Regulation of the Germinal Center Reaction and Somatic Hypermutation Dynamics by Homologous Recombination

This information is current as Gianna Hirth, Carl-Magnus Svensson, Katrin Böttcher, of September 30, 2021. Steffen Ullrich, Marc Thilo Figge and Berit Jungnickel J Immunol published online 9 August 2019 http://www.jimmunol.org/content/early/2019/08/09/jimmun ol.1900483 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 9, 2019, doi:10.4049/jimmunol.1900483 The Journal of Immunology

Regulation of the Germinal Center Reaction and Somatic Hypermutation Dynamics by Homologous Recombination

Gianna Hirth,* Carl-Magnus Svensson,† Katrin Bo¨ttcher,* Steffen Ullrich,* Marc Thilo Figge,†,‡ and Berit Jungnickel*

During somatic hypermutation (SHM) of Ig genes in germinal center B cells, lesions introduced by activation-induced cytidine deaminase are processed by multiple error-prone repair pathways. Although error-free repair by homologous recombination (HR) is crucial to prevent excessive DNA strand breakage at activation-induced cytidine deaminase off-target genes, its role at the hypermutating Ig locus in the germinal center is unexplored. Using B cell–specific inactivation of the critical HR factor Brca2, we detected decreased proliferation, survival, and thereby class switching of ex vivo–activated B cells. Intriguingly, an HR defect allowed for a germinal center reaction and affinity maturation in vivo, albeit at reduced amounts. Analysis of SHM revealed

that a certain fraction of DNA lesions at C:G bp was indeed repaired in an error-free manner via Brca2 instead of being processed Downloaded from by error-prone translesion polymerases. By applying a novel pseudo-time in silico analysis of mutational processes, we found that the activity of A:T mutagenesis during SHM increased during a germinal center reaction, but this was in part defective in Brca2- deficient mice. These mutation pattern changes in Brca2-deficient B cells were mostly specific for the Ig V region, suggesting a local or time-dependent need for recombination repair to survive high rates of SHM and especially A:T mutagenesis. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/

