Volume 162, Issue 4, Pages 727-737 (August 2015) Plasmodium Infection Promotes Genomic Instability and AID-Dependent B Cell Lymphoma  Davide F. Robbiani, Stephanie Deroubaix, Niklas Feldhahn, Thiago Y. Oliveira, Elsa Callen, Qiao Wang, Mila Jankovic, Israel T. Silva, Philipp C. Rommel, David Bosque, Tom Eisenreich, André Nussenzweig, Michel C. Nussenzweig  Cell  Volume 162, Issue 4, Pages 727-737 (August 2015) DOI: 10.1016/j.cell.2015.07.019 Copyright © 2015 Elsevier Inc. Terms and Conditions

Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 B Cell Responses to Plasmodium Infection (A and B) The total spleen cellularity, the total B cells (A), and the germinal center B cells (B), following Pc infection. The mean with SD is shown; at least five mice were evaluated for each time point. (C) AID expression is confined to germinal center B cells. Representative flow cytometry plots of splenocytes from Plasmodium-infected AIDGFP mice. The top row shows the relative expansion of germinal center (green) over non-germinal center (red gate) B cells over time (gated on B220+). The bottom row shows the expression of AIDGFP in non-B cells (B220−, gray), non-germinal center B cells (B220+CD38+CD95−, red), and germinal center B cells (B220+CD38−CD95+, green). At least three mice were evaluated for each time point. (D) AID protein in malaria GC B cells. Gating strategy (left) and semiquantitative western blot analysis (right) identifying AID in both light zone (LZ) and dark zone (DZ) cells sorted 3 weeks post-inoculation. The triangles indicate 3-fold serial dilution. AID−/− and wild-type (WT) control lanes are from in vitro-activated B cells of the respective genotypes. One of two independent experiments is shown. See also Figure S1. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Translocations in Malaria GC B Cells (A) A schematic diagram of the protocol to identify genomic rearrangements induced by Plasmodium in vivo. (B) The chromosome distribution of rearrangement-associated reads captured by I-SceI breaks on chromosome 15. (C) The profile of rearrangements around the I-SceI site. The dotted lines represent AID-deficient samples. (D) The proportion of genic rearrangements. (E) The frequency of rearrangements at genes with various levels of transcription. The empty bars represent AID-deficient samples. The dashed line represents the expected frequency based on random model. For all, rearrangements from malaria GC B cells are compared to cultured B cells (Klein et al., 2011). See also Figure S2. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 AID-Independent DNA Damage in Regions of DNA Replication-Associated Fragility (A) The observed number of chromosome rearrangements within hotspots of viral integration at early replication fragile sites (ERFSs, red dot), as compared to a random Monte-Carlo simulation (p < 0.001 for both). (B) The number of chromosome rearrangements within common fragile sites (CFSs, red dot), as compared to a random Monte-Carlo simulation. See also Figure S3. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 AID Contributes to Plasmodium-Induced DNA Damage (A) Translocations at the physiologic AID target Igh. The red rectangles indicate the location of AID hotspots, and 4-kb regions at these hotspots are magnified on top, where vertical lines each represent a unique translocation event. The numbers on top indicate the total of translocations at each hotspot region. The rearrangements obtained in cultured B cells retrovirally expressing AID are shown for comparison (Klein et al., 2011). TPKT is the normalized number of translocations per kilobase per 1,000 translocations in the library. (B) Examples of non-Igh hotspots of AID-dependent translocation induced by malaria. A 1-kb region is shown, with each vertical line representing a unique translocation. The numbers on the left indicate the total number of translocations. (C) A mutational analysis of malaria GC B cells DNA by MutPE-seq. For c-myc, two adjacent regions in intron 1 were analyzed. ∗p < 0.000001 for all (one-tailed Student’s t test). See also Figure S4 and Table S1. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 P53 Suppresses and AID Promotes Plasmodium-Induced Lymphoma (A) Survival of Plasmodium-infected mice. All mice are also CD19cre/+. (B) Spleen histology of Plasmodium-infected CD19cre/+p53lox/lox mice. L4 is AID-proficient lymphoma and S2 is AID-deficient benign B cell hyperplasia with marked extramedullary hematopoiesis. (C) Lymphoma versus benign hyperplasia in Plasmodium-infected mice. Lymphoid tissues were evaluated by histology, immunohistochemistry, and flow cytometry. “Benign hyperplasia” indicates mice with splenomegaly, but with normal B cell distribution and B220+ cells confined to follicular areas. “Atypical hyperplasia or early neoplasia” denotes splenomegaly and B220+ cells expanding into the periarteriolar lymphoid sheats (PALS). “Lymphoma” defines abnormal lymphoid tissue architecture and/or dissemination to multiple organs. (D) Extramedullary hematopoiesis in Plasmodium-infected mice. Spleen sections were evaluated for the degree of extramedullary hematopoiesis. See also Figure S5 and Table S2. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Genomic Rearrangements in Plasmodium-Induced Lymphomas (A) Distribution of lymphoma phenotypes in Plasmodium-infected and control uninfected CD19cre/+p53lox/lox mice. (B) Representative M-FISH images of metaphases from Plasmodium-induced CD19cre/+p53lox/lox lymphomas. Arrows point to chromosomes with detectable translocations. (C) Circos diagram of the L23 genome. Red arches represent interchromosomal rearrangements, and green arches intrachromosomal ones. For genic rearrangements, the name of the gene is indicated. Asterisks indicate if the recombined site is a known AID target (red; Hakim et al., 2012; Klein et al., 2011) or within hotspots of viral integration at ERFSs (black; M.J. and I.T.S., unpublished data). See also Figures S6 and S7 and Tables S3, S4, and S5. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S1 Kinetics of Parasitemia and AIDGFP Expression, Related to Figure 1 (A) Parasitemia over time in wild-type mice inoculated with Pc. Left: representative flow cytometry plots showing parasitized erythrocytes (gate). Right: summary plot of parasitemia over time. Mean value with SD is shown; at least five mice were evaluated for each time point. (B) Representative flow cytometry plots of Plasmodium-infected AIDGFP spleens at different time points after inoculation. See also Figure 1C. At least two mice were evaluated for each time point. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S2 In Vitro Validation of the ROSAAIDer and ROSAerISCEI Alleles, Related to Figure 2 (A) Class switch recombination in vitro. ROSAAIDer/+ B cells and controls were stimulated for 4 days with LPS and IL4, in the presence or absence of 4OH-Tamoxifen, prior to flow cytometry for IgG1. The gate indicates IgG1 switched B cells, FSC is forward scatter for cell size. (B) Western blot detection of erISCEI. B cells of the indicated genotypes were cultured for 4 days in the presence of LPS, IL4, and anti-CD180. The erISCEI protein was detected with anti-HA antibodies recognizing this N-terminal tag in erISCEI (arrow). B cells infected by an erISCEI expressing retrovirus were used as positive control. (C) erISCEI induces the recombination between I-SceI target sequences on the same chromosome. The ROSAerISCEI transgene was bred to AID−/− and to the IgHI−96k allele to generate ROSAerISCEI/+IgHI−96k/+AID−/−. The IgHI−96k/+ allele bears two I-SceI sites 96kb apart at the IgH locus on chromosome 12. Recombination between the I-SceI sites is achieved following I-SceI mediated cleavage, and can be detected by PCR using primers adjacent to the sites (see diagram; Bothmer et al., 2010). Shown is an ethidium bromide stained agarose gel with PCR products on cells cultured for four days under the indicated conditions. (D) erISCEI induces the recombination between I-SceI target sequences on different chromosomes. Similar to C, The ROSAerISCEI transgene was bred to AID−/− and to the MycI and IgHI alleles to generate ROSAerISCEI/+MycI/IIgHI/IAID−/−. The MycI and IgHI alleles each bear a single I-SceI site at the c-myc (chromosome 15) and Igh (chromosome 12) loci. Recombination between I-SceI sites can be achieved following I-SceI mediated cleavage, and can be detected by PCR using primers adjacent to the sites (see diagram; Robbiani et al., 2008). Shown is an ethidium bromide stained agarose gel with PCR products on cells cultured for four days in the presence of 1 μM 4OH-Tamoxifen. Recombination between I-SceI sites was confirmed by direct sequencing of the amplicons. (E) Relative mRNA expression for the indicated genes, normalized with Tubulin (Hogenbirk et al., 2013). Numbers indicate the fold difference between samples (in vivo/in vitro). Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S3 Features of Translocations within Hotspots of Viral Integration at ERFS in Malaria GC B Cells, Related to Figure 3 (A) Proportion of genic rearrangements within ERFS. (B) Frequency of rearrangements within ERFS at genes with various levels of transcription. Empty bars represent AID-deficient sample. Dashed line represents the expected frequency based on random model. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S4 Genomic Damage by AID in Malaria Germinal Centers, Related to Figure 4 (A) Mutational analysis by MutPE-seq. Coverage (top) and relative mutation frequency (bottom) at each position for the indicated amplicons. Blue is AID−/−, red is IgκAID transgene. See also Figure 4C. (B) Proportion of genic rearrangements within off-target AID hotspots. (C) Frequency of rearrangements within off-target AID hotspots at genes with various levels of transcription. Dashed line represents the expected frequency based on random model. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S5 Lymphoid Development in AID-Proficient and AID-Deficient CD19cre/+p53lox/lox Mice and Impaired Ability to Control Plasmodium in AID−/− Mice, Related to Figure 5 (A) Flow cytometry of lymphoid tissues shows no substantial developmental differences in CD19cre/+p53lox/lox compared to controls. Two experiments. (B) qPCR on genomic DNA shows efficient deletion of the floxed p53 locus in purified CD19cre/+p53lox/lox B cells (CD19+ fraction). Wild-type control is shown alongside. Mean with SD of two independent experiments. (C) Flow cytometry of lymphoid tissues shows no substantial developmental differences in CD19cre/+p53lox/loxAID−/− compared to controls. Two experiments. (D) Survival of immunodeficient NRG mice inoculated with 5-10 mio CD19cre/+p53lox/loxAID−/− splenocytes from sick mice. At signs of distress mice were sacrificed and analyzed. Splenomegaly was present in all, and lymphoma was confirmed by flow cytometry. (E) Hematologic values in CD19cre/+p53lox/loxAID−/− mice (12-14 months old) are consistent with increased erythrolysis as a consequence of chronic Plasmodium infection. Controls are same age-uninfected mice of the same genotype. (F) Parasitemia over time upon a single dose of Pc administration. Mice with p53lox/lox are also CD19cre/+. Representative of three independent experiments. (G) Hematologic values in representative mice 17 weeks after Pc infection. (H) Splenomegaly in AID-deficient mice 21 weeks upon infection with Pc. Extramedullary hematopoiesis was confirmed histologically (data not shown). (I) Decreased survival in the absence of AID in p53-proficient mice infected with Pc. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S6 Lymphomas in CD19cre/+p53lox/lox Mice, Related to Figure 6 and Table 1 (A) Survival of Plasmodium-infected, SRBC-immunized, and control mice. All mice are also CD19cre/+. (B) Representative flow cytometry plots of CD19cre/+p53lox/lox lymphomas. L4 and L23 are post-GC B cell lymphomas that arose in Plasmodium-infected mice. L22 is a pre-GC B cell lymphoma that developed in uninfected control. Numbers are the percentage of cells within the respective gate. FSC is forward scatter for cell size. (C) Distribution of lymphoma phenotypes in SRBC immunized CD19cre/+p53lox/lox mice. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure S7 Deep Sequencing of Lymphoma L23, Related to Figure 6 (A) Deep sequencing of malaria-associated lymphoma L23. Left: schematic of paired-end library preparation. Middle: ethidium bromide stained agarose gel with sonicated and adaptor-ligated tumor DNA (smear). Right: Agilent Bioanalyzer results of excised and pre-amplified ∼537–575 bp and ∼576–612 bp bands to verify size and purity before paired-end sequencing. The ∼537–575 bp bands were used for deep sequencing on Illumina platform (see Methods for details). (B) Ethidium bromide stained agarose gels with PCR products to confirm and identify translocation breakpoints in L23. Left panel: amplicon generated with primers 5-GTGGAGGTGTATGGGGTGTAGAC-3 and 5- CCTCAGTCACCGTCTCCTCAGGTA-3 to amplify c-myc/Igh of T(12;15). Middle panel (from left to right): amplicon for T(18;3) with primers 5-GCTAGGCCCAAGAATAGCCTC-3 and 5-CAGCTGCGACTGAACCATTG-3; amplicon for T(11;15) with primers 5-CCTAGCCAGTGGACCTTGTC-3 and 5-GAGGATGAGGGGACAGACAG-3; amplicons for T(8;17) with primers 5-GCACCGTGCGTTCCGCGTCTC-3 and 5-GGCTCTGGAGGTATTTAGGG-3 or with primers 5-GCCTTTCTCCCCCAACCCCCC-3 and 5-CAGGAGCTATGAGATCCCGG-3. Right panel: DNA quality control PCR at the 53bp1 locus with primers 5-GTGCCTCATTGTTGGGGAGAG-3 and 5- TGTGTGGTTCACCTTCTCTCTATGG-3. DNAs from tumor L4, wild-type tail (WT), and water (w) are controls. (C) Breakpoint analysis of translocations in lymphoma L23. Fifty nucleotides surrounding each breakpoint (middle) are shown, with the homology to germline sequence (top and bottom) indicated by vertical bars. Microhomologies are yellow; insertions green. Cell 2015 162, 727-737DOI: (10.1016/j.cell.2015.07.019) Copyright © 2015 Elsevier Inc. Terms and Conditions