of April 5, 2018.This information is current as

Activityζof DNA Polymerase Frequency in Complementary Mouse Models Altered Ig Hypermutation Pattern and

and Marilyn DiazMadhumita Ray, W. Glenn McGregor, Thomas A. KunkelKathleen Richter, Chuancang Jiang, Ming-Lang Zhao, Janssen Daly, Katarzyna Bebenek, Danielle L. Watt,

ol.1102629http://www.jimmunol.org/content/early/2012/04/30/jimmun

published online 30 April 2012J Immunol 

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The Journal of Immunology

Altered Ig Hypermutation Pattern and Frequency inComplementary Mouse Models of DNA Polymerase z Activity

Janssen Daly,* Katarzyna Bebenek,† Danielle L. Watt,† Kathleen Richter,*

Chuancang Jiang,* Ming-Lang Zhao,* Madhumita Ray,* W. Glenn McGregor,‡

Thomas A. Kunkel,† and Marilyn Diaz*

To test the hypothesis that DNA polymerase z participates in Ig hypermutation, we generated two mouse models of Pol z function:

a B cell-specific conditional knockout and a knock-in strain with a Pol z mutagenesis-enhancing mutation. Pol z-deficient B cells

had a reduction in mutation frequency at Ig loci in the spleen and in Peyer’s patches, whereas knock-in mice with a mutagenic Pol

z displayed a marked increase in mutation frequency in Peyer’s patches, revealing a pattern that was similar to mutations in yeast

strains with a homologous mutation in the gene encoding the catalytic subunit of Pol z. Combined, these data are best explained by

a direct role for DNA polymerase z in Ig hypermutation. The Journal of Immunology, 2012, 188: 000–000.

The genes encoding the Ag-interacting portions of Absand IgRs are subjected to a process of deliberate hyper-mutation during immune responses leading to enhanced

affinity of Abs to a specific Ag (1, 2). This mechanism, termedsomatic hypermutation (SHM), is triggered by the activation-induced deaminase (AID), a molecule expressed when B lym-phocytes are activated by foreign Ag (3, 4). AID deaminatescytosines in the DNA encoding the Ig variable (V) regions (5).Mice and humans defective in AID lack SHM and class switchrecombination (CSR), as AID is also required to generate switchedAbs such as IgG, IgA, and so on (3, 4). A subset of hyper IgMsyndrome patients are defective in AID; these patients lack CSRand SHM, and they suffer from lymph node hyperplasia (6).Despite the fact that AID is a cytosine deaminase, mice deficient

in AID lack mutations at both G-C and A:T bp (3). This paradox isin part explained by the hypothesis that AID-mediated deamina-tion of cytosines in Ig V regions triggers the recruitment oftranslesion synthesis DNA polymerases to Ig loci (7). These DNApolymerases have relaxed geometric requirements and thus areprone to inserting incorrect bases during DNA synthesis (8). Onesuch polymerase is DNA polymerase h, which has been shown toplay a role in the misinsertion of bases at A:T sites during SHM (9,10). Other DNA polymerases have also been implicated in SHM,

but because they have important roles in other cellular functionssuch as cell division and DNA repair, discerning whether they playa direct or indirect role in SHM has been difficult (11–13).The mutations made during SHM of Ig V genes are predomi-

nantly base substitutions. The pattern of hypermutation suggeststhat a putative error-prone DNA polymerase must not only insertthe incorrect base but also extend from a mismatched terminus,a very difficult task for most DNA polymerases. Confounding thisis the fact that some of the base substitutions in SHM occur intandem, suggesting multiple misinsertion and mismatch extensionevents during a single DNA transaction (14). This led to the hy-pothesis that DNA polymerase z plays a direct role in SHM, be-cause it is a robust mismatch extender, alone and in conjunctionwith other translesion synthesis DNA polymerases, including Pol h(9, 14–16). However, demonstrating this has been difficult be-cause mice deficient in DNA polymerase z are early embryoniclethals (17–19). A mouse expressing antisense RNA against Rev3(encoding the catalytic subunit of Pol z) experienced decreasedSHM frequency and severely impaired affinity maturation (20).However, because all cells, not just B cells, expressed the anti-sense transcript, it remained possible that the phenotype was dueto indirect effects, such as diminished T cell function. In addition,SHM was reduced in human B cell lines in which the Rev3 geneencoding the catalytic domain of DNA polymerase z was inhibitedby antisense oligonucleotides, suggesting a direct role for thispolymerase in an in vitro model of hypermutation (21). A con-ditional knockout mouse model of Rev3 using the CD21 promoteralso resulted in a reduced SHM frequency that was difficult todiscern from a proliferation defect (22).To circumvent the problem of embryonic lethality and non-

specific effects from Pol z deficiency in non-B cells, we generatedmice with B cell-specific deletion of Rev3. We also constructedmice with a knocked-in leucine (L) to phenylalanine (F) mutationat residue 2610 in Rev3 (Rev3L2610F mice). The homologouschange in Saccharomyces cerevisiae increases spontaneous andultraviolet light-induced mutagenesis and is associated with aspecific error signature (23, 24). We reasoned that if Pol z plays adirect role in SHM, a more mutagenic variant would increase thefrequency of mutation at Ig loci and its error signature would beaccentuated. Indeed, we show in this article that Rev3 knockoutmice experienced a dramatic reduction in SHM frequency,whereas Rev3L2610F mice showed a significant increase in SHM

*Somatic Hypermutation Group, Laboratory of Molecular Genetics, National Insti-tute of Environmental Health Sciences, National Institutes of Health, Department ofHealth and Human Services, Research Triangle Park, NC 27709; †Laboratory ofMolecular Genetics and Laboratory of Structural Biology, National Institute of En-vironmental Health Sciences, National Institutes of Health, Department of Health andHuman Services, Research Triangle Park, NC 27709; and ‡Department of Pharma-cology and Toxicology, James Graham Brown Cancer Center, University of Louis-ville School of Medicine, Louisville, KY 40202

Received for publication September 13, 2011. Accepted for publication April 3,2012.

