Genetics of Antigen Receptor Diversity – I & II

Antibodies and T Cell Receptor Genetics Learning Objectives To understand mechanisms for creating antigen receptor (BCR/TCR) diversity Be able to identify changes at the DNA level required to produce a functional immunoglobulin gene To understand isotype switching at the molecular level To recognize basic differences between antigen receptors (structure and function) on B and T cells

VARIABILITY Variability in the antibody variable region Variability in the antibody heavy chain constant region – isotype switching Variability in the T cell antigen receptor variable region No T cell isotypes

Immunoglobulins Have Two Roles As an Antigen Receptor To selectively recognize and bind antigens These include toxins, viruses, and exposed molecules on the surface of pathogenic organisms V domain function (VH and VL) As an Effector Molecule To eliminate or inactivate the foreign antigen or the cell that bears that antigen C domain function (CH)

Light chain gene in a B cell CDR CL VL L CDR Sequence myeloma light chain proteins. Bence-Jones. With more sequences, C and V, then V hypervariable or CDR 1-3. CDR3 most variable. Why? Mechanisms restricted to CDR3. Another mechanism effects entire V region. CDR

1920’s Karl Landsteiner Analysis of antibody specificity in “Specificity of Serological Reactions” Antibodies can distinguish between chemically identical molecules that differ only in the position of the COOH group – stereoisomers Immunologists struggled for many years to figure out how it is possible to make antibodies to any substance Landsteiner was a busy guy. He also discovered ABO blood groups (Nobel Prize) and poliovirus

Generation of Antigen Receptor Diversity “The Repertoire” Survival requires B and T cell receptor diversity to respond to the diversity of pathogens The immune system must “Be Prepared” to respond to antigens it has never encountered

Generation of Diversity One to 100 million different antibodies with different specificities can be produced Nine antibody isotypes IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE Similar numbers of T cell receptors for antigen 10 x 106 genes X 103 base pairs DNA/gene = 10 x 109 bp DNA No isotypes with TCR

Generation of Diversity Cellular Solutions Diversity operates at the level of the lymphocyte. Each lymphocyte has a single receptor specificity Clonal selection: 1. Generate one million different lymphocytes per day 2. Antigen selects cells by binding to a complementary receptor and stimulating cell division and differentiation Cell that don’t meet antigen die.

Clonal selection Plus CD4 T cell help IgM, IgG, IgA or IgE Unselected cells die. T cells in thymus, >95% die. Clonal selection

Generation of Diversity Genetic Solutions Genes for antigen receptors do not exist until they are generated during the development of lymphocytes Light chain gene CL VL L Variable regions exons are formed by splicing together segments of genes inherited through the germline

Generation of Diversity Genetic Solutions Functional genes for antigen receptors do not exist until they are generated during the development of lymphocytes Variable regions exons are formed by splicing together segments of genes inherited through the germline The process is called Ig or TCR gene rearrangement and generates tremendous diversity without monopolizing the genome

Anatomy of Immunoglobulin Genes in B Lymphocytes Intron V Region Exon V Region Exon C Region Exon C Region Exons DNA mRNA Protein Light chain Heavy chain

Variable region genes are constructed from gene segments Germline DNA Stem cell B Cell DNA mRNA Protein J here is not J in J chain of IgM and IgA.

Germline Ig Genes

V-region Gene Segments are Joined by Somatic Recombination VL Exon Deletion Circle Light chain gene CL VL L

Visualization of deletion circles by electron microscopy

VL VH VH IgM B Cell IgM B Cell VH VL

Somatic recombination at the Ig heavy chain locus VH gene segments DH segments JH segments Germline DNA * D  JH rearrangement No Ig produced at this point * V  DJH rearrangement Primary RNA transcript Splicing and polyA  mRNA Nascent protein Mature μ heavy chain protein Same thing as with light chain where we have juxtaposition of the segments and looping out of intervening DNA.