mmunoglobulin diversification in germinal center B cells is Processing of the U:G lesion caused by AID-mediated cytosine based on a unique combination of targeted introduction of deamination may lead to one of five following possible outcomes I DNA lesions by activation-induced cytidine deaminase (1, 3): 1) during class switch recombination of the Ig C region, (AID), followed by mostly error-prone processing via several of processing to double-strand breaks leads to constant module the major DNA repair pathways of the cell (1). At the same time, deletion and use of downstream modules to produce Abs with damage introduced by AID into several non-Ig genes may be variant effector functions; 2) during Ig gene conversion occur- repaired in an error-free manner, ensuring genome maintenance ring in several farm animals, but not in mice and humans, lesions (2). The molecular basis of this phenomenon is quite elusive, but in the Ig V region trigger HR-based sequence transfers from by guest on September 30, 2021 locus-specific malfunction of a major error-free repair pathway, upstream pseudogenes (4); 3) during somatic hypermutation such as homologous recombination(HR),maybeinvolved. (SHM) of Ig genes (in, e.g., humans and mice), lesions can be processed by a) replication over the uracil, leading to transitions *Department of Cell Biology, Institute of Biochemistry and Biophysics, Faculty at C:G, b) translesion synthesis (TLS) over abasic sites formed of Biological Sciences, Friedrich Schiller University, 07745 Jena, Germany; by uracil excision, allowing also for C:G transversions, or c) † Research Group Applied Systems Biology, Leibniz Institute for Natural Product nonconservative mismatch repair of the U:G pair, triggering A:T Research and Infection Biology, Hans Kno¨ll Institute, 07745 Jena, Germany; and ‡Faculty of Biological Sciences, Friedrich Schiller University, 07743 Jena, Germany mutagenesis via Polh (1); 4) misprocessing of the lesions may ORCID: 0000-0002-9723-9063 (C.-M.S.). lead to chromosomal translocations (5); and finally, 5) base ex- cision repair (BER) may restore the original sequence (6). The Received for publication April 29, 2019. Accepted for publication July 4, 2019. pathways governing the choice between these options are largely This work was supported by grants from the Deutsche Forschungsgemeinschaft (JU2690/4-1, JU2690/1-2) and the Jena School of Microbial Communication enigmatic to date. Most importantly, the question of why error- (all to B.J.), CRC/TR124 FungiNet Projects B4 (to M.T.F.) and C4 (to B.J.), and free processing (e.g., by BER) does not occur at the normal high the Deutsche Krebshilfe (Grant 70112155 to B.J.). efficacyinIggenesisamystery. G.H. designed and performed all experiments, analyzed the data, and wrote the In mammalian cells, lesions that fail to be repaired by BER manuscript. C.-M.S. designed and performed the bioinformatics analysis of the pos- terior probability density function of the relative mutation frequency by Bayes the- before entrance into S phase can then be processed by HR (7). orem and edited the manuscript. K.B. established the mouse experimental methods In fact, in this case the HR pathway becomes essential for and provided expert technical help with the experiments and analyses. S.U. created the prevention of replication collapse, genetic instability, or the Python algorithm for pseudo-time mutation analysis. M.T.F. supervised the bio- informatics analysis, analyzed data, and edited the manuscript. B.J. designed and cell death, as double-strand breaks formed upon encounter of a supervised the entire study, analyzed data, and wrote the manuscript. single-strand break by the replication fork can only be processed Address correspondence and reprint requests to Prof. Berit Jungnickel, Department in an error-free manner by HR (8). A reminiscent scenario has of Cell Biology, Institute of Biochemistry and Biophysics, Faculty of Biological been shown for AID-induced DNA double-strand breaks occur- Sciences, Friedrich Schiller University Jena, Hans Kno¨ll Strasse 2, 07745 Jena, Germany. E-mail address: [email protected] ring in G1 in non-Ig genes (off-target sites) of activated B cells, The online version of this article contains supplemental material. which require HR for resolution in S/G2 (9, 10). Unless com- Abbreviations used in this article: AID, activation-induced cytidine deaminase; BER, pletely resolved in the G1 phase of the cell cycle, remaining base excision repair; CI, confidence interval; HR, homologous recombination; NP, AID-induced lesions in the Ig genes should thus in part also be nitrophenylacetyl; NP-CGG, NP chicken g globulin; PDF, probability density func- processed by HR. However, the profound toxicity shown before tion; TLS, translesion synthesis. for HR defects in AID-expressing cells (10) may raise concerns Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 that the investigation of HR function during SHM in mouse cells,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900483 2 HOMOLOGOUS RECOMBINATION AND THE GERMINAL CENTER which requires a functional germinal center response, might be analysis of light and dark zone germinal center cells, splenic cells were a risky endeavor. stained with a-B220-BUV395 (563793; BD), PNA-FITC, a-CD95-PE, Gene-targeting studies in the chicken DT40 cell system, which is a-CD86-allophycocyanin (561964; BD), a-CXCR4-BV421 (562738; BD), and Fixable Viability Stain 780 (565388; BD). Flow cytometry was an established model for Ig gene conversion (4), have led to an performed on an LSR Fortessa (BD Biosciences) and analyzed in FlowJo intriguing concept of the relationship between HR and SHM. In (FlowJo, LLC). Catalog numbers are given for each Ab. particular, inactivation of HR leads to abolishment of Ig gene Immunization, germinal center cell sorting, and analysis of conversion and a concomitant switch to SHM based on TLS (11). J 4 and pre-Sm mutagenesis We have previously shown that checkpoint signaling via Chk1 H decreases SHM in chicken and human B cells, and our data in Eight- to twelve-wk-old male and female mice were immunized by DT40 cells imply a role of HR regulation via Chk1 in this peritoneal injection of 100 mg of alum-precipitated nitrophenylacetyl (NP) chicken g globulin (NP-CGG; Biosearch Technologies). Blood phenomenon (12). Conceptual application of this model to the samples for ELISA analyses were taken from the cheek 7 and 10 or 7 and mammalian situation is limited until a role for HR in Ig gene 14 d after injection. Splenic B cells were sorted for germinal center cells SHM is shown in vivo. (B220+,PNAhigh, CD95+) in a FACSAria (BD Biosciences) 14 d after To clarify this issue, we generated a conditional knockout of the immunization, followed by genomic DNA isolation and amplification of early recombination factor Brca2 in mouse B cells (13, 14). Brca2 the IgH JH4intron(usingprimersspecificforVH186.2andJH4) or the pre-Sm region. All primers and PCR programs are listed in Supplemental is crucial for the loading of Rad51 onto resected DNA to promote Table I. Gel-purified PCR fragments were excised and cloned into HR at double-strand breaks or stalled replication forks (15). We pGEM-T (Promega). Individual colonies were picked and sequenced by show that ex vivo–activated Brca2-deficient B cells proliferate the Sanger method using the JH4 reverse or the Sm reverse primer. The less and die more and are impaired in class switch recombina- so-obtained sequences had to be inverted to analyze the actual coding Downloaded from strand. Sequence alignments were done in Geneious (Biomatters), and tion, although to a milder extent than observed before in Xrcc2- mutational analyses were performed using the SHMTool (http://shmtool. deficient cells (10). Strikingly, we found that Brca2-deficient montefiore.org/cgi-bin/p1) (16). Whenever clonally identical mutated murine B cells may survive AID expression and form germinal sequences were found, all but one were excluded from the analysis to centers where SHM and affinity maturation do occur. Although rule out the influence of highly abundant B cell clones on the mutational the overall mutation load was lower in Brca2-deficient cells, pattern. pattern analysis showed a significant relative increase in C:G Analysis of non-Ig mutagenesis http://www.jimmunol.org/ transversion mutations but surprisingly also a drop in relative Twenty-seven– to thirty-three–wk-old mice were sacrificed and a single- A:T mutagenesis. In silico analysis of changes in mutation pat- cell suspension of Peyer patches was prepared, which was then sorted for terns upon mutation accumulation indicated that the relative germinal center and nongerminal center cells as described above. Ampli- probability of A:T mutator activity increases over the course of the fication of the Cd83 gene was done from genomic DNA, and the Cd83 germinal center reaction in normal but not Brca2-deficient ger- forward primer was used for Sanger sequencing. The resulting sequences minal center B cells. This increase of A:T mutagenesis seemed to were analyzed as described for JH4 and pre-Sm. be specific for the IgH V region and to pose a serious threat to ELISA genomic stability that is normally counterbalanced by HR. IgG1 titers of activated B cell culture supernatants were determined by guest on September 30, 2021 by coating plates with a-IgG1 (BD Pharmingen) and detecting with Materials and Methods a-IgG1–biotin (BD Pharmingen) using an O-phenylenediamine substrate (Sigma) and purified IgG1 (BD Pharmingen) dilutions as standard. Ab- Mice sorbance was measured at 492 nm after stopping the reaction with 3 N Brca2fl/+ mice on a C57BL/6 background were obtained from P.O. Frappart HCl. Analyses were done in duplicates with three different sample with permission of Dr. J. Jonkers (13). Mb1-cre mice on a C57BL/6 dilutions each. background were obtained from Dr. C. Kosan with permission of For measuring the amount of NP-binding IgG1 Abs in mouse sera, plates M. Reth (14). Brca2fl/flMb1cre/+ and Brca2+/+Mb1cre/+ littermates were were coated with NP3 or NP15 (Biocat), and pooled serum from immunized generated from intercrosses of Brca2fl/+Mb1+/+ and Brca2fl/+Mb1cre/+ mice. mice was used as standard. Serum samples were serially diluted starting Genotyping was performed with the primers and conditions indicated in with 1:100 (day 7), 1:400 (standard), and 1:500 (day 14). Detection was Supplemental Table I. Mice were bred in specific pathogen-free conditions, performed as described above. and all animal experiments were approved by the Thu¨ringer Landesamt fu¨r Verbraucherschutz. For the immunization experiments, five mice per ge- Statistical and bioinformatics analysis notype per addressed question were chosen to meet the requirements of the For mutation pattern analyses, p values were calculated using the two-sided principle for reduction of animal numbers while at the same time mini- x2 test for 2 3 2 contingency tables. When the prerequisites for x2 were mizing variations caused by individual immunizations. No randomization not given, a two-sided Fisher exact test was used instead. For all other or blinding was done. analyses, p values were calculated using a two-sided Student t test with *p # 0.05, **p # 0.01, ***p # 0.001 after checking for homo- or het- B cell isolation and stimulation eroscedasticity by an F-test. Bar graphs show the mean with error bars B cells were isolated from splenic single-cell suspensions of 8–16-wk-old depicting the SD; n refers to the number of mice per genotype. male and female mice using MACS depletion with a-CD43 beads (Mil- The algorithm used for pseudo-timing of mutation accumulation was tenyi Biotec). A total of 2 3 105 cells/ml were cultured in RPMI 1640 implemented in Python. Starting from the fasta file assembly required for (Invitrogen) with 10% FCS (Sigma), 10 mM HEPES (Thermo Fisher the SHMTool, it generates bins of sequences with k = 1, 2, 3, 4, etc. Scientific), 50 mM 2-ME (Sigma), and 0.2 U/ml penicillin/streptomycin mutations, and subsequently analyzes the mutational pattern in these se- (Invitrogen) and stimulated with 1 mg/ml a-CD40 and 20 ng/ml IL-4 (both quences as the total number of each type of base change in the respective eBiosciences) 1 h after seeding. To assess proliferation, B cells were bin, divided by the total amount of bases in the bin for which such a stained with 1 mM CFSE (Invitrogen) prior to seeding and stimulation. change would be possible (frequency of original base in the consensus sequence times number of sequences in the bin). Visualization of the Flow cytometric analyses results was achieved using the Matplotlib library. The relative mutation frequency was denoted w, and for a given number Activated B cells were stained with a-IgG1-PE (550083; BD Pharmingen) of mutations we calculated the posterior probability density function (PDF) or a-IgG1-DyLight405 (409109; BioLegend) when combined with CFSE of w by Bayes theorem as follows: and TO-PRO3 (Invitrogen). Splenic B cell subsets were stained with a-B220-FITC (553088; BD), a-CD3-PE (553064; BD), a-CD21-FITC pðfjDÞ } pðDjfÞpðfÞð1Þ (561769; BD), a-CD23-PE (561773; BD), and DAPI (Sigma). For sort- ing, B cells were stained with a-B220-PerCP (553093; BD), PNA-FITC Where D represents the data (i.e., sequences with k mutations), and the (Vector Laboratories), a-CD95-PE (554258; BD), and DAPI. For the likelihood function is written as follows: The Journal of Immunology 3

