing a conserved heptamer and nonamer with a 12/23 bp spacer
    • These are located upstream or downstream of gene segments 

14

What are the two types of turns? 

  • Two turn contains a 23 base pair spacer
  • One turn contains a 12 base pair spacer

15

Why do we have these two types of different turns located in the recombination signal sequences (RSS)? 

One turn/ Two turn rule (12/23) 

  • DNA recombination only occurs between a segment with a 12bp spacer and a segment with a 23bp spacer
    • Essentially a one turn can only recombine with a two turn
      • This is known as the one-turn/two-turn rule (12/23 rule)

16

How can we generate antibody diversity

  • Multiple germline V, D and J gene segments
  • Combination of V-J and V-D-J joining
  • Junctional flexibility
    • P-nucleotide addition
    • N-nucleotide addition (Tdt nucleotide addition) 
  • Combinatorial association of heavy and light chains (5 classes of heavy chain and 2 classes of light chain)
  • Somatic hypermutation during affinity maturation

17

What are the pros and cons of generating junctional diversity through V(D)J recombination and P and N nucleotide additions? 

Pros = Antibody diversity 

Cons = Can get disadvantageous combinations for example autoantibodies 

 

18

Explain the mechanism of recombination 

  • If you want to recombine the V17 segment with J3 the dark and light blue triangles will represent the turns
    • The enzymes RAG1 and 2 will bind to the turn and pull them together to form a hairpin
  • The DNA will be nicked and will automatically form a minor hair pin at the ends of the gene segment
    • RSS ends will be joined forming a signal joint which will play no role in recombination process 
  • The hairpin will be opened by the enzyme artemis 
  • The DNA ends will be processed by Tdt (P/N nucleotide addition) and exonucleases (junctional flexibillity) 
    • These will add or remove DNA nucleotides respectively 
  • The ends will then be joined together by another series of enzymes known as Ku70, DNA-PK, XRCC4

19

Explain how P/N nucleotide addition occurs

  • Once hairpin is formed artemis will randomly nick one end of dsDNA 
  • Nicked ends will linearise and form overhanging ends
  • Repair enzyme will fill these gaps = P-nucleotide addition 
  • Terminal deoxynucleotidyl transferase (Tdt) will add the N nucleotides into the two ends before they are ligated again
    • The addition of P and N nucleotides before joining of segments together will cause addition of extra amino acids or shifting of the whole reading frame

      • So if by chance the B-cell selects the same VDJ or VJ segment they will end up with different Abs due to P/N nucleotide addition 

20

What is junctional flexibillity? 

  • This involved the removal of mismatched nucleotides during V(D)J recombination 
  • Involves EXONUCLEASES 

21

What is the mechanism of junctional flexibillity? 

  • As mentioned before artemis will create the overhanging ends
  • In some these ends will overlap if there are enough complementary sequences
  • However, if there are mismatched nucleotides these will have to be removed by the exonucleases before the repair enzyme can work
    • Note the joining of the signal joins are always precise

22

What is allelic exclusion? 

  • Even though we have two copies of each Ig gene (one from each parent) and we end up with 2 x kappa and lambda VJC segments and 2 x VDJC heavy chain segments 
    • In most genes both genes will be expressed but in antibody genes in B cells 
      • Only one heavy chain allele and one light chain allele out of 6 possible alleles is expressed

23

How does allelic exclusion take place? 

  • There is a particular order which the loci will undergo rearrangement
    • HEAVY > KAPPA > LAMDA, 1st allele then 2nd
  • Essential the B cell will try the first allele of the heavy chain if it successfully produces a full heavy chain it will move onto the kappa chain allele, if not then it will try the second heavy chain allele
  • Again for the light chain the first kappa allele will be recombined if successful then all recombination will stop immediately if it is not successful in producing a light chain it will move onto the second kappa allele etc
    • Heavy or light chain recombination is not successful the cell will undergo apoptosis
  • This allelic exclusion mechanisms ensures that each B-cell will only make one type of antibody!!

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