This work was supported by the National Institutes of Health, National Institute ofEnvironmental Health Sciences, Division of Intramural Research, Project Z01ES101603 (to M.D.) and Project Z01 ES065070 (to T.A.K.).

Address correspondence and reprint requests to Dr. Marilyn Diaz, NIEHS/NIH, D3-01, Building 101, 111 TW Alexander Drive, Durham, NC 27709. E-mail address:diaz@niehs.nih.gov

The online version of this article contains supplemental material.

Abbreviations used in this article: AID, activation-induced deaminase; CGG, chickengamma globulin; CSR, class switch recombination; GC, germinal center; HC, Hchain; NP, 4-hydroxy-3-nitrophenylacetyl; SHM, somatic hypermutation; V, variable;WT, wild-type.

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frequency and an altered SHM specificity. The results indicatea direct role for DNA polymerase z in SHM.

Materials and MethodsGeneration of B cell-specific Rev3 knockouts and Rev3L2610Fknock-ins

A linearized targeting vector containing loxP sites flanking exon 26 of Rev3was generated and electroporated into embryonic stem cells from C57BL/6mice (Supplemental Fig. 1A, 1B). Exon 26 of Rev3 encodes the metalbinding domain of DNA polymerase z and it is essential to its DNAsynthesis function (25, 26). Recombinants were electroporated with cre-recombinase to eliminate the loxP site-flanked neo site. Clones stillretaining the loxP sites flanking exon 26 of Rev3 (Rev3-floxed mice) wereidentified by Southern hybridization. Recombinant clones were confirmedby PCR analysis using primers N17delckF: 59-GTTTGGGGCATTGGTTT-ACAGGTG-39 and N17del3R: 59-CTCCTTACTGCTGGGGATACTCAT-GTG-39. Several clones were selected for blastocyst injection resulting inmale chimeras. Chimeras were bred with C57BL/6 Taconic mice and het-erozygotes obtained.

Rev3-floxed mice were crossed with the cre-recombinase transgenic lineC.Cg-Cd19tm1(cre)Cgn (BALB/c) (27), which we will refer to as CD19+,and reporter mice B6.129 3 1-Gt(Rosa)26Sortm1(EYFP)Cos/J (C57BL/6), which we will refer to as YFP+, strains purchased from The JacksonLaboratory (Bar Harbor, ME). CD19+ mice were crossed with YFP+ miceto give CD19+/2, YFP+/2; these mice were then backcrossed to the Rev3F/Fbackground (10 generations) to give CD19+/2, YFP+/2, Rev3F/F mice ina C57BL/6 background. The resulting mice had B cell-specific deletion ofRev3 exon 26, and the recombinant B cells can be collected by FACS byvirtue of their YFP expression (Supplemental Fig. 1B).

The targeted exon 26 of the Rev3 gene was examined by PCR withprimers N1753F (59-TTTCTATCAGCTTGTGCCCTTATCCTTACT-39) andN17cKR (59-ACAAGATTTCTTTGTGTAACAGCCCTGG-39) for the 59loxp site, giving WTand mutant products of 474 bp and 624 bp, respectively.Genotyping of the whole exon 26 region, using N1753F and N17del3R,resulted in a WT product of 1330 bp and a mutant product of 1630 bp(Supplemental Fig. 1C). Upon cre-mediated deletion of the floxed region,the above primers resulted in a product of 632 bp (Supplemental Fig. 1C).

To detect the CD19 cre-recombinase transgene primers, CD19 59 (59-CTATCTGAAAAATATTTAACAGGTGCCAC-39), CD19 39 (59-CAC-TATCCTCCACGTTCACTGTCCA-39), and CD19 cre (59-GGCAAATT-TTGGTGTACGGTCAGTAA-39) were used, resulting in a WT product of950 bp and a transgene product of 850 bp (Supplemental Fig. 1D). YFPgenotyping primers were 0IMR0316 (59-GGAGCGGGAGAAATGGATAT-39), 0IMR0883 (59-AAAGTCGCTCTGAGTTGTTAT-39), and 0IMR4982(59-AAGACCGCGAAGAGTTTGTC-39), which amplify a WT fragmentof 600 bp and a mutant fragment of 320 bp (Supplemental Fig. 1E).

To generate Rev3L2610F (mutator knock-ins), a second vector wasmade, with a mutation replacing the leucine at position 2610 with phe-nylalanine in exon 23 (Supplemental Fig. 2A). The homologous mutation inyeast reveals a 2- to 3-fold increase in Rev3-mediated mutagenesis (23, 24).Rev3L2610F cells with the L to F mutation that had undergone homolo-gous recombination were identified by Southern hybridization and con-firmed for the mutation by PCR, using primers N29B5PCRF: 59-CTTT-CATGTTCTCCATCAGCGTTTCC-39 and N29B5PCRR: 59-GGTTAGCT-GGGCTACATTCCAATTCATC-39, followed by sequencing with primer:N29B5PCRR: 59-GGTTAGCTGGGCTACATTCCAATTCATC-39 (Sup-plemental Fig. 2B, 2C).