Nk pðDjfÞ¼∏ pðmijf;k;S;MÞð2Þ i¼1

In Eq. 2, mi denotes the number of mutations of type M from sequence i BKO originating from either S ¼ Ctrl or S ¼ Brca2 ,andNk is the number of sequences with exactly k mutations. The number of mutations follows a binomial distribution governed by the relative mutation frequency as follows:   k mi ðk2miÞ pðmijf;kÞ¼ f ð1 2 fÞ ð3Þ mi

We chose a flat, uninformative prior,  1if0 f 1 pðfÞ¼ ð4Þ 0 otherwise; which does not bias the value of w. To quantify changes in the relative mutation frequency as a function of the number of overall mutations, we performed linear fits to values drawn from the posterior PDF using Monte Carlo simulation (17, 18). Each fit is parametrized by the slope (aj) and intercept (bj). Values of w were drawn 1000 times and 95% confidence intervals (CIs) for the differences of slope Downloaded from and intercept between Ctrl and Brca2BKO were calculated. Differences in intercept reveal whether the basal relative mutation frequencies were dif- ferent between Ctrl and Brca2BKO, whereas differences in slope indicate differences in acceleration or retardation of the respective mutation. Data and computer code availability

The JH4 sequences presented in this article have been submitted to Gen- Bank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers http://www.jimmunol.org/ MN098330 to MN098764. The Python code for the pseudo-time analysis and the underlying JH4 alignments are available at https://github.com/ noxxi/bioinfo-pseudotiming-somatic-hypermut. The code for the Bayes- ian inference pipeline is available at https://github.com/applied-systems- biology/Bayesian-Analysis-SHM.

