Antibody Biology 2

Question 1

How is antibody diversity achieved?

Answer 1

• Ig genes are organised differently to other genes
• In all cells except B cells, Ig genes are in fragmented form that cannot be expressed
• Ig heavy and light chain loci consist of families of gene segments, arrayed sequentially along the chromosome
• They are inherited in this form through the germline. Therefore their arrangement is termed “germline configuration”
• In developing B cells Ig gene segments must be rearranged to assemble functional light and heavy chain gene

Question 2

Ig gene rearrangements occur at defined points in B cell development
TRUE OR FALSE

Answer 2

TRUE

e.g. V-DJ rearranging at “Late pro-B cell”

Question 3

Pre-B cells randomly recombine heavy chain gene segments

TRUE OR FALSE

Answer 3

FALSE
PRO-B cells randomly recombine heavy chain gene segments

1. D gene segment is joined to a J gene segment
2. V gene segment joined to DJ gene segment
   “Functional VDJ gene”
   Transcription, splicing, and translation of this VDJ gene will produce a heavy chain polypeptide (e.g. mu, delta)

Question 4

Pre-B cells rearrange light chain genes

TRUE OR FALSE

Answer 4

TRUE

Repeated light chain arrangements are possible (i.e. multiple recombinations.)

Question 5

Rearrangement guided by conserved non-coding sequences known as BLANKS

Answer 5

Rearrangement guided by conserved non-coding sequences known as Recombination Signal Sequences (RSS)

Question 6

The 12/23 Rule:

Answer 6

• 12 and 23 refer to the lengths (in bp) of the spacers in RSSs
• RSSs flank recombination sites
• Genes with a 12bp spacer can recombine with one with a 23bp spacer. This is the 12/23 rule

Question 7

RSS structure:

Answer 7

Conserved Heptamer
Nonconserved spacer (12 or 23bp)
Conserved Nonamer

Question 8

Enzymatic requirements for recombination:

Answer 8

Set of enzymes needed to recombine V, D, and J = V(D)J recombinase

Two components are made only in lymphocytes:
- Products of the recombination-activating genes RAG-1 and RAG-2

RAG-1 and RAG-2 associate with each other and other proteins known as high mobility group of proteins. This is the RAG complex

Question 9

RAG complex action on RSSs:

Answer 9

1. One RAG complex binds 23 spacer RSS, another binds 12 spacer RSS
2. RAG complex interaction aligns RSSs and cleaves DNA at ends of gene segments
3. DNA hairpin formed at end of each gene segment, and clean break at heptamer ends
4. RAG complexes hold DNA in place while broken ends rejoined by DNA repair enzymes = non-homologous end-joining
5. Coding joint formed in chromosome, signal joint in removed piece of circular DNA

Question 10

Enzymes that open hairpins and form coding
joint introduce additional sequence diversity
into CDR3 in several ways:

Answer 10

• The nick that opens hairpin can occur at several
  different positions
• Ends variably modified by exonucleases (remove
  nucleotides) or terminal nucleotidyl transferase
  (TdT) which randomly adds nucleotides
• Forms single-stranded end in which
  complementary bases that were originally on
  two DNA strands are now on same strand (Creates sequence that would form palindrome
  in dsDNA)

Question 11

Diversity of Ig repertoire generated by four main processes:

Answer 11

1. Combinatorial diversity - many copies of each gene segment type which can combine in many different ways
2. Junctional diversity - nucleotides added or subtracted at joins between gene segments during enzymatic steps
3. Many different combinations of light and heavy chains V regions that pair to form antigen binding site
4. Somatic hypermutation introduces point mutations into rearranged V region genes of activated B cells

Question 12

Developing and naïve B cells use alternative splicing
to express both IgM and IgD
TRUE OR FALSE

Answer 12

TRUE

naïve B cells = Circulating B cells that haven’t yet encountered an antigen

Question 13

Each B cell can produce Igs of any antigen specificity

Answer 13

FALSE
Each B cell produces Ig of a single antigen specificity

Ig gene rearrangement is tightly controlled.
Ensures only one heavy chain and one light chain expressed.
Known as allelic exclusion - only one copy or allele of heavy chain locus .
and one of the light chain locus are rearranged to form functional genes.
Thus each B cell produces Ig of a single antigen specificity.

Question 14

Membrane and secreted forms are generated by alternative RNA processing of the heavy chain
TRUE OR FALSE

Answer 14

TRUE

Transmembrane Ig has a hydrophobic transmembrane domain of around 25aa that acts as an anchor in plasma membrane

Secretory Ig has a hydrophilic secretory tail

The 2 carboxy termini are encoded in separate exons and selected by alternative RNA processing

Question 15

Ig is first made in a membrane-bound form that is present on B cell surface
TRUE OR FALSE

Answer 15

TRUE

B cell receptor (BCR) is made up of cell surface IgM with invariant proteins Ig’alpha’ and Ig’beta’

Invariant chains are important for signaling once antigen binds to IgM

Ig heavy chains have hydrophobic sequence near C terminus that anchors them in membrane