For Rev3L2610F mice, detection of the mutant allele was done by PCRwith primers N29BNeodelF: 59-GTTAAATCAGCTTCCGTTGCAGCA-CT-39 and N29BNeodelR: 59-GCTTCCACAAGTGTTTCCTATGAGAG-TTG-39. The presence of the mutation in mRNA encoding Rev3 in thesemice was confirmed using cDNA as template and using primers F59-ATgAgAgCCCCACAgTgTgTT-39 and R59-CAACCCTAgCACCCCATTT-CT-39 and the nested primers 59-gTCTCgTTTCTATAgCAACTCTg-39 and59-CTggCTTTgTgAACAATgCTATC-39. The nested primers were alsoused for sequencing (Supplemental Fig. 2D). All mice were kept at thepathogen-specific–free animal facility at National Institute of Environ-mental Health Sciences and were maintained in microisolator cages onhardwood bedding, and provided with autoclaved food and reverse-osmosis, deionized water.

Confirmation of Rev3 exon 26 deletion in YFP-expressingB cells by PCR

For detection of WT transcripts, cDNA from Rev3-deleted B cells wasamplified using Taq DNA polymerase (Invitrogen) and using Rev3 junct

25/26 F (59-GCCGTGCATTGAGGTTGGTGATA-39) and Rev3 exon 27 R(59-CTTCAGTTTCACTGGCCTAGGATTAGTA-39), resulting in a 229-bpWT fragment. Mutant Rev3 transcripts were detected using Rev3 junct 25/27 F (59-TGCCGTGCATTGAGTATGTTTGTACTACT-39) and Rev3 exon30 R (59-CAGCGTTTCATACATGTAGCCCACAT-39), resulting in a 191-bp mutant Rev3 transcript (Supplemental Fig. 1F). The RT-PCR of mouseGAPDH was performed with GAPDH F primer (59-ACCACAGTCCAT-GCCATCAC-39) and GAPDH R primer (59-TCCACCACCCTGTTGCT-GTA-39).

Immunizations

Mice 8–12 wk old were immunized by i.p. injection of 100 ml (0.1 ml of1 mg/ml) of 4-hydroxy-3-nitrophenylacetyl (NP) hapten, or 2,4,6-trini-trophenyl hapten, conjugated to chicken g-globulin (NP-chicken gammaglobulin [CGG], N-5055-5, and trinitrophenyl-CGG, T5052-1; BiosearchTechnologies, Novato, CA) prepared in an alum-precipitated suspension.Mice were euthanized and their spleens and serum recovered for analysis14–15 d post immunization.

Preparation of bone marrow B cells and of splenic and Peyer’spatch germinal center B cells

Spleen and bone marrow cell suspensions were created as described inRef. 28. Peyer’s patches were collected from mice at 8, 12, and 26–52 wkof age and cell suspensions generated as described previously (29).

Flow cytometry analysis

Bone marrow progenitor B cells were enriched using allophycocyanin-labeled rat anti-mouse B220/CD45R (clone RA3-6B2). Combined stain-ing with either PE-labeled rat anti-mouse CD117/c-kit (clone ack45),CD25/IL-2Ra-chain (clone Pc61), or IgM (clone R6-60.2) isolated pre-BI,pre-BII, or immature B cells, respectively. Splenic B cells were isolatedusing PE-labeled rat anti-mouse CD19 (1D3) and IgM (clone R6-60.2), plusallophycocyanin-labeled rat anti-mouse B220/CD45R (clone RA3-6B2),all from Pharmingen (San Diego, CA). Activated splenic B cells from NP-immunized mice were stained for allophycocyanin-labeled rat anti-mouseB220/CD45R (clone RA3-6B2) (BD Pharmingen) and biotinylated rat anti-mouse Ig l1, l2, and l3 L chain (clone R26-46) (BD Pharmingen). Bio-tinylated Abs were revealed using streptavidin-PE (Southern Biotechnol-ogy, Birmingham, AL), streptavidin-allophycocyanin (BD Pharmingen), orstreptavidin-tricolor (Caltag Laboratories, Burlingame, CA). Peyer’s patchB cells were enriched by staining with PE-labeled rat anti-mouse B220/CD45R (clone RA3-6B2) and allophycocyanin-labeled anti-mouse Ly-77(clone GL7) from eBioscience (San Diego, CA).

Cell sorting was carried out using a Becton Dickinson FACSVantage SEFlow Cytometer or FACSDiva (Franklin Lakes, NJ). Data were analyzedusing FlowJo (Ashland, OR).

PCR cloning and sequencing of the intronic region downstreamof rearranged V genes and of Vh186.2 regions

RNA from B220+ YFP+ l+ sorted cells was prepared in TRIzol. Onemicrogram of RNAwas used as template for cDNA synthesis in the reversetranscriptase reaction, using a SuperScript II First-Strand Synthesis Sys-tem (RT-PCR; Invitrogen). cDNA (2 ml) was amplified using Phusion DNApolymerase (New England BioLab, Ipswich, MA), with the VH186.2specific primers VH186.2 F (59-AGCAGCCTGGGGGCTGAGCTT-39)and IgH Cg1 R (59-CAGGGGCCAGTGGATAGACAGA-39) and the fol-lowing PCR conditions: 94˚C for 2 min; 35 cycles of 94˚C for 1 min, 60˚Cfor 45 s, and 72˚C for 45 s; and 72˚C for 5 min, resulting in an ∼400-bpproduct. Sorting for l+ cells and amplifying the VH186.2 region enrichesfor Ig sequences that have experienced SHM during the NP response (30).The reverse primer was anchored in the g constant domain to ensureamplification of the Ig HC from B cells likely to have participated in thegerminal center (GC) reaction. The IgH intronic region downstream ofrearranged V genes was amplified by PCR, as previously described (28).