Results We inactivated HR specifically in B cells by crossing the previously described Brca2fl/fl mice (13) to Mb1-cre mice (14) and used by guest on September 30, 2021 Brca2fl/flMb1cre/+ (henceforth termed Brca2BKO)andBrca2fl/flMb1+/+ or Brca2+/+Mb1cre/+ (henceforth termed Ctrl) mice for further FIGURE 1. Normal splenic B cell composition in Brca2BKO mice. (A) analyses. As expected for the Mb1-cre driver (14), B cell–specific Schematic representation of the modified Brca2 locus with PCR primers deletion of Brca2 occurred at close to 100% efficiency (Fig. 1A). used for the determination of deletion efficiency in purified B cells. LoxP Despite the reported essential function of HR for normal B cell sites flank exon 11 (approximately 4800 bp), encompassing the BRC re- development (19), FACS analyses revealed that splenic T versus peats responsible for Rad51 binding. Arrows mark the PCR products in- B B cell ratios as well as relative numbers of marginal zone versus dicative of wildtype, floxed, or deleted alleles. ( ) Representative FACS plots and percentages of splenic B and T cells and marginal zone versus follicular cells were not significantly altered in Brca2BKO as follicular B cells in Brca2BKO and Ctrl mice. Cells were pregated on alive compared with Ctrl mice (Fig. 1B). We thus conclude that (DAPI-negative) single cells. Mean and SD determined for five and three BKO Brca2 mice are suitable for the analysis of the influence of mice of each genotype are given, two-sided Student t test. p . 0.5. HR on late B cell maturation events. Stimulation of isolated Brca2BKO and Ctrl splenic B cells with a-CD40/IL-4 to induce AID expression and initiate class switch Brca2BKO B cells was observed when comparing populations with recombination from IgM to IgG1 revealed a substantial defect of the same number of cell divisions (Supplemental Fig. 1C, 1D). Brca2BKO B cells in survival, leading to an increasing loss of Brca2 deficiency thus leads to reduced class switching, suppos- living cells of up to 50% until day 4 after stimulation (Fig. 2A). edly by impairing proliferation and survival of ex vivo–activated This was congruent with a previous study (10) although less B cells, confirming the cytotoxic phenotype of AID expression pronounced. The survival defect was accompanied by decreased in HR-deficient cells (10). proliferation of the cells stimulated with a-CD40/IL-4 (Fig. 2B). We next asked whether Brca2BKO mice are capable of forming CSR is known to be dependent on proliferation. Accordingly, the normal germinal centers and conducting successful affinity mat- appearance of class-switched surface IgG1-positive cells was re- uration despite survival and proliferation defects. Therefore, we duced by half in Brca2BKO B cells (Fig. 2C), as was IgG1 se- immunized Brca2BKO and Ctrl mice with the model Ag NP-CGG cretion into the culture supernatant measured by a-IgG1 ELISA and analyzed the frequency of germinal center cells as well as the (Fig. 2D). As CSR is mechanistically based on nonhomologous distribution into light and dark zone at day 10 and 14 post- end-joining processes, in which Brca2 is not known to play a role, immunization by FACS. PNAhighCD95+ germinal center cells we presumed that the lack of class-switched cells was due to could clearly be observed in the Brca2BKO mice with approxi- the observed proliferation and survival problems. Stimulating mately the same frequency as in Ctrl mice at day 10 (Fig. 3A, 3C) with a-CD40 only, and thereby not inducing proliferation, but with reduced frequency at day 14 (Fig. 3B, 3D, no statistical resulted in significant but milder survival defects in Brca2BKO B cells significance because of one outlier). This implied ongoing de- (Supplemental Fig. 1A, 1B). A mitigated reduction of class-switched pletion of HR-deficient germinal center cells over the time course 4 HOMOLOGOUS RECOMBINATION AND THE GERMINAL CENTER

for the telltale W33L mutation indicative of increased Ab af- finity. Intriguingly, the frequency of this mutation in germinal center cells of Brca2BKO mice reached the level of some of the Ctrl mice (Fig. 3F), implying that the decrease seen in serum high-affinity IgG1 Abs (Fig. 3E) may be due to survival defects during class switching rather than the actual hypermutation and selection processes. To directly analyze the role of HR in somatic hypermutation, we performed sequence analysis of the nonselected JH4intron flanking the V186.2 gene rearrangement used in the NP response from splenic germinal center cells (Fig. 4). Previous studies in DT40 cells with a defect in HR or HR-activating pathways showed an increase of mutagenesis or point mutations (11, 12, 21, 22). However, the overall mutation load in the Brca2BKO B cells was lower than in Ctrl mice with a reduction of the mutation frequency of ∼50% (Fig. 4A, 4B). This coincided with a lower accumulation of highly mutated sequences in Brca2BKO mice (Fig. 4A, 4C). Because the Brca2 defectledtoanin-