VL VH IgM B Cell VH VL

Recombination Activating Genes Critical protein complex for VDJ and VJ recombination Two closely-linked genes RAG-1 and RAG-2 required for activity “Knock-out” of either gene leads to total absence of B and T cells How do RAG1 and RAG2 know where to cut the DNA? Baltimore. Found in all species with Gene rearrangement, not in others. The recombination activating gene 1 component is thought to contain most of the catalytic activity, while the N-terminal of the recombination activating gene 2 component is thought to form a six-bladed propeller in the active core that serves as a binding scaffold for the tight association of the complex with DNA.

RAGs Recognize Recombination Signal Sequences DNA sequences that flank rearranging gene segments of all immunoglobulin and T cell receptor loci Highly conserved palindromic heptamer and A/T-rich nonamer separated by a 12 or 23 bp spacer

Recombination Signal Sequences

RAGs Recognize Recombination Signal Sequences DNA sequences that flank rearranging gene segments of all immunoglobulin and T cell receptor loci Highly conserved palindromic heptamer and A/T-rich nonamer separated by a 12 or 23 bp spacer 12 and 23 bp correspond to one and two turns of DNA helix 12/23 bp rule: only RSS with 12 and 23 bp spacers can recombine with each other

Benefits of Antigen Receptor Gene Rearrangement Combinatorial joining The random somatic recombination of gene segments (VJ and VDJ) to create a variable region exon Vκ: 40 Vκ X 5 Jκ = 200 κ V region exons Vλ: 32 Vλ X 4 Jλ = 128 λ V region exons Light chain genes VH: 39 VH X 27 DH X 6 JH = 6,318 V region exons Heavy chain genes How many antibodies can be made? Combinatorial Association (6,318 HC) x (200 κ LC) = 1.3 x 106 IgM κ antibodies (6,318 HC) x (128 λ LC) = 0.8 x 106 IgM λ antibodies >2 million total

Benefits of Antigen Receptor Gene Rearrangement Combinatorial joining The random somatic recombination of gene segments (VJ and VDJ) to create a variable region exon Vκ: 40 Vκ X 5 Jκ = 200 κ V region exons Vλ: 32 Vλ X 4 Jλ = 128 λ V region exons Light chain genes VH: 39 VH X 27 DH X 6 JH = 6,318 V region exons Heavy chain genes How many antibodies can be made? Combinatorial Association (6,318 HC) x (200 κ LC) = 1.3 x 106 IgM κ antibodies (6,318 HC) x (128 λ LC) = 0.8 x 106 IgM λ antibodies >2 million total Is this enough diversity? Apparently not!

Strategies to Increase Diversity at the Site of V(D)J Joining (CDR3) Junctional flexibility – imprecise ligation of V, D, J segments Exonuclease nibbling P-nucleotide addition N-nucleotide addition P-nucleotide (palindromic) addition Non DNA template-encoded nucleotides generated during resolution of the hairpin at the coding end N-nucleotide addition Non DNA template-encoded nucleotides added to the free 3’ ends of the cleaved DNA of the coding joints More common in heavy chain VDJ rearrangements than in light chain VJ rearrangements Catalyzed by the enzyme terminal deoxynucleotidyl transferase (TdT)

Strategies to Increase Diversity at the Site of V(D)J Joining (CDR3) V(D)J Joining is imprecise Junctional flexibility – imprecise ligation of V, D, J segments Exonuclease nibbling

Frame shift more common. Genetic code is read 3 nucleotides at a time

Addition of N and P nucleotides to rearranging gene segments Occurs in CDR3 of H and L, but that is often the main determinant of Ag binding DJ

Benefits of Junctional Flexibility Increased diversity - only in CDR3 - CDR3 major contributor to antigen binding

CDR3 often directly involved in binding antigen CDR3 focus of diversity. Usually H more than L HWS

