Antigen Receptor Genetics: Somatic Recombination

Lecture 10: Antigen Receptor Genetics

• The objectives of this lecture are to review general biology, BCR: somatic recombination, BCR: combinatorial diversity, BCR: junctional diversity and T Cell Receptor Rearrangement

The Central Dogma of Biology (A Review)

• Replication of DNA results in DNA. DNA is nucleic acids
• Transcription of DNA results in RNA. RNA is nucleic acids
• Translation of RNA results in Protein. Proteins are amino acids
• Introns are transcribed from the leader sequence, exon 1, exon 2, exon 3, and exon 4
• Splicing results in only the exons
• Translation results in Cytoplasmic tail (Exon 4), Transmembrane portion (Exon 3), Domain 2 (Exon 2), Domain 1 (Exon 1)

A Brief Review of Recombination

• DNA Recombination: The exchange of genetic material either between multiple chromosomes or between different regions of the same chromosome
• Homologous Recombination: Occurs between identical (or nearly identical) sequences. (e.g. meiosis)
• Non-homologous Recombination: Occurs between non-identical sequences:
  • Site specific recombination
  • Translocation
  • Nonhomologous end joining
• All types of recombination are mediated by enzymes

Differences in Innate and Adaptive Immunity

• Innate immunity has a response time of minutes/hours, whereas adaptive immunity is days
• Innate immunity is specific for molecules and molecular patterns associated with pathogens and molecules produced by dead/damaged cells, while adaptive immunity is highly specific and discriminates between even minor differences in molecular structure of microbial or nonmicrobial molecules
• The diversity of innate immunity is limited number of conserved, germ line-encoded receptors, whereas adaptive immunity is highly diverse and has a very large number of receptors arising from genetic recombination of receptor genes in each individual
• Innate immunity memory responses are observed in invertebrate innate responses and mouse/human NK cells and adaptive immunity has a persistent memory, with faster response of greater magnitude on subsequent exposure
• Self/nonself discrimination in Innate Immunity is very good as there are no microbe-specific self/nonself patterns in host
• Self/nonself discrimination in Adaptive Immunity is very good, but occasional failures of discrimination result in autoimmune disease
• Soluble components of blood in innate immunity are are many antimicrobial peptides, proteins, and other mediators, including cytokines. Soluble components of blood in adaptive immunity are antibodies and cytokines
• Major cell types in innate immunity are Phagocytes (monocytes, macrophages, neutrophils, dendritic cells), natural killer (NK) cells, other leukocytes, epithelial and endothelial cells. Major cell types in adaptive immunity are T cells, B cells, antigen-presenting cells

Pattern Recognition Receptors vs. Adaptive Immunity Receptors

• Receptors of innate immunity are directly encoded in the germline
• Receptors/molecules of adaptive immunity can't be directly encoded in the germline as there is a limited number of receptor genes

Antigen Receptor Diversity

• Survival requires B and T cell receptor diversity in order to respond to all potential pathogens
  • The immune system must be prepared to recognize and respond to antigens it has never before encountered
• Diversity operates at the level of the lymphocyte:
  • There can be Up to 100 million different antigen receptors (BCR/Ig on B cells, TCR on T cells)
  • Each lymphocyte has a unique receptor for antigen
• The antigen 'selects' the lymphocyte by binding to a cognate receptor and stimulating cell division and differentiation (antibody-secreting plasma cells or effector T cells); called the clonal selection theory
• Assuming it meets the criteria, virtually any substance can elicit an antibody response, therefore: The recognition machinery (receptors) must be very diverse and the effector response to that antigen must also be diverse

The Antibody Diversity Problem

• Approximately there are millions of different lymphocyte receptors (and lymphocytes)
  • 100 million different antibodies can be produced
  • 9 antibody isotypes (and subtypes): IgM, IgG1, IgG2, IgG3, IgG, IgA1, IgA2, IgE, IgD (Human)
  • Similar numbers of T cell receptors for antigen
• There are approximately 20,000 genes in the human genome

The Problem (and the Solution)