creasing loss of cells over time (Figs. 2A, 3C, 3D), the more Downloaded from pronounced reduction in the mutation frequency in the JH4intron may be attributed to the loss of highly mutated clones at later time points of the germinal center reaction. Strikingly, Brca2BKO mice also showed significant differences in the mutation pattern of JH4 sequences compared with Ctrl

mice (Fig. 4B, 4D, Supplemental Fig. 2A). To get an idea about http://www.jimmunol.org/ changes in the different mutator systems described in the model of Neuberger and Di Noia (1), we looked more closely at tran- sitions and transversions at C:G and mutations at A:T (Fig. 4D). FIGURE 2. Reduced survival, proliferation, and class switching in At C:G residues, Brca2BKO mice displayed a relative increase in Brca2BKO B cells. (A) Survival of B cells isolated from spleens upon transversion mutations (Fig. 4D), indicating that TLS over the stimulation with a-CD40 and IL-4 as determined by FACS analysis on abasic site generated by uracil excision (1) is proportionally day 0, day 3, and day 4 (d 0/3/4). Living cells were defined as TO-PRO– increased. Concomitantly and unexpectedly, relative mutagen- negative signals of total events. Mean with SD of five mice per genotype esis at A:T residues was significantly decreased. This differ- is shown. Significance was calculated using a two-sided Student t test. ence also remains significant when the data of the five mice per by guest on September 30, 2021 *p # 0.05, **p # 0.01, ***p # 0.001. (B) Proliferation of purified CFSE- genotype were not compiled (Supplemental Fig. 2B). stained B cells stimulated with a-CD40 and IL-4 measured by FACS on day 1, 3, and 4. Representative experiment with n = 2 mice per genotype. An increase in transversions at C:G, which are mostly caused (C) Percentage of cells with surface IgG1 expression among alive cells at by activity of the TLS polymerase Rev1 (23) upon HR inactiva- the indicated days after stimulation measured by FACS. Cells were pre- tion, would be consistent with observations in the DT40 system gated on single cells. Mean and SD of seven mice per genotype are shown. (11, 12). This implies that a certain fraction of DNA lesions is in (D) IgG1 secretion of stimulated B cells, as determined via a-IgG1 ELISA fact repaired in an error-free manner via HR, thereby preventing in culture supernatants at day 4. Shown is the mean and SD of five mice per the processing of these lesions via TLS. genotype, analyzed in duplicates. At the same time, the observed changes in A:T mutagenesis, which is mediated by noncanonical MMR and TLS Polymerase of the germinal center reaction. Comparing the amount of cen- h (24), were unexpected and could, in fact, not be studied in the troblasts in the dark zone or of centrocytes in the light zone, DT40 system before. Considering the lower amount of highly BKO indicative of cells undergoing high rates of proliferation and mutated JH4 sequences in Brca2 cells (Fig. 4A, 4C), we mutagenesis or selection and differentiation, respectively (20), wondered whether this might be linked to the decreased A:T revealed a normal distribution of germinal center cell subsets in mutagenesis. We first compared the Rev1 and Polh hallmark BKO Brca2 compared with Ctrl mice (Fig. 3A–D). To assess the mutations in lowly and highly mutated JH4 sequences (Fig. 5A). In output of the germinal center reaction, the accumulation of Brca2BKO cells, the relative frequency of G.C and C.G muta- NP-specific Abs in blood was analyzed at day 14 (Fig. 3E). tions, indicative of Rev1 activity, is higher in both cases, implying Brca2BKO mice showed a strongly reduced amount of overall that a preference for inserting transversions instead of transitions (a-NP15) and of high-affinity (a-NP3) IgG1 Abs compared with at C:G occurs throughout the process. Intriguingly, in case of the Ctrl mice. Despite the differences between individual immuni- Polh hallmark mutation A.G (or T.C on the other strand), a zation experiments, the overall decrease in serum NP-specific very different picture emerged: Brca2BKO cells with lowly mu- IgG1 Abs approaches significance. However, the presence of tated sequences have as many or even more mutations caused by NP3-binding high-affinity Abs in Brca2BKO mice implied that Polh than the Ctrl, but in highly mutated sequences, the relative the germinal center response, albeit impaired, is in principle amount of these mutations decreased significantly compared with functional. the Ctrl. To evaluate the ability of Brca2BKO mice to undergo the full Because this finding suggested a differential evolution of affinity maturation program more clearly, we sorted PNAhighCD95+ mutation accumulation, we decided to stage mutational processes germinal center cells and PNAlowCD952 nongerminal center cells in the germinal center reaction on the basis of the sequence data at day 14 postimmunization from five Brca2BKO and five Ctrl mice obtained. B cells entering the germinal center undergo multiple and assessed the selected V186.2 gene implied in the NP response cycles of proliferation, mutation, and selection with an overall The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 3. Germinal center formation and affinity maturation in Brca2BKO mice. (A and B) Representative FACS plots of B220+CD95+PNAhigh ger- minal center cells, CXCR4highCD86low dark zone (DZ), and CXCR4lowCD86high light zone (LZ) cells in spleens of Ctrl and Brca2BKO mice 10 (A) and 14 (B) d after immunization with NP-CGG. Cells are pregated on alive (DAPI-negative) single cells. (C and D) Percentages of germinal center or light and dark zone cells in immunized Ctrl and Brca2BKO mice and nonimmunized (n.i.) Ctrl mice on day 10 (C) and day 14 (D) after immunization. n = 5 (day 10), n = 8 (day 14, among which n = 3 for the DZ/LZ staining). Significance was calculated using a two-sided Student t test. ***p , 0.001. (E) Serum titers of IgG1 Abs specific for NP 14 d after immunization from five mice shown in (D). Binding to NP15 detects most NP-specific Abs, whereas binding to NP3 indicates high affinity. The values represent arbitrary units relative to a pooled serum control of NP-immunized mice, which was defined as 500 U. Each dot represents the mean of technical replicates derived from serially diluted serum of one mouse. There was no detection of NP-specific Abs in n.i. mice in any of the experiments. Significance was calculated using a two-sided Student t test. (F) Frequency of the W33L exchange at day 14 after immunization indicative of affinity maturation in the NP-immunized mice shown in (D). Data are based on a total of 34 and 53 V186.2 sequences of Ctrl and Brca2BKO mice, respectively; each dot represents data acquired from one mouse. mutation rate of ∼1 mutation per V gene sequence per cell di- To more clearly evaluate this idea, the posterior PDF of the vision (25, 26). B cells with sequences harboring more mutations relative mutation frequency, denoted by F, was calculated using have thus undergone more “productive cycles” of mutation in- Bayesian methods (27, 28) (Fig. 5C). The relative mutation sertion, and the history of mutational events can therefore be frequency F describes the probability to observe a certain base traced by sequentially evaluating mutation patterns in sequences change in sequences with k =1,2,3,4,5mutations,thusnow carrying k = 1, 2, 3, 4 or more mutations, establishing an in silico depicting the relative activity of the respective mutator system at pseudo-timing of mutation accumulation. any given step in our pseudo-timing approach, rather than the Using a custom Python algorithm, we visualized mutation result (as in Fig. 5B). Complete data from this simulation are frequencies for each possible base change depending on their shown in Supplemental Fig. 3B, whereas Fig. 5C focuses on the appearance in sequences with certain numbers of mutations (k) Rev1 and Polh hallmark mutations. This analysis revealed that (Fig. 5B, see Supplemental Fig. 3A for every base change). Most for most base changes/mutator systems, the activity is relatively types of base changes showed a relatively steady mutation ac- constant throughout the process of mutation accumulation. For cumulation in sequences from both Ctrl and Brca2BKO B cells, Polh-mediated A.G mutations, an increase in relative activity is whereas the situation for the Polh hallmark mutation A.G(e.g.) seen at later pseudo-timepoints in Ctrl samples, whereas for was strikingly different. Although a steep increase was observed Brca2BKO cells, the activity remains constant at the initial level in Ctrl sequences, in case of Brca2BKO, these base changes were throughout the process. To determine whether there are signifi- inserted with similar efficiency during early productive cycles cant differences in the mutation accumulation curves of Ctrl but then failed to further accumulate. and Brca2BKO clones, we calculated 95% CIs of the slope and 6 HOMOLOGOUS RECOMBINATION AND THE GERMINAL CENTER Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 5. In silico staging and mutation classification in Brca2BKO mice. (A) Relative frequency of Rev1 (G.C, C.G) and Polh (A.G, T.C) hallmark mutations as percentage of a total of 76 (Ctrl) or 122 (Brca2BKO) mutations found in lowly mutated sequences (one and two mutations per sequence) and as percentage of a total of 140 (Ctrl) or 111 (Brca2BKO)

