B cell effector functions II Overview of antibody response and changes in antibodies Antibody class switching -brief review of effector functions Class switching mechanism- part 1 overview and targeting -distinction from V(D)J recombination -targeting of switch recombination by cis acting elements -relationship of targeting to sterile transcription Somatic mutation -the striking restriction of mutation -mutation or lack of repair? -models of mutation with relation to DNA repair -paradoxical effect of repair mutants AID and a unified model of somatic mutation and class switch -initial lesion -repair and resolution

The purpose of the antibody response is to tag specifically the microbe, marking it for appropriate disposal. The variable portion of the antibody confers affinity/specificity The constant portion regulates the effector function A high affinity and appropriate effector class must be established

Low affinity IgM class High affinity IgG class IgM Antibody protein sequence and bioactivity changes during the immune response

Antibody protein sequence is altered in several independent ways (Pre-immune) membrane IgM/IgD coexpression RNA splicing Regulation of membrane vs secreted IgM RNA splicing Switch to IgG, IgA, IgE antibody H-chain C exons DNA class switch recombination Affinity maturation Somatic hypermutation (in some species, gene conversion)

VDJ joining here creates heavy chain variable region domain VD Note: there are exons encoding the membrane and secreted forms of each of the antibody heavy chains. a

The first antibody heavy chain type to be expressed is membrane IgM Note: there are membrane and secreted forms of each of the antibody heavy chain classes.

Prior to activation, B cells express two forms of antibody as membrane receptors, IgM and IgD. On any given cell, the antigen specificity is identical. This is accomplished by differential RNA splicing. IgM IgD

VDJ recombination RNA splicing controls expression of membrane vs secreted IgM Class switch is a DNA recombination, distinct from VDJ recombination, that occurs in immune responses Antibody heavy chain locus

Immunoglobulin classes

Information/ Specificity Effector/ triage function

Antibodies come in different classes

V(D)J recombination Short, conserved recognition sequence RAG1/2 cleaves DNA Resolved by DNA repair proteins that specialize in non-homologous end joining. “Calculated imprecision” introduced to create diversity.

Terminal transferase (TdT) can add untemplated nucleotides here

VDJ CC CC SS SS Joining can occur anywhere within the large, repetitive S-regions. There is no obvious conserved sequence motif. Unlike V(D)J recombination, this gene splicing occurs between, rather than within, coding sequences. Like V(D)J recombination, targeting of elements for rearrangement is correlated with prior “sterile transcription”

Like V(D)J recombination, class switch recombination is regulated by 1) expression of a recombination machine 2) targeted “accessibility” mediated by nearby enhancers and promoters

Promoter upstream of human IgE I-region

Accumulation of V- region point mutations during the antibody response.

From Gearhart and Bogenhagen PNAS 80:3439, 1983 Somatic mutations are focussed on the variable region exon Promoter Enhancer elements

Patricia J. Gearhart and Richard D. Wood

One proposed way that a mutator could have localized action

Clues from the pattern of somatic mutation Strand bias, transitions>transversions, bias vs pyrimidines (as assessed on coding strand). From Betz et al, PNAS 90:2385.

Is antibody mutation induced? Natural mutation rate in the absence of repair is high ~10 -5 /bp/generation With repair, spontaneous mutation rate is ~10 -9 or less. Repair pathways -DNA polymerase 3’-->5’ exonucleolytic proofreading improves fidelity ~100X -mismatch repair system improves fidelity ~100X Initial estimates found values of /bp/generation in clonally related B cells carrying mutations. Suggested that repair is either turned off or mutation is induced.

Characteristics of Somatic Mutation 1. Occurs at high rates: /bp/generation. 2. Occurs by untemplated single base substitutions. 3. Restricted to a brief period of B cell differentiation. 4. Restricted to the rearranged V region and its immediate flanking sequences. 5. Occurs in germinal centers with T cell help. 6. Occurs throughout the V region but more frequently in RGYW ( A/G G C/T A/T ) motifs. 7. Mutations in kappa light chain transgenes require intronic and 3’ enhancers but not in the V region promoter or V coding region. BioEssays 20:227–234, 1998

-If mutations are routinely removed from replicating DNA, a process that prevents repair locally would target mutation. If so, knockouts of DNA repair genes would have little or no effect. -If mutations are introduced by massive local DNA damage, possibly needed to overwhelm the normal repair mechanisms, then repair mutants would have increased mutation rates in the targeted regions (near assembled VDJs). -Alternatively, DNA repair enzymes may be needed to generate mutations.