• We need to make a lot of proteins (receptors), but we don't have the room (genes) to do it
  • The "one gene → one [protein]" hypothesis doesn't work
  • These receptors are NOT DIRECTLY ENCODED IN THE GERM-LINE
• Recombination: We take a few basic building blocks (gene segments) and arrange them in various ways to create different results
  • Example: 26 letters in the English language; 218, 632 words in the English language (Oxford Dictionary)

Proteins Necessary for Lymphocyte Receptor Gene Expression

• The Lymphoid-Specific Proteins: Antigen receptor gene recombinase complex. DNA cleavage is mediated by RAG1. Epigenetic targeting is directed by RAG2
• Reduced N-nucleotide addition is seen at coding joints is Terminal deoxyribonucleotidyl transferase (TdT)
• Artemis-deficient B and T cells have blocked formation of coding joints and accumulation of hairpin-sealed coding ends. Mice lacking Artemis have severely impaired B- and T-cell development is Artemis

B Cells, B Cell Receptors, and Antibodies

• Membrane-bound form (BCR) has a spacer, hydrophobic segment, cytosolic segment and Iga,Igß
• Secreted form (antibody) has a Hydrophilic segment

Complementarity-Determining Regions

• Complementarity-determining regions are found in both B cells and T cells
• Hypervariable regions work together to help determine complimentarity

How Do We Generate Receptor Diversity?

• Somatic Recombination, for VDJ recombination
• Combinatorial Diversity: Randomness of heavy and light chain pairing
• Junctional Diversity: Insertion of new nucleotides
• Somatic Hypermutation: Occurs during B cell development

Mechanism 1: Somatic Recombination

• Genomic Ig Gene has Gene Segments
• Gene rearrangement, resulting in V exon and C exon
• With Production of primary transcript which becomes: Ig mRNA
• Followed by translation

The Puzzle of Immunoglobulin Gene Structure

• V segment contains 45 amino acids (amino acids 1-101)
• D segment contains 23 amino acids (amino acids 102-106 approx.)
• J segment contains 6 amino acids (amino acids 107-123 approx.)

VDJ Recombination

• Germ-line heavy chain (DNA), V cluster, D cluster (12-14 segments) and J cluster (4 functional segments)
• After which D-J and V-D rearrangement

Recognition Signal Sequences

• The sequences of non-coding DNA are adjacent to coding DNA
• Contains: A heptamer (7 bp), Spacer sequences (12 or 23 bp), A nonamer (9bp)
• Every single gene segment contains an RSS

Recombination Signal Sequences and the 12/23 Rule

• The recombination machinery is designed to pair one RSS bearing a 12 bp spacer with another bearing a 23-bp spacer; it is a mechanism by which the cell ensures/promotes the correct pairing of gene segments, and D only pairs with J, not with another D

The RAG1/2 Enzyme Complex

• The RAG1/2 Enzyme Complex picks and recombines/assembles various gene segments into a segment of DNA that can be expressed
• It 'chooses' by random (as far as we know)
• Defective RAG → Severe Combined Immunodeficiency (SCID)

The BCR/Antibody Gene

• Light Chains Contain: Variable Regions, Joining Regions, Constant Regions
• Heavy Chains Contain: Variable Regions, Diversity Regions, Joining Regions, Constant Regions
• These genes are each on different chromosomes; processes occur independently

Allelic Exclusion and the Order of Ig Locus Rearrangement

• Each gene has two copies, and if they each arrange randomly and independently, how does each cell have only one specificity? (and not two-one for each allele)
• Each B-cell will make only ONE functional heavy chain rearrangement and only ONE functional light chain rearrangement. Thus, each B cell produces antibodies with only a single variable region (i.e. single specificity)
• Once a successful in-frame heavy chain rearrangement has been made, rearrangement at the other heavy chain locus stops and that allele is no longer expressed. It is excluded (allelic exclusion). The same is true for both the к (Igk) and λ (Igl) light chain loci

Igl. If the Igk rearrangement is successful, there is no need for an Igl rearrangement (therefore most antibody light chains are kappa)

• For whatever reason, the Igk locus is rearranged before the

Mechanism 2: Combinatorial Diversity

• Is the randomness of heavy and light chain pairing
• Heavy and light chains are on different chromosomes and are assembled post-translationally
• pairing heavy chain CDR and light chain CDR will cause randomness

