Antigen Receptor Genes

Inheritance of DNA (Most Genes)

Maternal and paternal DNA come together to form the maternal and paternal forms of proteins

• maternal DNA and paternal DNA contribute one allele each

Issue: B cells are clonal:

One B cell produces only 1 Ab (e.g. monoclonal Ab production)

Issue: Limits

There is no limit to the number of antigens that can be recognized by B cells

DNA Recombination of Immunoglobulin Genes Occurs in Somatic Cells

• Hozumi and Tonegawa performed a paradigm-shifting experiment in 1976.

• Showed that multiple gene segments encode the antibody protein chains

What are antibodies?

Immunoglobulin proteins made by B cells that bind antigen

Antibody Structure

• Consists of heavy chains and light chains:

  • heavy chains are longer and on the inside

  • light chains are shorter and on the outside

• The heavy chains ‘hinge’ to form a V-shape at the top of the antibody

• Disulfide bonds connect the heavy chains and the light chains

Papain Digestion of an Antibody

Results in Fab (2) and Fc (1) subunits

Pepsin Digestion of an Antibody

Results in F(ab’)2 and Fc fragments OR mercaptoethanol reduction (splits up heavy (2) and light (2) chains completely)

Secreted and Membrane Ig:

have the same Fab but a different C-terminus

Complementarity-determining Regions (CDR)

the loops in the Fab region where antibodies interact with Ag

Framework Regions

the other parts of the Fab region where there is no antibody-Ag interaction

Non-covalent Bonds

mediate the interaction between antibodies and antigens

Isolation and Discovery of Antibodies

• Serum is isolated from rabbits that have been injected with ovalbumin

• Electrophoresis separates proteins by charge and mass

• Agglutination reactions demonstrate that Ab are substances:

  • Agglutination = aggregation or clumping of particles or cells

  • Exp.: Test a series of dilutions (serial dilution) of anti-SRBC serum, Abs to SRBCs added to wells

Reactions between Ab (or Ab fragments) and Ags:

Using agglutination as the read-out, it is clear that the “valency” of antibodies and antigens is important:

• i.e. univalent Ags will not cross-link

Neutralization

Ab binding blocks binding of toxin or virus to cell

Opsonization

promotes phagocytosis of Ag via interaction with Ab

Activation of Complement

directly destroys cells

Antibody-dependent Cell-mediated Cytotoxicity (ADCC)

killing by NK cells

Granule Release

release of granules into the extracellular spac

Transcytosis

Moving Ab across epithelial layers (ie. placenta)

Generation of Ab Diversity

Occurs in the bone marrow independent of Ag

2 types of light chain:

• Lambda λ

• Kappa κ

2 parents = 4 possible C gene alleles:

Everyone inherits 2 copies of light chain genes, one from each parent:

4 possibilities:

• κ/κ

• κ/λ

• λ/κ

• λ/λ

The same CH or CL region can be connected to millions of different VH or VL regions

Ag-binding Region (V Domain)

• The light and heavy chains are encoded by multiple gene segments that form 1 gene

• The shuffling of these gene segments results in the formation of this region

Light chain V region gene segments in embryo (germline DNA):

1. V segment (amino acids 1-97)

2. J segment (amino acids 98-110)

Downstream to the C region gene segment

Heavy chain V region gene segments in embryo (germline DNA):

1. V segment (amino acids 1-101)

2. D segment (amino acids 102-106 (approx.))

3. J segment (amino acids 107-123 (approx.))

Downstream to the C region gene segment

κ-chain DNA

• shortest

• LVκ(n) regions

• Jκ region

• Cκ region

λ-chain DNA

• mid-length

• LVλ(n) regions

• Jλ(n) regions

• Cλ(n) regions

Heavy-chain DNA

• longest

• LVH(n) regions

• DH(n) regions

• JH(n) regions

• C(n) regions (different kinds)

Rearrangement of the light chain:

The cell loses genetic material between the gene segments

• rearrangement between V and J clusters

Rearrangement of the heavy chain:

The cell loses genetic material between the D and J gene segments and then the DJ and V

Early B cell development:

Occurs in the Bone Marrow independent of antigen:

1. lymphoid stem cell (germ line)

2. pro-B cell - (Ig-α/Ig-β)