Spleen sectioning and immunohistochemistry

GC morphology was examined via biotin-labeled peanut agglutinin (VectorLaboratories, Burlingame, CA), as previously described (20). Briefly, spleensfrom 8- to 12-wk-old mice were frozen in Tissue-Tek OCT (Sakura, Tor-rance, CA) and sectioned on a Leica CM 3050 S cryostat (6 mm) andaffixed to slides with Rapid Fix solution (Shandon Lipshaw, Pittsburgh,PA). Protein blocking was carried out with an avidin-biotin blocking kit(Vector Laboratories). Incubation with peanut agglutinin was done at a 1/1000 dilution and labeling with a Biogenex streptavidin label. The stain wasdeveloped with diaminobenzidine chromogen (DakoCytomation, Glostrup,

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Denmark), and the slides were counterstained with hematoxylin and visu-alized with a fluorescence microscope.

Lymphocyte proliferation assay

For CFSE analysis, splenic B cells from 8- to 12-wk-old C57BL/6J, CD19+

(B6), Rev3L2610F, and Rev3-deleted mice were CD43 depleted usingCD43 MicroBeads and MACS cell separator magnetic columns, accordingto the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach,Germany). Cells were incubated with 5 nM CFSE for 15 min at 37˚C,following the manufacturer’s instructions (Invitrogen, Molecular Probes,Carlsbad, CA). Cells were washed twice with PBS and incubated in RPMI1640 medium. Cells (106 cells per milliliter) were then stimulated over 4 d,using LPS alone (20 mg/ml; Sigma-Aldrich, St. Louis, MO) or togetherwith IL-4 (25 ng/ml; R&D Systems, Minneapolis, MN). All samples wereanalyzed as duplicates. The absorbance (Ex. 492 nm/Em. 517 nm) wasmeasured using BD Biosciences FACSVantage SE Flow Cytometer. Ac-tivated B cells were determined using flow cytometry with allophyco-cyanin-Cy7–labeled rat anti-mouse B220/CD45R (clone RA3-6B2) andPE-labeled IgM (clone R6-60.2) and IgG1 (clone 15H6), all from BDPharmingen. A total of 105 cells were analyzed for each sample; five miceper group were used.

Statistics

The difference in the distribution of mutational classes (number of cloneswith a particular number of mutations) among groups was examined forsignificance using the Kolmogorv–Smirnov test. The differences in themutation frequency among genotypes in the spleen and Peyer’s patch wereexamined using Mann–Whitney statistic and Kolmogov–Smirnov. Thedifference in the proportion of insertions and deletions, of tandem multi-ples, or of mutations from deoxythymidine among all mutations was testedfor significance among groups, using Fisher’s exact test or x2 statistic. Allprobability values were considered significant if ,0.05.

ResultsTo test the hypothesis that DNA polymerase z directly inducesmutations at Ig loci during SHM, we generated mice with B cell-specific deletion of Rev3 exon 26, which encodes the metalbinding domain, and mice with a mutation in Rev3 that, in yeast,renders the polymerase more mutagenic (23, 24) (SupplementalFigs. 1–3). Rev3 knockout mice were immunized with the NPhapten, and mutations at the Vh186.2 H chain (HC) were analyzedusing cDNA from splenic B cells. To sort the cells, we used B220+,l+, and YFP+B cells to make the cDNA and amplified rearrangedVh186.2 regions that had switched to IgG. This regimen enrichesfor NP-reactive, isotype-switched B cells. YFP is a marker forexpression of cre-recombinase activity and thus strongly corre-lates with Rev3 deletion, as YFP+ cells lacked the floxed exon(Supplemental Figs. 1A, 1B, 1F). Vh186.2 is the Ig HC associatedwith enhanced affinity to NP, and Igs bearing Vh186.2 HCs and llight chains experience a 10-fold increase in affinity when tryp-tophan at position 33 is mutated to leucine (30). Affinity matu-ration to a specific Ag such as NP can profoundly influence themutational pattern and frequency at the relevant Ig loci. Therefore,we also examined the accumulation of mutations in the intronicregion downstream of rearranged J558 Ig HC V genes in B cellsfrom the ileal Peyer’s patches of Rev3-altered mice. Because theregion analyzed is noncoding, mutations here are a more accuraterepresentation of the intrinsic SHM machinery in the absence ofselection.

B cell development in the bone marrow and the spleen appearsnormal in Rev3-altered B cells

Impaired Pol z function did not impact B cell development in thebone marrow or the spleen compared with CD19-driven cre-recombinase transgenics and C57BL/6 controls (Fig. 1A, 1B).More than 90% of B220+ B cells were also YFP+, the marker forRev3 deletion (Fig. 1C, 1D). The number of GC B cells (GL7+) inB cell-specific Rev3 knockout mice was similar to that in C57BL/6

controls. Although .90% of all splenic B cells were YFP+, thepercentage decreased to 56% among GL7+ B cells (Fig. 1B). Thisfinding was accentuated dramatically in Peyer’s patches, whereonly 3% of the GL7+ B cells were YFP+ (Fig. 1B). Splenic orPeyer’s patch B cells from Rev3L2610F mice displayed no de-fects in B cell development or in the number of GC cells (Fig.1A; data not shown).

