L7 - Genetics of antigen receptors

Question 1

Recognition of pathogens: what two types are there and what do they produce?

Answer 1

Pattern recognition - PAMPS/MAMPS/DAMPS
* receptors are encoded in the germline (inherited)

BCR/TCR - rearrangement of genes within B-cells and T-cells to recognise more specific epitopes
* Somatically generated
* Needs no prior exposure to pathogens
* Great for achieving broad recognition
* Difficult for pathogens to predict and evolve to avoid
* potential for Autoimmunity

Question 2

B-cell receptor: how is it???

Answer 2

Recruited by FcR interactions of different soluble antibody isotypes

(“whatever that means”)

Question 3

T-cell receptor: how is it????

Answer 3

Mediated by cell-cell contact or local secretion of cytokines

(“whatever that means”)

Question 4

T-helper cell 1: what is it used for and what example is there?

Answer 4

Th1 Intracellular pathogens

• Increase macrophage killing

Question 5

T-helper cell 2: what is it used for and what example is there?

Answer 5

Th2 Extracellular parasites

• Eosinophils, mast cells, IgE (activation (??))

Question 6

T-helper cell 17: what is it used for and what example is there?

Answer 6

Th17 (Inflammatory) extracellular bacteria/fungi

• Recruit neutrophils

Question 7

T-follicular helper cell 1: what is it used for?

Answer 7

Localise to B cell follicles to support them

Question 8

T-regulatory cell: what is it used for?

Answer 8

Tolerance/Immune regulation

Question 9

T-cytotoxic cell 1: what is it used for?

Answer 9

Killer T cells

Question 10

Antibody typical structure

Answer 10

2 heavy chains and 2 light chains

Each heavy site forms both the constant region and the hypervariable region, with the two regions separated by a hinge and the two chains joined by disulfide bridges

The light chains only form the hypervariable region and work with the heavy chain to form antigen-binding sites

Question 11

Antibody/TCR/BCR genes: why are they strange?

Answer 11

They contain V-gene segments and J-gene segments which can recombine to give rise to various manufactured proteins

A typical protein contains a Vₗ chain and a Cₗ chain

Question 12

Light chains of antibodies: what type of segments in their genes do they have?

Answer 12

• V-segments
• J-segments

Question 13

Heavy chains of antibodies: what type of segments in their genes do they have, how does rearranging occur, what is the exon, where are CDR1/2/3 located, and what gene is the extra gene segment found?

Answer 13

V, D, and J segments

Two gene arrangements occur sequentially,
D → J and then V → DJ

VDJ = V exon

CDR1 and CDR2 are contained in V and V, D and J all contribute to CDR3

IgH gene

Question 14

Combinatorial diversity: how are the high levels of diversity established?

Answer 14

V/D/J (and sometimes H) differential joining results in some of the diversity

Junctional diversity - imprecise joining of gene segments causes lots more diversity

Question 15

RSS: what are they, what do they do, and what use do they have?

Answer 15

Recognition signal sequences

Border gene segments and enforce the 12/23 rule (recombination can only occur between segments with different spacer lengths - only either 12/23 or 23/12 may be formed)

Allows controlled diversity - helps keep the immune system functional

Question 16

Molecular mechanics of V(D)J recombination

Answer 16

1 - Initiation:
* RAG1/RAG2 - bind to RSS and initiate a cleavage event, generating a DSB in the DNA, and allowing recombination to occur

2 - DNA-damage recognition and hairpin opening:
* The cleavage results in hairpin structures at the ends of the gene segments which are opened by enzymes and other cellular components and generate free ends which can be ligated

3 - DNA repair:
* TdT and ligase activity repair DNA and finish the recombination

Question 17

TdT: what is it and what does it do?

Answer 17

Terminal deoxynucleotidyl transferase

Adds nucleotides to hairpins on VDJ recombinations

Question 18

Combinatorial and junctional diversity: the difference between the two?

Answer 18

Combinatorial Diversity  multiple gene segments

		 any H with any L

Junctional Diversity  P-nucleotide addition

		 N-nucleotide addition			 exonuclease activity

Question 19

T cells: what types are there?

Answer 19

Gamma/delta - play roles in immune responses at epithelial surfaces (innate)

Alpha/beta - most abundant (~95%)

Question 20

B cells/T cells: where do they develop?

Answer 20

B cells - bone marrow
T cells - thymus

Question 21

T cells

Answer 21

Follow the same recombinant machinery as BCRs

Question 22

Immunoglobulin vs TCR recombination diversity

Answer 22

Both are assembled by somatic recombination of multiple gene segments - Similar levels of combinatorial diversity

> Greater junctional diversity in TcR;
- D segments read in all reading frames
- TdT activity at all junctions
- TCR diversity is focused on CDR3, which contacts the antigenic peptide

> More J gene segments

• Footprint of αβTCRs docking down onto MHC class I and II peptide complexes

Question 23

Mature activated b cell gene expression

Answer 23

Only productively rearranged genes are expressed, everything else is excluded

Question 24

Clonality: what is required?

Answer 24

• Only one antigen specificity to be expressed

Question 25

Gene rearrangements: which steps occur at which time?

Answer 25

Early pro b-cell:
* H-chain gene rearrangement
* D-J rearrangements on both chromosomes

Late pro b-cell:
* H-chain gene rearrangement
* D-J rearrangements on first chromosomes
* D-J rearrangements on second chromosomes
* Cell loss

Pre-b cell:
* L-chain gene rearrangement
* Rearrange the k gene on the first chromosome
* Rearrange the k gene on the second chromosome
* D-J rearrangements on second chromosomes
* Cell loss

Immature b-cell:
* Rearrangement ceases

Question 26

Are secreted forms of antibodies the same as their forms expressed at the transmembrane?

Answer 26

They differ by the secreted forms having a reduced carboxy terminus

AAA region is cut off

Google or ask whatever the TM forms of antibodies are (and AAA region.)

Question 27

Antibody expression: is only one antibody expressed by a cell at one time?

Answer 27

Yes, mostly

IgM and IgD may be co-expressed and this is regulated by alternative RNA processing

Question 28

How does class switching occur?

Answer 28

Isotypes are formed by recombinations between switch regions

Question 29

Are the antibodies produced in the bone marrow (primary repertoire) the only antibodies produced throughout their cell’s lifespan?

Answer 29

Not usually, as the cells move from the BM to the germinal centre in lymph nodes, they first have somatic hypermutation for affinity maturation, and then may later class switch

Question 30

V-region assembly: what does it result in, is it reversible and does it occur in T/B-cells?

Answer 30

Somatic recombination of DNA

No

Both

Question 31

Junctional diversity: what does it result in, is it reversible and does it occur in T/B-cells?

Answer 31

Imprecise joining, N-sequence insertion

No

Both

Question 32

Transcriptional activation: what does it result in, is it reversible and does it occur in T/B-cells?

Answer 32

Activation of promoter by proximity to the enhancer

No but regulated

Both

Question 33

Switch recombination: what does it result in, is it reversible and does it occur in T/B-cells?

Answer 33

Somatic recombination of DNA

No

B-cells

Question 34

Somatic hypermutation: what does it result in, is it reversible and does it occur in T/B-cells?

Answer 34

DNA point mutation

No

B-cells

Question 35

IgM/IgD expression on cell surface: how is it formed, is it reversible and does it occur in T/B-cells?

Answer 35

Differential splicing of DNA

Reversible, regulated

B-cells

Question 36

Membrane/secreted antibody form: how is it formed, is it reversible and does it occur in T/B-cells?

Answer 36

Differential splicing of DNA

Reversible, regulated

B-cells