3. pre-B cell - (surrogate light chain of pre-BCR)

4. immature B cell - (IgM)

5. naive B cell - (IgM)

6. mature B cell - (IgM and IgD)

RSS

Recombination signal sequence:

• DNA sequence motif recognized by recombination enzymes

• Regulates joining

• The region is on the DNA that flanks the gene segments

12/23 Rule

a 12 RSS can only join with a 23 RSS

Productive Rearrangement

a protein can be made

Non-productive Rearrangement

no protein can be made

Combinatorial Diversity

V x D x J or V x J

• E.g. 100 V x 10 D x 50 J = 50,000 different combination

Junctional Diversity

• Flexible joining at RSS sequence

• P and N nucleotide addition

Where does VDJ and VJ rearrangement in B cells occur?

The bone marrow

How is the DNA rearranged at the molecular level?

Enzymes join and cut the DNA:

• RAG1 and RAG2

• Recombination-Activating Gene

• RAG1/2 recognize the RSS sequences to join them and cut the DNA

• Other proteins repair the cut ends

• Result: New VJ gene and signal joint (garbage)

RAG1/2

• Recombination-Activating Genes

• Recognize the RSS sequences to join them and cut the DNA

Mechanism of V(D)J Recombination

1. RAG1/2 and HMGB1/2 proteins bind to the RSS region and catalyze synapse formation between a V and a J gene segment

2. RAG1/2 performs a single-stranded nick at the exact 5’ border of the heptameric RSSs bordering both the V and the J segments

3. The hydroxyl group that was liberated by the nick at the 3’ end of the coding strand attacks the corresponding phosphate group on the noncoding strands of both the V and the J segments to yield a covalently sealed hairpin coding end and a blunt signal end

4. Signal end joining ligates the ends of the two RSS heptameric sequences that were originally in contact with the V and J coding sequences:

• Sequence at the signal junction results from the joining of the two heptameric regions

5. Opening of the hairpin can result in a 5’ overhang, a 3’ overhang, or a blunt end:

• The most common result generated by Artemis is a 3’ overhang

6. Cleavage of the hairpin generates sites for P nucleotide addition

• DNA repair enzymes fill in complementary strands

7. Ligation of light chain V and J regions:

• Ligation of complete segments by DNA ligase IV and XRCC4

STEPS 8-10 ONLY OCCUR IN Ig HEAVY CHAINS

8. Exonuclease cleavage can result in the loss of nucleotides on either or both sides of the coding joint

9. Nontemplated nucleotides are added to the coding joint by TdT or occasionally by pol μ

10. Ligation of heavy chain by NHEJ DNA ligase complex

V and J recombination if V and J segments lie in the same direction:

• signal and coding joints form

• formation of a signal joint results in circularization of intervening DNA into an episome that is lost upon subsequent cell divisions

If V and J gene segments are in opposite directions:

It is possible for the signal joint to remain in the genomic DNA:

1. Nicking and hairpin formation

2. Inversion and ligation of the central fragment

• The signal joint remains in the DNA upstream of the recombined antibody gene

• The direction of transcription is now consistent between the recombined V and J segments

How does the cell deal with the extra DNA that does not contribute to the Ig?

It “ignores” it through RNA processing:

• Precursor mRNA is spliced to form Processed mRNA

RNA Processing:

It is also how one B cell can express the membrane and secreted forms of the same Ab

• alternative splicing

Where is diversity generated?

at the level of DNA at the IgL locus and IgH locus independently

Recombination between gene segments:

is required to generate complete variable light-and-heavy-chain gene

If a new species of mammal has 10 V, 5 J, and 10 D, how many possible antigen-binding sites are possible?

• Higher than 500, due to additional factors like junctional diversity and combinatorial pairing of heavy and light chains

• infinite antigen-binding sites are possible

How does a Pro-B Cell transform into a Pre-B Cell?

Surrogate Vpre-B and λ5 Germ-line κ and λ:

• Rearrange H chain → IgM (μ)

How does a Pre-B Cell transform into a Immature B Cell?

