Immunological diversity Gilbert Chu January 2004

Discovery of antibody diversity 430 BCThucydidesOn bubonic plague: ”It was with those who had recovered from disease that the sick and the dying found most compassion. These knew what it was from experience, and had now no fear themselves; for the same man was never attacked twice - never at least fatally.” 1796JennerNoted that cowpox was rarely followed by smallpox Showed that cowpox innoculum protected from smallpox Pasteur coined “vaccine” from vacca, cow in Latin 1901LandsteinerDiscovered antibodies against ABO blood antigens Made antibodies against many organic molecules: specificity and diversity Discovered antibodies against the red blood cell antigen in paroxysmal cold hemoglobinuria: autoimmunity

The antibody molecule

Mouse immunoglobulin genes V1-V500D1-D12J1-J4 H chain locus (Chr 12) CC CC C3C3 C1C1 C  2bC  2aCC CC V1-V250J1-J5 CC V 2J 2C 2  chain locus (Chr 6) chain locus (Chr 16) C 3C 1V 1J 3J 1

Mechanisms for generating antibody diversity l V(D)J recombination l Somatic hypermutation l Class switch recombination V1-V500D1-D12J1-J4constant regions

l V(D)J recombination Mechanisms for generating antibody diversity V1-V500D1-D12J1-J4constant regions Hozumi and Tonegawa, PNAS 1976

l V(D)J recombination Mechanisms for generating antibody diversity V1-V500D1-D12J1-J4constant regions

l V(D)J recombination Mechanisms for generating antibody diversity V1-V500D1-D12J1-J4constant regions

l V(D)J recombination D to J joining Mechanisms for generating antibody diversity

l V(D)J recombination D to J joining Mechanisms for generating antibody diversity

l V(D)J recombination V to DJ joining Mechanisms for generating antibody diversity

l V(D)J recombination l Somatic hypermutation ** * Somatic mutations Mechanisms for generating antibody diversity

** * l V(D)J recombination l Somatic hypermutation l Class switch recombination Class switch Mechanisms for generating antibody diversity

l V(D)J recombination l Somatic hypermutation l Class switch recombination ** * Class switch Mechanisms for generating antibody diversity

l V(D)J recombination l Somatic hypermutation l Class switch recombination ** * V(D)J recombination and class switch recombination involve double-strand breaks Mechanisms for generating antibody diversity

Recombination signal sequence (RSS) direct V(D)J recombination

V(D)J recombination involves DNA cleavage and end-joining

Cleavage is initiated by RAG1/RAG2 (recombination activating genes) van Gent, Gellert et al. Cell 1995

DNA ends are modified by addition and deletion l N-nucleotide addition by terminal deoxynucleotidyl transferase (TdT) l P-nucleotide addition by asymmetric opening of hairpin coding ends l Nucleotide deletion

Addition at DNA ends

Deletion at DNA ends

DNA pathways in V(D)J recombination

Evolution of V(D)J recombination l RAG1 and RAG2 contain no introns and are tightly linked on the same chromosome l RAG1 and RAG2 are conserved back to the evolution of jawed fish l Evolutionary hypothesis: a transposon with RAG1, RAG2, and associated RSSs infected a precursor of jawed fish

RAG1 and RAG2 do not exist in jawless fish hagfish lamprey

Hypothetical RAG transposon

Transposon integration Agrawal, Eastman and Schatz Nature 1998

Origin of the immunoglobulin genes

The scid mouse l Mouse with severe combined immunodeficiency, lacking mature B and T cells l Defective in the joining of coding ends Normal in the joining of signal ends l Hypersensitive to ionizing radiation The scid mouse suggested a link between V(D)J recombination and the repair of DNA double-strand breaks

Mutant nonlymphoid cells can be tested for V(D)J recombination l Mutagenesis of Chinese hamster epithelial cells generated several X-ray sensitive cell lines l These cells were co-transfected with RAG1, RAG2, and V(D)J recombination substrates l The cells were then assayed for either coding joint formation or signal joint formation

V(D)J recombination substrates Lieber, Gellert et al. Cell 1998

Mutants in V(D)J recombination and X-ray resistance Taccioli, Alt et al. Science 1993

Mechanisms for repairing DNA double-strand breaks l Homologous recombination l Non-homologous end-joining V(D)J recombination mutants are defective in non-homologous end-joining

Proteins involved in non-homologous end-joining

Human diseases with defects in non-homologous end-joining l Severe combined immunodeficiency with radiation sensitivity (Artemis) l Ataxia telangiectasia-like disorder (Mre11) l Nijmegan breakage syndrome (NBS1)

Ku recruits DNA-PKcs to DNA ends DNA-PKcs Ku DNA-PKcs DNA-PKcs then brings DNA ends together

Stoichiometry of the synaptic complex

Kinase inhibition does not affect synapsis

DNA-PK is activated cooperatively by DNA (Phosphorylation occurs after synapsis)

Leuther, Hammarsten, Kornberg, and Chu, EMBO J 1999

DNA with single-stranded ends activates DNA-PKcs most efficiently Hammarsten, DeFazio and Chu, J Biol Chem 2000

DNA ends with single-strand loops fail to activate DNA-PKcs

DNA ends blocked with streptavidin fail to activate DNA-PKcs

Model for activation of DNA-PKcs

Smider, Rathmell, Lieber, and Chu, Science 1994 Non-homologous end-joining

Hammarsten and Chu, PNAS 1998 Non-homologous end-joining

DeFazio, Stansel, Griffith, and Chu, EMBO J 2002

Non-homologous end-joining

A jawed fish (trout)

Alex’s model for end-joining, 1995

Questions about end-joining Protein questions What are the DNA polymerases? What are the nucleases? Phosphorylation questions Which proteins are targeted by DNA-PK? How does phosphorylation regulate these proteins? How does DNA-PK phosphorylate these proteins before phosphorylating itself?

Somatic hypermutation (SHM) SHM targets immunoglobulin genes (but not T cell receptor genes) SHM requires active transcription SHM involves DNA single-strand breaks

Model for somatic hypermutation Activation-induced deaminase (AID) Expressed only in activated B cells Converts C to U in single-stranded DNA Other proteins insert mutations Uracil DNA glycosylase converts U to an apurinic site AP endonuclease nicks the DNA adjacent to the AP site Exonuclease removes the AP ribose An error-prone polymerase fills in the gap

Model for somatic hypermutation How is C mutated on both strands with the same frequency? How does SHM target the Ig locus, but not other loci?

Class switch recombination (CSR) CSR rearranges the constant regions to generate different antibody isotypes CSR regions located 5’ to each C H gene, except for C  consist of repeats of GAGCT and GGGGGT; e.g.,  switch region is [(GAGCT) n GGGGGT] 150 CSR requires active transcription AID initiates CSR

CSR occurs via double-strand breaks CSR requires Ku and DNA-PKcs CSR junctions show characteristics of non-homologous end-joining Deletions to regions of microhomology Duplications from DNA polymerase activity

Model for class switch recombination How does AID initiate CSR at one locus and SHM at another? (The C-terminus of AID is required for CSR, but not SHM.)

Summary l Diversity is generated by multiple mechanisms l V(D)J recombination l Somatic hypermutation l Class switch recombination l Some components are lymphocyte-specific l RAG1/RAG2, TdT, AID l Other components are ubiquitous l Double-strand break repair, base excision repair