Rearranged Vh186.2 HCs from Rev3-deleted splenic B cellsdisplayed a reduction in mutation frequency

Following immunization with NP-CGG in alum, YFP+, Rev3-de-leted B cells from Rev3 conditional knockouts that had beencrossed to CD19 promoter-driven cre-recombinase transgenicswere examined for mutations at the rearranged Vh186.2 locus.More than 40% of the clones from these mice had either zero orone mutation compared with ,15% of the clones from eitherC57BL/6 controls or CD19 cre-recombinase transgenics withoutthe floxed Rev3 gene (Fig. 2A, 2B, Supplemental Fig. 4). Thisdistribution of mutational frequencies was significantly differentfrom that in controls (Mann–Whitney and Kolmogorov–Smirnovtest, p = 0.00041 and p = 0.001, respectively). In addition, amongVH186.2 clones with at least one mutation, 46% had the affinity-enhancing mutation W to L at amino acid 33 (Fig. 2B). Re-markably, in some clones this was the only mutation. GCs fromthese mice were indistinguishable from those of control mice (Fig.1B), in terms of both morphology and abundance, 15 d followingimmunization. The fact that GC morphology was intact and thatthe same proportion of clones from these mice acquired the NPaffinity-enhancing mutations as in controls suggests that the de-crease in the mutation frequency is not due to a defect in prolif-eration in these cells (see below).

Altered immune responses to NP in Rev3L2610F mice

Following immunization, most of the unique clones derived fromRev3L2610F mice B cells contained slightly fewer mutations thandid C57BL/6 controls (Fig. 2C). However, the fraction of clonescontaining $10 mutations increased, with a few clones harboring.15 in the rearranged V region (Fig. 2C). Although significanceat 0.05 was not achieved, a trend was noted for an increase in thefraction of Vh186.2 clones with the characteristic W to L changeat amino acid 33 that is associated with increased affinity to NP(.60% of the clones, Fig. 2C). These data may be explained by anincreased rate of SHM due to an enhanced mutagenic potential oractivity of DNA polymerase z. For example, B cells from thesemice may acquire the affinity-enhancing mutations in VH186.2more efficiently, and thus terminate SHM early in the GC reaction.The accumulation of mutations in conditions in which the impactof selection is minimized (see below) helps clarify these data.

Proliferation among Rev3-altered B cells

Given that only 3% of GL7+ B cells in Peyer’s patches and 56% ofGL7+ B cells from the spleen were YFP+ (compared with .90%of all B cells), and to rule out a defect in proliferation from Rev3deficiency, we measured proliferation in Rev3-deleted B cells.Following activation with LPS, Rev3-deleted B cells consistentlyexhibited a significant defect in proliferation compared with CD19transgenic WT controls (Fig. 3A), as LPS only-treated Rev3-de-leted B cells appeared to have an excess of cells in earlier stages ofdivision. This defect was less obvious in LPS plus IL-4–treatedcells when compared with CD19 transgenics with normal Rev3function (Fig. 3B). Rev3L2610F mice activated with LPS and IL-4displayed normal proliferation (Fig. 3B). As CSR is tied to pro-liferation, we examined the switch to IgG1 following activationwith LPS and IL-4 and found that Rev3 knockout cells displayed

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decreased numbers of IgG1+ cells when compared with CD19transgenic controls, whereas Rev3L2610F had levels that weresimilar to those in C57BL/6 controls (Fig. 3C).

Mutation accumulation at the intron downstream of rearrangedV genes in Peyer’s patch B cells of Rev3-deleted B cells isdramatically reduced

Given the profound impact of Ag-driven selection on the frequencyand pattern of SHM, we examined SHM in Rev3-altered mice inthe intronic region downstream of rearranged VDJ segments fromPeyer’s patch B cells. This region has been characterized as atarget of the SHM machinery, with a reported peak mutationfrequency of 0.01–0.15 mutation per base pair (31, 32). Peyer’spatch B cells are chronically stimulated and accumulate mutationsover time, but by 5 mo of age most mice reach their peak mutationfrequency (33). All but one of the clones from mice with B cell-

specific deletion of Rev3 were not mutated, even at 6 mo of age(Fig. 4A). In contrast, .75% of the clones from control mice hadat least one mutation by 6 mo of age. Because only 3% of GL7+

B cells in Peyer’s patches were YFP+ in the Rev3 knockout mice,it is possible that the low mutation frequency of Rev3-deletedB cells places them at a selective disadvantage in the GC envi-ronment of Peyer’s patches, especially given the pressure exertedby microbial flora in the small intestine. Alternatively, a prolifer-ation defect may place these cells at a competitive disadvantage.

Mutation accumulation at the intronic region downstream ofrearranged V genes in Peyer’s patch B cells of Rev3L2610Fmice is increased and the pattern is significantly altered

Rev3L2610F mice were also examined for mutations at the in-tronic region downstream of rearranged V genes. A highly signif-icant increase in the mutation frequency in GC B cells from

FIGURE 1. Normal B cell development in Rev3 knockout and Rev3L2610F mice. (A) B cells from 8- to 12-wk-old mice were sorted and examined by

FACS. Development of Rev3 knockout and Rev3L2610FB (Rev3L2F) cells in bone marrow stage pre-BII (left, B220+ CD25+) and in the immature B cell

stage (middle, B220+ IgM+) is comparable to CD19 promoter-driven cre-recombinase transgenics (heterozygotes) and C57BL/6 controls (not shown). As

the B cells mature and migrate to the spleen (right, B220+ IgM+), normal numbers of mature/resting splenic B cell populations are observed in both the

Rev3 knockout and Rev3L2610F. (B) Unimmunized 12-wk-old mice were NP-CGG injected and their B cells examined 15 d later. The activated splenic GC

B cell population in Rev3 knockout mice undergoing SHM (B220+ GL7+) appears normal in numbers, with 42–56% of this gated population being YFP+.

