Wk3 Antibody Genetics

BCR

gene that encodes B cell receptors

VDJ recombination

A single gene from V (variable), D(diversity), and J(joining) regions are spliced in from the vast array to create the mRNA transcript

Where are the VDJ genes located

Upstream of C (conserved regions) in gene

Light Chains vs Heavy Chains recombination

Light: VJ only

Heavy: DJ join, then V added

Kappa vs Lambda light chain rearrangements

Kappa recombination happens first and has more diversity, and any V gene can join to any J gene

Lambda rearrangement occurs if it fails in the heavy chain pairing, then lambda is used. Specific V genes are restricted to specific J genes, limiting diversity

Heavy chain rearrangement

DJ recombination, V added at 5’ end, then spliced with a single C region which codes for the isotype of the immunogloubulin at 3’ end

Rescue attempt

If multiple VJ doesn’t work out with the produced immunoglobulin, cells can carry out a rescue attempt in the light chains, using any leftover (downstream and upstream) elements that exist in the genome and are inactive due to distance from each other

RSS

Recombination sequences at 5’ and 3’ ends of VDJ exons, which are heptamer CACAGTG and nonomer ACAAAAACC motifs separated by 12 or 23 spacer sequences. Each exon has a heptamer and nonamer end, but orientation varies allowing for flipping of the genome

what does recombination occur between

12 and 23 spacer, regardless of orientation, allowing reversal of exon

RAG1 and RAG2

Enzymes that work together to cleave a 12 spacer to a 23 spacer by:

• binding to two different RSS

• forming a loop in between them

• cutting off the loop leaving DNA “hairpin” ends

• opening of DNA hairpins by Artemis

• TdT completes sticky ends with complementary nucleotides

• TdT adds random nucleotides & ligates DNA

Result: exons have some random nucleotides in between which affects final receptor structure

CDR Regions

3 regions that compose the antigen binding site of an antibody

CDR 3

• 3rd, variable section on both heavy and light chains

• End of V section, junction to J section and possibly D, so has the most variability

CDR 1 and 2

• Vary based on the V region spliced in

• comprise two regions of antigen binding site

• Both heavy and light chains

Somatic Hypermutation

• Process that occurs after B cell activation and after clonal expansion

• B daughter cells use AID to enact single base pair changes in the antigen binding site

• B cells with increased affinity (binding strength) of immunoglobulin survive better, multiply, and avoid apoptosis

• Mutations occur with much higher frequency than regular body cells

FRs

Framework regions between CDRs which provide the antibody structure and position the CDRs, composed of B-pleated sheets. Less variable than CDR, and mutations can be helpful or detrimental

Class switching

When T helper cell prompts antibody production, the C region, the constant region which contains the Fc region is selectively spliced to promote a certain function (ie. secretion, opsonization, agglutination, membrane binding)

• first cut: between Mu and Delta chain (after VDJ region and before C regions)

• second cut: Somewhere between C exons so the last included gene determines the Fc region

• second cut placement is determined by local cytokine activity from a connected T helper cell

• Rejoined by VDJ looping mechanism (double strand breakage & rejoin)

This can happen to daughter B cells so that multiple classes of antibodies can be produced

Allelic exclusion

The original, unmodified gene of each of heavy and light chain can come from the mother or father, adding new combinations

• 2 chances for heavy chain, 2 chances for Kappa, 2 chances for lambda + VDJ recombination

Enhancer

light and heavy chain genes are activated by the same enhancer, so equal amounts of each are made before they complex (post VDJ recombination)

Pre B cell receptor

After VDJ recombination has occurred in the heavy chain, the heavy chain presents on the B Cell membrane stabilized with surrogate (immature) light chain

• Lets the cell check heavy chain viability before committing to light chain VJ recombination = antibody completion

B cell life cycle

• Hematopoietic Stem cell

• Lymphoid cell

• Pro-B cell (heavy chain VDJ complete)

• Pre-B cell (light chains VJ complete)

• Immature (Naive) B cell (presents antibody) = IgM

• Mature B cell

• Antigen Stimulation —> Activated B cell

• Plasma cell + Clonal Expansion + Somatic Hypermutation

• Class switching creates different isotypes of the same antibody