Benefits of Junctional Flexibility Increased diversity - only in CDR3 - CDR3 major contributor to antigen binding Cost Most V(D)J rearrangements are non-functional Solutions Rearrange on second chromosome if first fails Generate lots of cells

Allelic and Isotypic Exclusion of Ig Genes

Ig gene rearrangement during B cell development

Generation of Antibody Variable Region Diversity Antigen Independent V(D)J rearrangement (Combinatorial joining) Junctional flexibility P nucelotides N nucleotides Exonuclease nibbling Combinatorial association Antigen and T Cell Dependent Somatic Hypermutation Changes the DNA sequence of Ig Vh and Vl exons

Primary and Secondary Serum Antibody Responses Higher titer Higher affinity Tetanus toxoid. Somatic mutation has produced antibodies that can bind better to the antigen. CSR has produced IgG memory B cells.

Immune response to the hapten 2-phenyl-5-oxazolone (phOx) Demonstration of somatic mutation of Ig Vh and Vl exons during an immune response Immune response to the hapten 2-phenyl-5-oxazolone (phOx) Immunize mice with phOx-protein carrier At various times isolate spleen cells and make hybridomas secreting anti-phOx antibodies Sequence Ig Vh and Vl exons and compare it to the germline gene sequence to look for any mutations Most antibodies to phOx use a particular Vh and Vl gene – VhOx1 and VkOx1 Hapten alone, no response.

Somatic Mutation of Variable Regions during an Immune Response Isolate hybridomas making anti-phOX monoclonal antibodies VDJ-Cm VJ-Ck 2-phenyl-5-oxazolone (phOx) Claudia Berek and Ceaser Milstein. Day 7 mostly IgM. Berek, et al., Nature 316:412, 1985

Antibody Affinity Actual data. 100 fold increase in affinity.

Somatic Hypermutation Introduction of random mutations in the variable regions (VH and VL) of B cells responding to antigen T cell help is essential CD40 ligand deficiency → hyper IgM immunodeficiency Rate ~ 10-3/bp/cell division ~ 10-8 – 10-9 for conventional genes Mutations random Silent Replacement Selection of B cells bearing mutated receptors with increased affinity for antigen Competition for help from follicular helper T cells (TFH) in the GC

Somatic Hypermuatation Is Targeted to Ig V genes

Somatic Hypermutation II Cell no longer able to bind antigen or get insufficient T cell help die by apoptosis SHM occurs in germinal centers of secondary lymphoid organs Exact mechanism unknown – requires transcription of targeted gene Requires the activation induced deaminase (AID) gene

Somatic hypermutation in the germinal center Summarizes a lot of events that occur during the generation of a T cell dependent antibody response. Microevolution, survival of fittest in this GC. Other GC, completion may be different.

IgG IgA or IgE Plus T cell Help Isotype switching

Switch Recombination Switch region Switch region VDJ C C C C VDJ DNA deletion Simplified Ig locus.

Switch Recombination Advantage No requirement for separate VDJH recombination for each isotype Only cells that switch will be those responding to antigen Features Irreversible Individual plasma cell produces one isotype one specificity Since VDJ recombination is so wasteful, don’t want to have to do it for each isotype.

Ig Class (Isotype) Switching Mouse

Human has duplicated part of the locus. One Ce is psuedogene Human has duplicated part of the locus. One Ce is psuedogene. Anthropomorphically, maybe not good to have two Ce.

Regulation of Isotype Switching IgM/IgD Dr. Chaplin mentioned. Different in humans. Directed by sterile transcription of target C gene

Switch Recombination II Not site specific recombination like V(D)J Recombination is in S-region in introns Targeted constant region genes must be transcribed Similar to somatic hypermutation and V(D)J rearrangement Activation-Induced Cytidine Deaminase (AID) required for both SHM and isotype switching Although many cytidine deaminases are RNA editing enzymes, AID edits DNA AID introduces single stranded nicks in DNA. Error prone repair get SHM AID introduces double stranded breaks in DNA switch regions. Ligation of two S-regions leads to isotype switching AID also required for gene conversion. Used in chickens and sheep, cows to diversify antibodies.