mutations found in highly mutated JH4 sequences (four and five mutations per sequence) compared with all observed base changes; significance was FIGURE 4. Altered SHM in Brca2BKO mice. (A) Mutated sequences calculated using the two-sided x2 or Fisher exact test comparing the absolute detected in the Jh4 intron in germinal center B cells isolated at day 14 post number of a certain base change to all other base changes among (e.g.) NP-CGG immunization from five mice of each genotype. Numbers of lowly mutated sequences. *p # 0.05. (B) In silico pseudo-timing analysis analyzed sequences are given in the middle of the pie charts; the fractions of Rev1- and Polh-mediated mutational events in Ctrl and Brca2BKO show the portion of sequences with 0, 1, 2, etc. mutations. The mutation mice. Mutation pattern of sequences containing k = 1–5 mutations are frequency was determined as mutated bases divided by all analyzed bases shown. The y-axis depicts mutation frequencies of the indicated base among all sequences (or among mutated sequences only, shown in pa- substitution, corrected for the abundance of the original base within the rentheses). Significance was calculated comparing the total number of respective sequences. (C) The posterior PDF of the relative mutation mutated versus unmutated bases of all mice per genotype with a two-sided frequency (w)ofRev1(G.C, C.G) and Polh (A.G, T.C) hallmark x2 test. ***p # 0.001. (B) Absolute numbers of nonunique base substi- mutations in pseudo-time (x-axis) for Ctrl and Brca2BKO as density tutions in the sequences analyzed in (A), compiled for all mice of one maps. The lines show the median fit based on 1000 Monte Carlo genotype. (C) Amount of JH4 intronic sequences with a certain number of simulations. For full details on significant differences between Ctrl and mutations. (D) Relative frequency of transition versus transversion muta- Brca2BKO see Table I. tions at C:G and mutations at C:G versus at A:T among a total of 578 (Ctrl) and 350 (Brca2BKO) mutations. Significance was calculated using a two- 2 sided x test comparing absolute numbers of the indicated types of mu- (see values for differences in intercept [bj]inTableI).ForPolh- tations [shown in (B)] between Ctrl and Brca2BKO (i.e., comparing TS at mediated A.G mutations, in contrast, the fitted slope is signif- C:G versus TV at C:G, or comparing TS at C:G versus all other mutations). icantly steeper in Ctrl than Brca2BKO (see values for differences *p # 0.05, **p # 0.01. No correction for base composition of the J 4 H in slope [aj]inTableI). intron was applied for this figure. To tackle the question why Brca2BKO mice fail to increase the A:T mutator activity at a certain (pseudo-)time point, we decided intercept of linear fits to the relative mutation frequency in to look at AID-induced mutagenesis in other gene loci: the pre-Sm pseudo-time using Monte Carlo simulations based on the pos- region and non-Ig genes targeted by AID like Cd83. Class switch terior PDFs (Table I). No significant difference in initial rela- recombination and thereby mutagenesis of the Sm region is con- tive mutation rate was detected between Ctrl and Brca2BKO sidered to be an early process in the germinal center reaction and The Journal of Immunology 7

