Exam 2 -- Generation of Lymphocyte Antigen Receptors

:: There are at least 10⁸ B-Cell variants in your average human

:: There is a TON of variability in antigen receptors

Who discovered primary immunoglobin gene rearrangements?

Susumu Tonegawa

By what process are Ig genes rearranged in B- and T-Cells?

Somatic Recomination

In germline DNA, where are constant (C) and variable (V) TYPICALLY regions found? (somatic rearrangement)

On different genes

In B-Cell DNA, where are the constant (C) and variable (V) TYPICALLY regions found? (somatic rearrangement)

On the same DNA fragment

Why is the placement of typical germline Constant and Variable regions compared to the placement of Antibody DNA relevant?

Science is built upon little experiments - The germline DNA undergoes somatic rearrangement so the C and V fragments end up on the same piece of DNA!

How do Hypervariable regions in antibodies take form?

HV regions form 3 discrete loops

What are the four regions that make up the HEAVY CHAIN of an antibody?

V - Variable Regoin

D - Diversity Region

J - Joining Region

C - Constant Region

What are the three regions that make up the LIGHT CHAIN of an antibody?

V - Variable Region

J - Joining Region

C - Constant Region

How many Hv (Hypervariable) regions are encoded in a single V-Region?

3

Where does the CDR3 region originate from

2+ gene segments joined together

In a light chain, which gene segments are rearranged to yield the CD3 region of the Ab?

The V (Variable) and J (Joining) Regions

In a heavy chain, which gene segments are rearranged to yield the CD3 region of the Ab?

The V (Variable), D (Diversity) and J (Joining) Regions

In a light chain, which gene segment encodes the CDR 1 and 2 regions? (BCR and TCR)

The V (Variable) Region

In a heavy chain, which gene segment encodes the CDR 1 and 2 regions? (BCR and TCR)

The V (Variable) Region

In a light chain, what processes generate the CDR3 region?

Recombination and Joining of the V (Variable) and J (Joining) regions

In a heavy chain, what processes generate the CDR3 region?

Recombination and Joining of the V (Variable), D (Diversity), and J (Joining) regions

What does CDR stand for?

Complementarity Determining Region

What does the C (Constant) Region determine?

The type of heavy chain

(IgG, IgM, IgA, IgE, IgD)

What does RSS stand for?

Recombination Signal Sequence

What is the RSS (Recombination Signal Sequence)?

A heptamer (7bp) and a nonamer (9bp) sequence separated by 12 or 13bp

Allow Rag-Mediated gene recombination

What is the 12/23 rule?

Recombination typically occurs between RSS’s with spacers that are 12bp OR 23bp long

What can we glean from the 12/23 rule?

RSS’s that are 12bp or 23bp apart are more likely to recombine with each other

Why do scientists believe the 12/23 rule arose?

The RAG1/RAG2 heptamer forms asymmetrically, allowing it to bind V units 12bp away and J units 23bp away with each arm

It also keeps D segments from recombining with each other (SOMETIMES this still happens)

TRUE or FALSE — D (Diversity) segments NEVER recombine

FALSE !!

What are the components of the V(D)J recombinase enzyme?

RAG 1

RAG 2

TdT

What do the components of the V(D)J recombinase enzyme stand for? (RAD and TdT)

RAG - Recombination-Activating Gene

TdT - Terminal Deoxynucleuotide Transferase

What are the enzymatic steps in RAG-Dependent V(D)J recombination?

1. TdT randomly creates diverse, imprecise ends

2. P&N nucleotides are added

In the first step of RAG-Dependent V(D)J recombination, TdT randomly creates diverse, imprecise ends of gene sequences?

Adds nucleotides to the chain, increasing diversity of gene segment

What are P-Nucleotides?

Palindromal sequences (Palindromal Nucleotides)

What are N-Nucleotides

Non-template encoded Nucleotides

TRUE or FALSE — 2/3 of TdT genetic rearrangements are nonproductive frameshift mutations

TRUE !

Why does the CDR3 region need to be so diverse?

This is the regions of the Ab that actually contacts the peptide

What deficient genes result in Severe Combined Immune Deficiencies (SCIDs)

RAGS, DNA-PKCs, Ku, Artemis

What do gene deficiencies reulting in Severe Combined Immune Deficiencies (SCIDs) do? What is the end product of these deficiencies?