Surrogate Vpre-B and λ5 Germ-line κ and λ:

• Rearrange L chain

Membrane-bound Ab is part of the B cell receptor (BCR):

This is how a B cell communicates with the rest of its world

Igα-Igβ Dimer (BCR)

This part carries the signal from the outside of the cell to the inside of the cell

Surrogate Light Chain (Placeholder)

The different gene segments (VDJ & Cμ) that make up the heavy chain are rearranged and expressed with this

If Heavy chain expression is successful:

The genes that make up the light chain (VJ) are rearranged

Surface expression of heavy chains (IgH) and light chains (IgL) form:

The mature BCR on an immature B cell

Apoptosis

programmed cell death (cell suicide)

12 RSS

one turn

23 RSS

two turns

Productive rearrangement and allelic exclusion:

ensures only one Ab per B cell is expressed

B Cell Development is composed of multiple steps/stages:

Successful completion of 1 stage is necessary before entering the next stage:

• Bone marrow, antigen-independent

  • Lymphoid stem cell →

  • Pro-B cell →

  • Pre-B cell →

  • Immature B cell

• Periphery, antigen-dependent

  • Naive B cell →

  • Mature B cell

The Microenvironment

Essential for early B cell development:

• IL-7 (a crucial cytokine and hematopoietic growth factor produced by stromal cells)

CD

Cluster of differentiation

CD Markers

• Named approximately in order of discovery, starting in 1982

• Now up to ~ 320

2 major types of T cells:

T helper cells (Th) and Cytotoxic T cells (Tc or CTL)

• Both recognize Ag via their TCR, but co-receptors regulate the APC that they interact with

Helper Cells

• CD4+

• TCR + Peptide

• MHC Class II

Cytotoxic Cells

• CD8+

• MHC Class I

The T Cell Receptor (TCR) and Associated Receptors

• Recognition of antigen in the context of MHC

• Transfers signals to nucleus

TCR Complex

TCR:

• 1 α chain, 1 β chain

CD3:

• 1 γ chain, 1 δ chain, 2 ε chains, 2 ζ chains

• all T cells

• Invariant

• ‘chaperone’ for TCR

• doesn’t bind Ag

• signal transduction

Valency and Conformation

BCR (Ab):

• 2 or more Ag-binding sites

• 4 chains

• Flexible

TCR:

• 1 Ag-binding site

• 2 chains

• Rigid

• TCR resembles Fab fragment of Ab

Differences between TCR and BCR

• Valency and conformation

• Antigen recognition

• Secretion of the receptor

  • TCR is not secreted

• No change in TCR during response to Ag

  • Affinity of TCR does not change

Generation of TCR Diversity

• Multiple germ-line gene segments

• Combinatorial V-(D)-J joining

  • RAG enzymes join and cut the DNA

• Random association of α and β chains

• Junctional and insertional diversity

• Receptor editing (uncommon)

NOT involved in TCR diversity:

• Somatic hypermutation

• Somatic gene conversion (not in humans)

Gene Rearrangement: αβ TCR

Similar to IgL:

• δ gene segments interspersed between α gene segments

Similar to IgH

Can T cells activate receptor editing?

Yes, to alter their antigen specificity, particularly to avoid self-reactivity

How is the generation of self-reactive (recognizes self/host proteins) prevented?

Negative selection

Central Tolerance or Negative Selection

Must eliminate B cells expressing Ab that is reactive to self-proteins:

• Occurs in the bone marrow (IgM= Ab found on immature B cell)

1. Apoptosis:

• Programmed cell death

• Interaction of the B cell receptor (BCR) on immature B cells “tells” the cell to die

• Composed of highly regulated series of events

2. Anergy

• State of unresponsiveness

• Anergic B cell (expressing self reactive Ab) emigrates to the periphery

3. Receptor Editing

• Interaction of the BCR with self-Ag in the bone marrow can ‘signal’ to the cell to rearrange the light chain again (“do-over”)

• Occurs on the light or heavy chains but much more frequently on light chain

Anergy

Long-term activation → inactivated B cell leaves the bone marrow

B cell expressing receptor for non-self-antigen:

leaves bone marrow

B cell expressing receptor for self-antigen:

Deletion → death of B cell expressing receptor for self-antigen

What happens if the RAG genes are mutated?

Severely impair V(D)J recombination, the process essential for developing mature B and T lymphocytes, leading to severe primary immunodeficiencies