Sterile transcripts Need to activate the B cell to get this process rolling. Here using LPS to polyclonally activate. Even though this is kind of TI response, in vitro can get switching to other isotypes by adding cytokines normally provided by T cell in a TD response

Ig Gene Diversification RAG 1/2 Summary of the action at the DNA level in IgH Teng and Papavasiliou Annu. Rev. Genet. 41:107 (2007)

Summary of the cellular level.

T CELL RECEPTOR DIVERSITY ANTIBODY DIVERSITY Variable region Antigen independent – bone marrow Ig gene segment rearrangements Antigen dependent – 20 lymphoid organs Somatic hypermutation Constant region Antigen dependent – 20 lymphoid organs IgM  IgG or IgA or IgE T CELL RECEPTOR DIVERSITY Finished with B, lets look at T. Only antigen independent VDJ recombination. Variable region Antigen independent – thymus TCR gene segment rearrangements

The T Cell Receptor for Antigen TCR

Comparison of the TCR and the BCR (Immunoglobulin) V regions encoded by rearranging genes Rearranging parts of two receptors

Structure of the T-cell Receptor Both chains TM proteins, C and V regions

The T Cell Receptor Heterodimer that only exists as a transmembrane antigen receptor It is not secreted since T cells function by direct cell contact The variable regions of the TCR are generated by somatic gene recombination as the T cells develop in the Thymus The process is identical to Ig gene rearrangement, but different genes are used Kill or help, cant do at a distance.

B cells recognize intact protein antigens Heavy Chain Lysozyme Ab recognize epitopes on intact molecules. Antigenic Determinant Epitope Light Chain Ab recognize epitopes on intact molecules.

T cells recognize processed (degraded) protein antigens Peptide-MHC T cell recognize peptide MHC.

Differences in antigen recognition by B and T lymphocytes B cell antigen receptor Immunoglobulin (Ig) B cells Transmembrane protein Secreted by effector cells (Plasma cell) Humoral Immunity T cell antigen receptor T cell receptor (TCR) T cells Transmembrane protein Transmembrane protein on effector cells CD4 Helper T Cells CD8 Cytotoxic T Cells Cell Mediated Immunity

Differences in T and B Cells Recognize native protein antigens in solution or on cell surfaces Secreted antibody is effector molecule Antibodies can operate at a distance T cells Recognize peptides from degraded antigens Peptides are displayed on cell surfaces in association with specialized proteins (MHC) Antigen-specific T cell functions require direct cellcell interactions

Here are the two receptors again.

T-cell Receptor - and -chain V Regions are Generated by Gene Segment Rearrangement B1 and B2 not isotypes.

T-cell Receptor - and -chain V Regions are Generated by Gene Segment Rearrangement How locus really looks. Delta locus embedded in alpha locus Va>Ja delete delta. Again B1 and 2 not isotypes.

The  TCR Basic structure similar to  TCR Encoded in separate gene loci Only a minor subpopulation of T cells in the blood are  T cells The  T cells may be enriched in tissues such as skin and intestinal epithelium A more primitive T cell? The first line of defense?

The T Cell Receptor Gene Loci Gamma a bit like Ig lamda of Ig. Multiple C with upstream V and J. No evidence that they are isotypes in either case.

Diversity Variable region Antigen independent [V(D)J] ANTIBODY DIVERSITY Variable region Antigen independent [V(D)J] Antigen dependent (SHM) Constant region Antigen dependent IgM  IgG or IgA or IgE T CELL RECEPTOR DIVERSITY Variable region Antigen independent

Dangers in Diversity Mechanism is essentially a random draw By chance, some Ig and TCR will react with self antigens Autoreactive B and T cells must be eliminated or silenced to prevent autoimmune diseases Chromosomal translocations arising during VDJ recombination or isotype switching may lead to lymphoid malignancies