Immunological diversity Gilbert Chu January 2004.

Immunological diversityGilbert Chu

January 2004

Discovery of antibody diversity430 BC Thucydides On 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.”

1796 Jenner Noted that cowpox was rarely followed by smallpox

Showed that cowpox innoculum protected from smallpox

Pasteur coined “vaccine” from vacca, cow in Latin

1901 Landsteiner Discovered 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-V500 D1-D12 J1-J4

H chain locus (Chr 12)

C C C3 C1

C2b C2a C C

V1-V250 J1-J5 C

V2 J2 C2

chain locus (Chr 6)

chain locus (Chr 16)

C3 C1V1 J3 J1

Mechanisms for generating antibody diversity

V(D)J recombination

Somatic hypermutation

Class switch recombination

V1-V500 D1-D12 J1-J4 constant regions

V(D)J recombination



Hozumi and Tonegawa, PNAS 1976

V(D)J recombination



V(D)J recombination



V(D)J recombination

D to J joining


V(D)J recombination

D to J joining


V(D)J recombination

V to DJ joining


V(D)J recombination


** *

Somatic mutations


** *

V(D)J recombination



Class switch


V(D)J recombination



** *

Class switch


V(D)J recombination



** *

V(D)J recombination and class switch recombination involve double-strand breaks


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

23

CACAGTG–––––––ACAAAAACC23

GGTTTTTGT–––CACTGTG12

heptamer nonamer nonamer heptamer

12

V J

12/23 : The rule recombination occurs between 12 23 a RSS with a bp spacer and a RSS with a bp spacer

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

coding join signal join

cleavage

end-joining

Cleavage is initiated by RAG1/RAG2 (recombination activating genes)

RAG1/RAG2 nick DNA at two RSSs....

then catalyze nucleophilic attack by 3' OH on the opposite strand

5' OH

5' OH

5'

5'

generating hairpin coding ends and blunt signal ends

van Gent, Gellert et al. Cell 1995

DNA ends are modified by addition and deletion

N-nucleotide addition by terminal deoxynucleotidyl transferase (TdT)

P-nucleotide addition by asymmetric opening of hairpin coding ends

Nucleotide deletion

Addition at DNA ends

TCAAGT

TCAAGT

N-nucleotide additionaddition at 3' ends by TdT

addition

Germ-line gene Rearranged gene Mechanism

GATCACTAGT

P-nucleotide additionhairpin formation andasymmetric cleavageaddition

TCAAGT

GATCAT

ATAGTTATCA

Deletion at DNA ends.

Germ-line gene Rearranged gene Mechanism

CAGT

TCAAGT

Nucleotide deletionrandom

deletion

TCAAGT

Nucleotide deletionmicrohomology directed

deletion

TCAAGT

CAGT

DNA pathways in V(D)J recombination

Evolution of V(D)J recombination

RAG1 and RAG2 contain no introns and are tightly linked on the same chromosome

RAG1 and RAG2 are conserved back to the evolution of jawed fish

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

hagfishlamprey

Hypothetical RAG transposon

RAG1 RAG2

excision

Transposon integration

5' 5'

5'

5' 5'

5'

OH OH

Agrawal, Eastman and Schatz Nature 1998

Origin of the immunoglobulin genes

D JV

ancient receptor gene

V

gene duplication

D×12 J ×4V×500 C

first transposon integration

second transposon integration

The scid mouse Mouse with severe combined immunodeficiency,

lacking mature B and T cells

Defective in the joining of coding ends Normal in the joining of signal ends

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

Mutagenesis of Chinese hamster epithelial cells generated several X-ray sensitive cell lines

These cells were co-transfected with RAG1, RAG2, and V(D)J recombination substrates

The cells were then assayed for either coding joint formation or signal joint formation

V(D)J recombination substrates

stopcatamp catamp

stopcatamp catamp

plasmid for coding joins coding join

signal join

Lieber et al. (1988) Cell 55, 7-16

plasmid for signal joins

Lieber, Gellert et al. Cell 1998

Mutants in V(D)J recombination and X-ray resistance

impairedX

impairedX

RAG1/RAG2 cleavage at RSS signal sequence

coding sequence

normal group 7 (scid) groups 4, 5, 6

precise precise

modified impairedX

signal joins

coding joins

Taccioli, Alt et al. Science 1993

Mechanisms for repairing DNA double-strand breaks

Homologous recombination

Non-homologous end-joining

V(D)J recombination mutants are defective in non-homologous end-joining

Proteins involved in non-homologous end-joining

Protein Enzymatic activity

Ku DNA end-binding

DNA-PKcs DNA-dependent protein kinase

XRCC4/ Ligase 4 DNA ligase

Artemis exonuclease

Rad50/ Mre11/ Nbs1 exo/endonuclease

Human diseases with defects in non-homologous end-joining

Severe combined immunodeficiency with radiation sensitivity (Artemis)

Ataxia telangiectasia-like disorder (Mre11)

Nijmegan breakage syndrome (NBS1)

Ku recruits DNA-PKcs to DNA ends

DNA-PKcsKu 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


Hammarsten and Chu, PNAS 1998



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



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 CH gene, except for C

consist of repeats of GAGCT and GGGGGT; e.g., switch region is [(GAGCT)nGGGGGT]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 Diversity is generated by multiple mechanisms

V(D)J recombination



Some components are lymphocyte-specific RAG1/RAG2, TdT, AID

Other components are ubiquitous Double-strand break repair, base excision repair

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Immunological diversity Gilbert Chu January 2004.

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