Table I. Slope and intercept for accumulation of defined mutations in Ctrl and Brca2BKO sequences

Median Slope Median Intercept Mutation (aCtrl 2 aBrca2BKO) (bCtrl 2 bBrca2BKO) G.C 0.013 (20.025, 0.050) 20.081 (20.215, 0.067) C.G 20.014 (20.049, 0.014) 0.026 (20.063, 0.146) A.G 0.050 (0.004, 0.093) 20.098 (20.250, 0.046) T.C 0.008 (20.033, 0.046) 20.010 (20.135, 0.143) For each mutation type, lines are fitted to values of w generated using Monte Carlo simulations based on the posterior PDFs. Fitted lines are described as fj = ajw + bj,where j indicates if we have Ctrl or Brca2BKO. The table lists the median difference between BKO Ctrl and Brca2 slopes (aj) and intercepts (bj), with 95% CIs calculated using boot- strapping in parentheses. Significant differences are marked in bold. even starts before the germinal center has fully matured (29, 30), whereas SHM, leading to V region mutagenesis, supposedly rea- ches its maximum when the dark zone is established where pro- liferation and AID and Polh expression are at height (20, 31, 32).

The mutation frequency in the pre-Sm region of Brca2BKO splenic Downloaded from germinal center cells was reduced by only ∼22%, and the lack of highly mutated clones was not as evident as among the JH4 se- quences (Fig. 6A, 6D). In contrast to the JH4 intron, no significant changes were detected in the mutation pattern of the pre-Sm re- gion (Fig. 6B), with A:T mutagenesis being generally lower than in JH4 sequences (Figs. 4D, 6B). To analyze the Cd83 gene, which http://www.jimmunol.org/ is a known off-target of AID (2), we isolated genomic DNA from Peyer patch germinal center cells of unimmunized Ctrl and Brca2BKO mice. In this study, Brca2BKO cells had a similar to somewhat higher (not significant) mutation frequency compared with Ctrl cells (Fig. 6C, 6D). The overall very low mutation fre- quency in non-Ig genes, however, did not allow a mutation pattern analysis because only 36 (Ctrl) and 51 (Brca2BKO) nonunique mutations were found in the Cd83 sequences out of which 10 (Ctrl) and 8 (Brca2BKO) were mutations at A:T. by guest on September 30, 2021 We may thus conclude that a deficiency in Brca2 does not affect the mechanism of A:T mutagenesis as such, but rather interferes with the accelerated manifestation of A:T mutations in more highly mutated JH4 sequences. This suggests that HR-deficient cells cannot survive once A:T mutagenesis has reached a certain threshold. The pre-Sm region, in contrast, showed an overall lower A:T mutagenesis than the JH4 intron (Figs. 4D, 6B). Thus, the critical threshold might not yet be exceeded dur- ing Sm mutagenesis. Likewise, non-Ig mutagenesis with even lower A:T mutagenesis did not show any of the effects ob- served in JH4 sequences, suggesting that the increase of the A:T mutator activity and the subsequent challenges for geno- mic stability are specific for the V region and later phases of the germinal center reaction.