However, although the percentage of GL7+ B cells in Peyer’s patches appears normal, only a tiny fraction of those (3%) were YFP+. (C) Enrichment for

activated splenic B cells from conditional Rev3 knockout, 15 d after immunization with 150 mg NP-CGG in alum. The postsort (B220+ YFP+ l+) GC

splenic B cell population is greatly enriched for the YFP (marker for Rev3 deletion). The l L chain was selected because it is associated with the Vh186.2

HC for the high-affinity response to the NP hapten. This pure Rev3-null population was used for subsequent RT-PCR and mutation analysis. (D) More than

90% of B220+ B cells in the spleen were YFP+ in Rev3 knockout mice.

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Peyer’s patches of these mice was noted (Fig. 4A). The numberof mutations per base pair in Rev3 knock-in B cells was ∼0.035,which represents nearly a 2-fold increase over the WT controls.Because of the elevated mutation frequency, for analysis, we di-vided the 1.2-kb region into proximal and distal regions, as definedby the distance from the rearranged V region, with the idea that thedistal region may be less saturated and a more accurate assess-ment of the mutational frequency and pattern will be possible. Thisanalysis revealed that the distal region from Rev3L2610F micehad experienced a 3-fold increase in mutation frequency (0.014)over the same region in the WT controls (0.004) that was alsostatistically significant (Fig. 4; Supplemental Fig. 3D). Strikingly,40% of the clones derived from Rev3L2610F mice had accumu-lated .15 independent mutations, with many of those having 30–40 mutations in the 420 bp of the proximal region alone (Fig. 4B).Highly significant changes in the pattern of mutation in B cellsderived from Rev3L2610F mice were also observed. Tandem

mutations, such as adjacent doublets and triplets, were increased3- to 4-fold, and insertions or deletions (mostly +1 or –1 bp) were5-fold higher than in controls (Fig. 4C; Fig. 5). Finally, exami-nation of the nonsaturated sequences (distal region or mutationsfrom 2-mo-old mice) showed that mutations at thymidine baseswere moderately increased in these cells (Fig. 6), suggestingstrand bias by Pol z in SHM at A:T bp.

DiscussionIn this study, we tested the hypothesis that DNA polymerase zplays a direct role in SHM, using mice with altered Rev3 function,the gene encoding the catalytic subunit of DNA polymerase z.Mice with a B cell-specific deletion of Rev3 displayed a significantreduction in the accumulation of mutations at the VH186.2 locusduring the immune response to the NP hapten, and Peyer’s patchB cells from these mice displayed a dramatic reduction in muta-tion frequency at the intronic region downstream of rearranged V

FIGURE 2. The immune response in mice with altered DNA polymerase z function. (A and B) Rev3 knockout B cells experienced a decrease in the

frequency of mutations at Ig V regions during the immune response to NP-CGG, compared with those in WT mice and CD19 transgenics. Close to 40% of

the clones in the Rev3 knockout had zero or one mutation, compared with,10% in the controls. Spleens from Rev3 knockout mice displayed normal GC as

shown by peanut agglutinin staining (white arrows), with 46% of the clones achieving the affinity-enhancing W to L mutation at position 33 of Vh186.2. (C)

Rev3 knock-in mice (Rev3L2610F) had a similar overall distribution of mutational classes to controls but had a higher percentage of clones with more than

nine mutations. These mice displayed normal GC, but also showed an increase in the fraction of Vh186.2 clones with the affinity-enhancing W to L change

(pie chart). The following mice were used for these experiments: C57BL/6 and CD19 promoter-driven cre-recombinase transgenics in the C57BL/6

background, n = 6 mice and 75 unique clones; Rev3 knockout, n = 6 mice and 59 unique clones; Rev3L2610F clones, n = 4 mice and 50 unique clones.

Unique clones were determined by the CDR3 amino acid sequence.

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genes. Indirect effects on SHM, a possibility plaguing previousstudies, are minimized in this study, as B cell-specific deletion ofRev3 eliminated the impact of Rev3 deletion in other cells, such asTh cells or APCs. Furthermore, in our model, Rev3-deleted B cellscould be specifically recovered because cre-recombinase deletionof Rev3 (under control of the CD19 promoter) coincides with YFPexpression (resulting from cre-recombinase–mediated deletion ofa floxed stop codon upstream of the YFP gene). Thus, Rev3-de-leted cells could be isolated based on YFP expression. This iso-lation minimized the impact of incomplete Rev3 deletion, particu-larly in the highly competitive GC environment. Indeed, although.90% of the splenic B cells were YFP+, only 56% of GL7+ Bcells, a GC marker, were YFP+. Peyer’s patch B cells with Rev3deletion displayed almost no mutation accumulation, even at 6 moof age. B cells from Rev3 knockout mice formed morphologicallynormal GCs, yet displayed a proliferation defect following acti-vation with LPS. Among those GC B cells that were mutated, asimilar proportion had acquired the affinity-enhancing tryptophanto leucine change at amino acid 33 of VH186.2 following immu-nization with NP, suggesting that eventual acquisition of high-affinity Abs was not significantly altered in these cells, althoughit is possible that the affinity maturation rate may have been al-tered. These results are similar to those of a previous study usingconditional Rev3 knockout mice, in terms of reduced mutationfrequency, but differ in that smaller GCs from Rev3 knockout micewere reported in the previous study, whereas GCs appeared nor-mal in our study (22). This discrepancy may be due to the differentcre-recombinase transgenic strains used in the studies (CD19versus CD21). For instance, with earlier Rev3 deletion (as theCD19 promoter is active earlier than the CD21 promoter), it ispossible Rev3-deleted cells may have partially compensated, interms of proliferative capacity from Rev3 deficiency, because this