Combinatorial Antibody Diversity in Humans

• Number of heavy-chain segments (estimated) V: 45, D: 23, J: 6
• Number of K-chain segments: V: 41, J: 5
• Number of A-chain segments: V: 33, J: 5
• Possible number of combinations: 45 × 23 × 6 = 6210, 41 × 5 = 205 and 33 × 5 = 165
• Possible number of heavy- and light-chain combinations in the human = 6210 × (205 + 165) = 2.3 × 10°

Mechanism 3: Junctional Diversity

• 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
• Opening of the hairpin can result in a 5' overhang, a 3' overhang, or a blunt end -The most common result generated by Artemis

Cont. - Mechanism 3: Junctional Diversity

• Nucleotide addition and/or exonuclease trimming at the V(D)J joints does not necessarily occur in sets of three nucleotides, and so can lead to out-of-phase joining
• If recombination has caused the loss of the correct reading frame for the transcription process cannot encode antibody molecules and such rearrangements are unproductive
• DNA (hairpins) from various segments (e.g. V, D, or J) are cleaved, Strands are filled in-added nucleotides are random,Segments are ligated to one another - This is Mediated by terminal deoxynucleotidyl transferase (TdT)

The Big Picture View

• Heavy Chain (Igh) Locus: D always joins with J first and V then rearranges to join with the DJ segment
• Light Chain (Igl) Locus: First kappa, then the lambda
• once rearrangement occurs, gene expression can then occur as normal (transcription, splicing, translation)

What About The Constant Region?

• Once VDJ recombination has occurred, the recombined variable region must be connected to a constant region
• Note which constant region (isotypes) are closest in proximity to the variable region
• B cell isotypes are expressed first
• Note that this is for the heavy chain

T Cell Receptor Diversity

• Two classes of T-cell receptors are αβ T-cell receptor and γδ T-cell receptor

VDJ Recombination in T Cells

• Mouse TCR a-chain and 8-chain DNA
• Mouse TCR B-chain DNA
• Mouse TCR y-chain DNA

• α-chain locus (Compare to the Immunoglobulin light chain; VJ)
• rearranged DNA
• β (Compare to the immunoglobulin heavy chain; VDJ)
• rearranged DNA

αβ νς. γδ Τ Cells

• Gene family V region segments D region segments J region segments: for α, β, Y, and δ

Cont. - αβ νς. γδ Τ Cells

• TCR genes (like BCR genes) are generated by the rearrangement of V, J, (and D) segments
• To become an aẞ T cell, the cell must successfully generate a TCR ẞ chain
• To become a үб T cell, the cell must successfully generate both a TCR y chain and a TCR &
• It's easier to successfully generate one chain than two, so most T cells are aẞ
• Once cells have successfully generated a ẞ chain, they're committed to the aß lineage-the a chain comes later
• Most 'early' T cells are γδ, but that quickly switches to aß during gestation
• γδ T cells are important in barrier immunity, and appear to recognize 'unconventional' antigens presented by ‘unconventional' MHC molecules
• Can secrete cytokines immediately after exit from the thymus
• Most T cells are aß and will be referred to as "T cells", and γδ T cells will be referred to as “γδ T cells"

The T Cell Receptor Complex

• The CD3 complex is CD3Y, CD36, CD3ε,the Z chain
• Its purpose is transport of the TCR to the cell surface and to perform Cell signaling

Comparison of B Cell and T Cell Receptor Generation

• Immunoglobulin: Heavy V segment: 40, D segment: 23, J segment 6, V gene pairs: 2×10^6, Junctional diversity: 3×10^7, Total diversity: 5×10^13
• Immunoglobulin: Light V segment: 70, D segment: 0, J segment 5k, 4λ, V gene pairs: 2×10^6, Junctional diversity: 3×10^7, Total diversity: 5×10^13
• T-cell receptor: β V segment: 52, D segment: 2, J segment 13, V gene pairs: 6×10^6, Junctional diversity: 2×10^11, Total diversity: 1×10^18
• T-cell receptor: α V segment: 70, D segment: 0, J segment 61, V gene pairs: 6×10^6, Junctional diversity: 2×10^11, Total diversity: 1×10^18