Block in mature lymphocyte development

No mature B and T cells

What are the four processes that prompt Ig diversity?

1. Combinatorial Diversity (VDJ combos)

2. Junctional Diversity (Addition/Subtraction of nucleotides P/N Nucleotides/Tdt)

3. Combinatorial Diversity (Light and Heavy chain combos - γ/κ and α/δ/γ/ε/μ)

4. Somatic hypermutation (Mutation in germinal centers)

How is TCR gene rearrangement similar to BCR gene rearrangement?

12/23bp rule

What does a TCR resemble?

A Fab!

How does TCR gene rearrangement differ from BCR gene rearrangement?

More α and β recombination segments

What are the two types of T-Cells?

α:β

γ:δ

What is the TCR α-chain related to in a BCR chain?

Light Chain

What is the TCR β-chain related to in a BCR chain?

Heavy Chain

α : β, γ : δ, light chain :

heavy chain

In a α / γ chain, what processes generate the CDR3 region?

The joining of the V (Variable) and J (Joining) segments)

In a β / δ chain, what processes generate the CDR3 region?

The joining of the V (Variable), D (Diversity), and J (Joining) segments)

Where is the δ locus contained?

Within the α locus

Why does it matter that the δ locus is contained within the α locus?

When the α locus undergoes somatic rearrangement, the δ locus is deleted!

What results from the δ locus being contained within the α locus?

When the α locus rearranges, the δ locus is deleted

FAR fewer γ:δ T-Cells than α:β T-Cells

TRUE or FALSE — Structural variants in Ig constant regions define their classes

TRUE !

What are Igs named for?

Their abundance in serum

What is IgM good at?

Activation of Complement

What are IgG1 and IgG3 good at?

Placental transfers

What is IgE good at?

Binding mast cells and basophils

TRUE or FALSE — Ig’s are all good at the same thing

FALSE !! —They all have differing abilities

How many kinds of constant region genes exist

5

One for each type of Ig (IgG, IgD, IgM, IgA, and IgE)

What do the constant region genes correspond to?

Cα, Cγ, Cδ, Cε, Cμ

IgA, IgG, IgD, IgE, and IgM

What are the constant region genes?

Cα, Cγ, Cδ, Cε, Cμ

Where are Ig isotopes encoded?

In a cluster of Ig heavy-chain C-Region genes

:: Ig isotypes and subtypes predated speciation

:: We should not attempt to extrapolate the function of Ig’s across species - Don’t assume!

How is IgD and IgM co-expression regulated?

Alternative RNA splicing

How are transmembrane versus secreted forms of Igs regulated?

Alternative RNA splicing

TRUE or FALSE — All Igs can be produced as membrane-bound secreted proteins?

TRUE !

What is the Membrane-Bound sequence for Igs?

M1 and M2

What is the Secretion sequence for Igs?

SC

TRUE or FALSE — IgM and IgD can form multimers

TRUE !

What multimer does IgM create most commonly?

IgM

What multimer does IgA create most commonly in plasma?

Monomers

What multimer does IgA create most commonly in mucous secretions?

Dimers

What is Affinity

Binding strength

What is Avidity?

Total binding strength (cumulative total of all binding interactions of a molecule)

:: Antibodies can have high affinity for one antigen, and low affinity for a different antigen

:: Affinity depends on the antigen type

TRUE or FALSE — Avidity is more than just the sum of individual affinities

TRUE !

:: Each Ab has at least 2 identical binding sites, each with a given affinity

:: Multiple binding sites increase avidity

What are the primary three diversifiers of antibody responses?

Somatic Hypermutation

Gene Conversion

Class Switch

TRUE or FALSE — Somatic Hypermutation, Gene Conversion, and Class Switch are all driven by antigens?

FALSE !! — Just Somatic Hypermutations and Class Switches

What happens during a class switch?

The kind of Ig is switched — Ex- IgM → IgG

What does AID stand for?

Activation-Induced Cytidine Deanimase

What does AID (Actication-Induced Cytidine Deanimase) do? (Broad)

Introduce mutations in genes transcribed in B-Cells

What are the key proteins involved in Gene Conversion?

AID, MSH

UNG

REV1

APE1