polymerase is not essential for DNA replication. To circumventthe proliferation issue, and to determine if the SHM frequencyreduction observed in both studies is the result of DNA poly-merase z participating in the SHM process rather than entirelyexplained by a defect in proliferation, we examined mutations atIg loci in Rev3 knock-in mice with a hypermutagenic polymerasez and an altered mutational spectra. An alteration in the patternof mutations, consistent with the observed error signature for theRev3Ki polymerase, is strong evidence for a direct involvementand unlikely to be explained by a defect in proliferation.In the Rev3Ki mice, the Rev3L2610F mice, a highly significant

increase in mutation frequency at the intronic region downstreamof rearranged V genes was seen in Peyer’s patch B cells. In yeast,the equivalent leucine to phenylalanine replacement in the Rev3gene increases the error rate of DNA polymerase z ∼2- to 3-foldwithout significantly altering its catalytic activity (23, 24). It isalso associated with an increase in complex mutations (more thanone mutation within a short patch presumed to occur in a singleevent) and with an increase in insertions and deletions. Given thehigh mutation frequency in the Rev3L2610F clones, it was diffi-cult to ascertain which complex mutations originated from anaccumulation of single mutational events or from a single DNApolymerase transaction that yielded complex mutational changes.However, limiting the analysis to tandem doublets or tripletsrevealed a 3-to 4-fold increase in these events. Noted also was a 5-fold increase in insertions and deletions, mostly adding or deletinga single nucleotide. These are similar to the mutational patternobserved in yeast (23, 24). Interestingly, an increase in mutationsfrom thymine was also observed, suggesting Pol z strand biasduring SHM. This is reminiscent of strand bias at A:T bp in Igtemplates from DNA polymerase h-deficient patients and mayimply complementary roles for Pol z and Pol h in SHM. Together,

FIGURE 3. B cell proliferation in Rev3 knockout (Rev3KO) and Rev3L2610F mice. CD43-depleted splenic B cells from 8- to 12-wk-old mice were

isolated and incubated with 5 nM CFSE in PBS to measure cell proliferation. B cells (106 cells/ml) were stimulated over 4 d, using LPS alone (20 mg/ml)

(A) or together with IL-4 (25 ng/ml) (B), to stimulate CSR. In vitro stimulation of B cells was determined at day 0 and day 4 of stimulation. Flow cytometry

was used to determine the B cell proliferation following in vitro stimulation, as depicted by histograms. The numbered gates represent the percentage of B

cells within sequential rounds of cell division. These proliferation peaks were based on 105 events, duplicate samples, and a total of five mice per group. (C)

Percentage of IgG1+ B cells following 4 d of activation with LPS and IL-4.

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the results from both mouse models can best be explained by adirect role for DNA polymerase z in SHM.Although significant changes in both the frequency and the

pattern of SHM in mice with altered Rev3 function were observed,no obvious difference between mutations at G:C and A:T bp wasnoted in either strain: All base pairs experienced either a decreasein Rev3-deleted B cells or an increase in Rev3L2610F B cells(with the exception of a modest increase in mutations from Tbut not from A in the Rev3L2610F mice). The mutation frequency

for Rev3 knockout B cells and the lack of G:C bias are consistentwith data from previously described models of Rev3 deletion,including mice expressing antisense transcripts to Rev3 (20, 22).These results are different from those with human B cells deficientin DNA polymerase h, in which the loss of mutations was pri-marily at A:T bp (10), and suggest that DNA polymerase z par-ticipates in both the G:C and the A:T phase of SHM.On the surface, it appears difficult to reconcile the fact that Pol z

is directly involved in introducing mutations at both G:C and A:T

FIGURE 4. Mutations at the intronic region downstream of rearranged V genes in Peyer’s patch B cells in mice with altered DNA polymerase z function.

The IgH intronic region downstream of rearranged V genes of GC B cells was assessed for accumulation of mutations. Peyer’s patch B220+ GL7+ (and

YFP+ for Rev3 knockout) B lymphocytes were isolated from mice aged 12 or 36 wk: C57BL/6, n = 20 mice and 76 unique clones (with 53% of the mutated

clones from 36-wk-old mice); Rev3L2610F, n = 8 mice and 84 unique clones (with 58% of the mutated clones from 36-wk-old mice); Rev3 knockout, n = 4

mice and 18 unique clones (with 100% of the clones from 36-wk-old mice). Because the rearranged CDR3 was included in the PCR fragment, unique

clones could be assessed, and those differing by more than five mutations were considered different clones, although shared mutations were counted only

once. (A) Mutations per nucleotide among mutated clones were calculated for both the proximal (∼430 bp, beginning immediately downstream of JH4) and

the distal ends of the IgH intronic region downstream of rearranged V genes (region was the last 590 bp of the 1.2-kb PCR fragment). The regions were

analyzed separately to examine mutation pattern and frequency in less saturated regions. In both the proximal and the distal regions, the mutation rate in the