Discussion In the current study, we identified Brca2floxMb1cre mice as a model system to study the role of HR on the germinal center reaction. BKO B cells lacking Brca2 showed a profound survival and prolifera- FIGURE 6. Analysis of pre-Sm and Cd83 sequences in Brca2 mice. (A) Mutation frequency of pre-Sm sequences 14 d after NP-CGG immu- tion defect in ex vivo experiments, leading to a reduced amount of nization of four mice per genotype among all sequences (and among mutated class-switched cells. We observed a functional germinal center sequences only, shown in parentheses), *p # 0.05 (two-sided x2 test of mutated BKO reaction in Brca2 mice but with reduced numbers of germi- versus unmutated bases of all Ctrl and all Brca2BKO mice). Numbers of ana- nal center cells as well as affinity-matured Abs. Importantly, we lyzed sequences are given in the middle of the pie charts; the fractions show the showed that error-free repair by HR is active at the hyper- portion of sequences with 0, 1, 2, etc. mutations. (B) Relative mutation fre- mutating V region of the Ig locus. Upon its inactivation, B cells quency as percentage of a total of 193 (Ctrl) and 151 (Brca2BKO)detected showed a higher propensity to process some C:G lesions by mutations among all pre-Sm sequences. No significances were detected in a two- 2 error-prone TLS, leading to a relative increase in C:G trans- sided x test. (C) Mutation frequency of Cd83 sequences from isolated Peyer A versions. Simultaneously, HR was not able to prevent the vast patch germinal center B cells analyzed as in ( ). Mice were 27–33 wk old, three D majority of AID-induced point mutations but is, on the contrary, mice per genotype. ( ) Comparison of mutation frequencies (among all se- quences) in the J 4 intron, the pre-Sm region, and the Cd83 gene for each mouse indispensable for the manifestation of highly mutated B cell H tested. Significance was calculated using a two-sided Student t test. **p # 0.01. 8 HOMOLOGOUS RECOMBINATION AND THE GERMINAL CENTER clones and a supposedly V region–specific increase in A:T of problems in A:T mutagenesis in BRCA2BKO is delayed. mutagenesis. Whether this reflects the timing of high Bcl-6 induction in The lesions introduced by AID, cytidine deaminations pro- mutating B cells (45, 46) which would interfere with checkpoint re- cessed to abasic sites, and single-strand breaks, are among the sponses (47, 48) or rather the onset of dark zone formation, of which most frequent spontaneous changes in our genome, occurring Polh is a signature gene (32), and where proliferation and AID ex- between 100 and 10,000 times per day in each cell of our body. pression is highest (31), will need to be investigated with tools en- The repair pathways dealing with them, BER and HR, are ac- abling the tracing of single mutator system activities in B cells cordingly robust, and their interplay is well described (7); lesions throughout the germinal center response. Further investigations into that are left unprocessed by BER will become targets of HR in the timing of mutational events and potential subsequent pattern replicating cells. Together with a previous study on XRCC1 (6), differences would also be of interest for the identification of germinal our analysis allows for the conclusion that both standard repair center exit time points of (e.g.) memory B cells. pathways for the critical lesions introduced by AID are active at As a general notion, based on our finding of differential hypermutating Ig loci in vivo yet fail to complete error-free pro- evolution of mutagenesis throughout the germinal center re- cessing. Although this may be due to a sheer cellular damage sponse, we would like to point out the importance of consid- overload, such a scenario is unlikely, as it has been shown directly ering proliferation or survival defects when interpreting SHM that the repair defect is locus-specific (2). Accordingly, so must be data, in particular concerning A:T mutagenesis. We also sup- the deregulating mechanism, whatever its nature. port the idea of exploiting the profound problems of HR- Landmark studies for the chicken B cell line DT40 (11, 33, 34) defective cells caused by AID-activity (10) for the treatment have shown that the repair process taking over after HR inacti- of AID-expressing cancer types, as it has already been Downloaded from vation is TLS, leading to transversions at C:G. Although our study approached (49), and would like to contribute as an addition now also shows for murine B cells a relative increase in C:G that specifically the A:T mutagenesis machinery may play an transversions, the effect is not as substantial as in DT40. Chicken important role in AID-induced toxicity under these conditions. cells normally diversify their Ig genes by gene conversion, a process taking place in the chicken’s bursa (35) using HR in cis Acknowledgments and presumably in G1 (36), whereas the error-free HR pathway http://www.jimmunol.org/ We thank J. Jonkers, M. Reth, and E. Hobeika for providing transgenic we have assessed is restricted to S/G2 in mammalian cells when a mouse models, the staff of the animal facility of the Faculty of Biological sister chromatid is available. This, in contrast, confirms that SHM Sciences for expert caretaking, and the FACS facility of the Fritz Lipmann via TLS over abasic sites partially takes place in S/G2, where it Institute for cell sorting. We thank all members of the Jungnickel laboratory competes with HR for the repair of these lesions, a hypothesis that and C. Kosan for critical reading of the manuscript, members of the Kosan has also been suggested by Sharbeen et al. (37). laboratory for sharing expertise and reagents, and A. Schmidt, J. Efremova, In the DT40 system, A:T mutagenesis is essentially absent (38). L. Giebeler, A. Mu¨ller, and P. Gruebner for expert technical assistance. In mice and humans, A:T mutations relying mostly on Polh can be observed (24, 39). Brca2 has been reported to interact Disclosures by guest on September 30, 2021 with Polh during repair synthesis by HR (40), so direct effects The authors have no financial conflicts of interest. of the Brca2BKO on A:T mutagenesis would be possible, pro- vided the A:T mutator were dependent on the HR machinery or replication. This has not been reported so far, although ca- References nonical mismatch repair is tightly associated with replication 1. Di Noia, J. M., and M. S. Neuberger. 2007. Molecular mechanisms of antibody somatic hypermutation. Annu. Rev. Biochem. 76: 1–22. (41). A:T mutagenesis during SHM is instead believed to 2. Liu, M., J. L. Duke, D. J. Richter, C. G. Vinuesa, C. C. Goodnow, happen in the G1 phase only (42). The results of our in silico S. H. Kleinstein, and D. G. Schatz. 2008. Two levels of protection for the B cell analysis also imply that HR is not required for A:T mutagen- genome during somatic hypermutation. Nature 451: 841–845. 3. Rada, C., J. M. 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