B Cell Diversity - Essay

Front 1

Structure

Back 1

Introduction

Combinationaldiversity: Light chain,Heavy chain, Immunoglobulin Loci,Control, Result, Issues with control

Class switching

Somatic hypermutation(in germinal centres) - Targeting,Mechanism, Issues

Front 2

introduction - what is the antibody repertoire and what are hypervariable regions

Back 2

· The total number of antibody specificitiesavailable to an individual is known as the antibody repertoire and in humans isat least 10^11.

· ­Hypervariable regions - Three CDRs from eachthe heavy and light chains

Front 3

Combinational diversity- what is the process?

Back 3

V(D)J recombination.

Front 4

Where does the process occur

Back 4

Only in B cells

Front 5

At what stage does Ab gene rearrangements start?

Back 5

Early pro-B cell stage: D-J in heavy chain

Late pro-B V-Dj in heavy chain

Front 6

Light chain, explain the gene segments

Back 6

· Up to 40 variable (V) gene segments. FiveJoining (J) Segments. One Constant (C) Segment

Front 7

What are the steps

Back 7

· Step 1: Somatic Recombination (occurs at the DNAlevel).

· V-J joining results in the excision of a whole seriesof V and J genes to create a new V-J combination

· Step 2: Transcription and Splicing. Thesomatically recombined DNA is transcribed into pre mRNA (primary mRNA). Theintrons are spliced out, combining the V,J and C regions.

· The primary RNA is processes to add apolyadenylated (poly-A) tail after Cu chain. · The message is translated into the light chainprotein.

Front 8

How is diversity created?

Back 8

Imprecise joining

Front 9

Explain the gene segments in the heavy chain

Back 9

Heavychains have up to 27 additional D (diversity) region genes

Front 10

Explain somatic recombination in the heavy chain

Back 10

· Somatic recombination of heavy chain genesinvolves the D-J regions, followed by V-DJ joining. The rest of the process issimilar to the kappa chain. All other genes are deleted from genome.

Front 11

What are the Immunoglobulin Loci?

Back 11

· kgenes are on chr 2 - gene segments for light chain.

· lgenes are on chr 22 – light chain

· The heavy chain genes are on chr 14.

Front 12

Explain the control - overview

Back 12

· Conserved DNA heptamer/nonamer sequences flankthe coding regions and form a recombination signal sequence (RSS)

Front 13

How is specificity of recombination maintained?

Back 13

Rag1and Rag2 recombinase enzymes binds an RSSs flanking a V, D and J gene segments– creates a single-strand nick in DNA between the first base of RSS and thecoding segment. This is essentially energetically neutral – no ATP and resultsin the formation of a free 3’ OH and 5’ phosphate group on the same strand.

Front 14

Explain the next step with the OH group

Back 14

· The reactive OH group is positioned by therecombinase to attack the phosphodiester bond of opposite strand, forming twoDNA ends: a hairpin on coding segment and a blunt end of the signal segment.Current model – DNA nicking and hairpin formation occur on both strands simultaneously.

Front 15

Next step

Back 15

Additional proteins (DNA-dependent protein kinase, Ku, Artemis and a dimer of DNA ligase and XRCC4) are incorporated into a large complex with the Rag proteins. These RSS ends are joined, forming what is called the Signal Joint, to create a closed circular DNA, that plays no further role in the recombination process.

Front 16

Explain RSS

Back 16

· RSS composed of three elements: heptamera of 7conserved nts, spacer region (12/23 bps) and nonamer – 9 conserved nts. Whilethe majority of RSSs vary in sequence, the consensus heptamer and nonamersequences are similar. Although the sequence of the spacer region is poorlyconserved, the length is highly conserved – corresponds to 1 or 2 turns of DNAhelix.

Front 17

location of gene segments to be recombined?

Back 17

adjacentto RSS of different spacer lengths- important feature in the regulator of recombination

Front 18

What repairs re-joined segments

Back 18

DNAligase

Front 19

Result

Back 19

· Highly variable antigen-binding region even whenthe same gene segments are recombined.

· Overall: costly process and is strictlyregulated and controlled.

Front 20

Issues with control- syndrome

Back 20

· Omenn Syndrome - Caused by a missense mutationin RAG1. Ab gene rearrangement cannot occur

Front 21

Other issues

Back 21

· Evidence that signal joints may re-enter thegenome and lead to pathologies by activing oncogenes or interrupting tumoursuppressor gene functions.

· Major caveat: the DNA sequence must remainin-frame in order to maintain correct aa sequence in the final protein product.Out-of-frame – development of cell will be arrested.

Front 22

Class switching: what determines class

Back 22

constant-regionexon next to the V(D)J exon.

Front 23

What determines which Ab to switch to

Back 23

· If activated B cells encounter specificsignalling molecules via CD40 and cytokine receptors – Ab class switching toIgG, IgA or IgE.

Front 24

What is the class switching recombination?

Back 24

· This 'class switch recombination' process resultsin the replacement of the Cmexon by Cg, andthe deletion of the intervening sequence in the heavy chain.

· IgG protein is then produced by the cell,instead of IgM.

Front 25

How is the variable domain rejoined?

Back 25

· Thevariable domain exon is rejoined through a process called non-homologous endjoining (NHEJ) to the desired constant region (γ, α or ε).

Front 26

What ensures only maternal or paternal Ab genes are expressed?

Back 26

· Allelic exclusion – ensures only maternal orpaternal Ab genes are expressed. Other switched off.

Front 27

Where does somatic hypermutation occur?

Back 27

in germinal centres

Front 28

What is the reason for somatic hypermutation?

Back 28

Antibodyaffinity for antigen increases during the course of an immune response.

Front 29

What is result ofsomatic hypermutation

Back 29

· BCR locus undergoes high rate of somaticmutation – 106 times greater than normal

· Variation mainly in the form of single basesubstitutions. Indels are less common

· Mutations occur mostly at hotspots in DNA –concentrated in hypervariable regions.

Front 30

What three proteins are involved?

Back 30

Activation-Induced (Cytidine) Deaminase- (AID)

uracil-DNAglycosylase (UNG) - removesuracil

APE1– excises ribose

Front 31

Explain the mechanism

Back 31

· Activated B cells make (AID) which converts c to u in Ig DNA variableregions.

· Hypermutation occurs when cells try to fix theerror.

· AID works on ssDNA so is restricted to activeloci that are being transcribed.

· (UNG)and APE1 remove the Uracil. Other nucleic acids can get inserted in the gap.

Front 32

What fills in gap?

Back 32

· Error-proneDNAP fills the gap. Replacement of U at deaminated cytosine or neighboring bp leadsto a new codon resulting a new aa at the Ab antigen recognition site.

Front 33

Which B cell is selected?

Back 33

greatest affinity will be selected todifferentiate into plasma cells producing Abs and memory B cells.

Front 34

Issues

Back 34

· Mistargeted somatic hypermutation is a likelymechanism in the development of B-cell lymphomas