Mutation models involving error-prone DNA polymerases

Martin and Scharff Nat. Rev. 2:605 (2002) Mismatch repair in bacteriaMismatch repair in eukaryotes Mut mutants in bacteria have “mutator” phenotype Marti et al. J. Cell. Physiol. 191:28 (2002) Msh2,6 +/- linked to hereditary non-polyposis colon cancer (HNPCC)

MSH2-/- mice have reduced antibody gene mutation The authors suggested that the mutator might target the “wrong” strand for repair, in effect co-opting the mismatch repair process to introduce mutations. Cascalho et al, Science (1998) 279:1207

Table 2. Distribution of Mutations in Hot Spots vs Background Mutations Position Msh2+/- Msh-/- 39TGT 2.1% (11)4.6% (7) 56 AGC 2.7% (14) 9.3% (14) 62 GCA 1.8% (9) 4.6% (7) 253 GCT 1.6% (8) 6.6% (10) 8.2% (42) 25.1% (38) Rada et al. (1998) Immunity, 9:135.

… but what would cause the initial mismatch? 

A surprising finding: these three processes are dependent on a single gene, activation induced deaminase (AID). AID-deficient mice do not switch to IgG, IgA, IgE or show much antibody hypermutation.

The closest homologue to activation induced deaminase (AID) is APOBEC-1, an RNA editase involved in lipid metabolism.

Figure 2. Occurrence of somatic mutation in one DNA strand in the G1 phase of the cell cycle. Somatic mutation was induced in BL2 cells in the G1 phase of the cell cycle. Single cells were either analyzed for mutations in the V4-39 gene after 90 min of stimulation or isolated in single wells and left for 24 or 48 h (one or two divisions) before analysis. (a) Three representative mutations in the V4-39 gene, which show a mixed sequence. (b ) Visualization of one, two and four cells. Note the streptavidin beads that cross-link the biotinylated anti-IgM bound at the cell surface. (c ) Three patterns were observed when two BL2 cells that differed at a single position in their V gene (nucleotide 57) were amplified. In addition to the expected configuration of amplification of a mixed sequence (left), cases of biased amplification were observed, which resulted in amplification of either of the two "alleles" (middle and right). (d) Schematic representation of mutation occurrence on a single strand of DNA, segregated in the same 50:50 proportion after cell division. Faili, A. et al. Nature Immunology 3, (2002)

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Point mutations in the Sµ region can occur under switching culture conditions, but prior to switching, consistent with a common mechanism for the two types of DNA modification.

Deletions, mutations and short duplications are associated with switch recombination

Mismatch repair Homologous recombination Non-homologous end joining DNA repair system involved

Relationship to disease The hypermutation/ class switch mechanism could be disastrous if not correctly targeted, leading to translocations, point mutations, in appropriate gene conversions. It is not know if the mutator can be activated in non-B cell tumors. Surprisingly, the gene of the germinal center B cell specific transcription factor Bcl6 is often mutated in normal B cells, but many other tested genes are not mutated at higher rates. Many lymphoid tumors involve breakpoints between Ig genes and oncogenes. Some of these are associated with V(D)J type recombination, others with class switch, others with functional V regions.

Modern drug research and development (duration ~10 years): random screening of millions of compounds in bioassay to find initial candidates refining candidates based on minor substitutions and selection for improved affinity and specificity toxicity and efficacy trials The antibody response (duration ~14 days): select a few thousand (or fewer) cells among millions of B cells point mutate and select to develop highest affinities* class switch to appropriate effector class *Important for recognition of microbes that mutate rapidly

Harnessing the power of the immune system Rapid generation of antibody reagents. AID can mutate many genes when transiently overexpressed in cell lines.Recruiting the mutator system to selected non-Ig genes may be useful for protein engineering. Understanding selection and how it is optimized.