Rev3L2610 F mice is at least twice that observed in controls, and this difference was highly significant. Rev3 knockout clones had an extremely low

mutation frequency that was highly significantly different from controls. All except one of the unique Rev3 knockout clones had no mutation; thus, all were

included in the analysis. (B) The increase in mutation frequency among mutated clones in Rev3L2610F mice was the result of clones with a large number of

mutations: A dramatic increase was noted in the fraction of Rev3L2610F clones with .15 mutations, some having $30 more in the proximal region alone

(p = 0.037). Of the Rev3 knockout clones, 98% were unmutated, even in 6 mo-old-mice, compared with 33 and 57% for the control and the Rev3L2610F

clones, respectively. (C) Tandem doublets and triplets and insertions and deletions were significantly increased in Rev3L2610F mice. The difference in

mutation frequencies was tested for significance using the Mann–Whitney U test, the difference in the distribution of mutations among clones was tested for

significance using the Kolmogorov–Smirnov test, and the difference in the frequency of tandems and indels among all mutations was analyzed using the

Fisher exact test (two-sided p values are given).

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bp with the fact that AID triggers SHM by deaminating cytosinesin both strands. One possibility, however, is that Pol z is involvedin the mutagenic bypass of the abasic sites generated by the re-moval of AID-generated uracils in the DNA of Ig V regions.Continued synthesis past the abasic site would also result inmutations at A:T bp, perhaps in complex with Pol h, and thiswould generate mutations at all base pairs. Pol z is uniquely ef-ficient at extending synthesis past a variety of DNA lesions, in-cluding abasic sites, alone or in complex with REV1, a cofactor

shown to contribute to mutations at AID-mediated abasic sitesin a cell line (34, 35). Furthermore, it has been shown to extendmismatches generated by other TLS polymerases (36, 37). In Polz-deficient B cells, AID-generated abasic sites may be repaired inan error-free fashion, reducing mutations at all base pairs, not justG:C bp. Inefficient recruitment of error-prone DNA polymerasesto compensate for Pol z ablation may occur, explaining why 15%of Rev3-deleted clones derived from splenic B cells had more thansix mutations. Alternatively, Pol z may assemble a targeted

FIGURE 5. Tandem and indel mutations in the proximal HC intronic region. B cells from Rev3L2610F mice (33 unique clones) reveal an increase in

tandem (squares) and indel mutations (+ or 4) when compared with controls (41 unique clones). A similar pattern is seen in the distal region of the

sequences (data not shown). Shared mutations from oligoclonality and single base substitutions are not shown.

FIGURE 6. Mutational pattern of Rev3L2610F

mice reveals a moderate increase in mutations at T

bases, indicating Rev3L2610F-mediated strand

bias. The mutation pattern at the IgH intronic

proximal regions of B cells from young mice is

depicted for controls (A) and Rev3L2610F (B)

mice. The IgH intronic distal region of all controls

(C) and all Rev3L2610F (D) mice B cells are also

shown. The combined increase at thymidine was

statistically significant (x2 test, p = 0.04). The base

composition in the proximal region is 57% A:T

and 43% G:C; for the distal region, it is 61% A:T

and 39% G:C.

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mutasome complex to Ig loci that include AID. This complexwould explain an impact on mutation at all base pairs, but itappears less likely because it would predict loss of all mutations,not just a reduction in the mutation frequency, with Rev3 deletion.In addition, the results using Rev3L2610F mice indicate a role forPol z as a DNA polymerase, not just as a targeting complex.It is interesting that Rev3-deleted B cells appear to be at a dis-

advantage in the Peyer’s patch environment, compared with WTB cells within the same animal. Although close to 90% of B220+

B cells were YFP+ and therefore Rev3 deleted, only 3% of GCB cells in Peyer’s patches expressed YFP. In the spleen, the re-duction was less marked (56% of GL7+ B cells) and is more inagreement with other models of CD19 promoter-driven cre-recombinase deletion (38). This defect may be due to impairedproliferation, as seen with splenic cells, or the result of intensecompetition in the GC environment for Ag binding and increasedaffinity through SHM. A reduced mutation frequency may impactthe rate at which affinity-enhancing mutations are acquired, andthis may place Rev3-deleted B cells in a competitive disadvantagein the ileum, which is chronically stimulated by microbial flora.Interestingly, B cells from Rev3L2610F mice in the spleen weremore likely to have affinity-enhancing mutations to NP hapten (60versus 40% of controls). This fact raises the possibility that theconverse is true: A higher rate of SHM with a mutagenic Pol z isassociated with an enhanced affinity maturation rate for thosecells. Having these novel strains that differ in the ability of theirB cells to undergo SHM will be useful in examining the impact ofSHM rate on the rate of affinity maturation to certain pathogens,especially those associated with specific Abs bearing a largenumber of mutations, as appears to be the case for HIV (39) andinfluenza (40).

AcknowledgmentsWe thank John W. Drake and Dmitry Gordenin for critical reading of the

manuscript, Natasha Clayton and Tiwanda Masinde for assistance with im-

munohistochemistry, Carl Bortner and Maria Sifre for assistance with flow

cytometry, Yingbin Ouyang and Leanne Zhu from Xenogen and Manas Ray

from National Institute of Environmental Health Sciences for chimera gen-

eration, and Scott Jenkins for generation of the Rev3-deleted yeast strain.

DisclosuresThe authors have no financial conflicts of interest.

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