Table S1. Summary of IgV gene sequence analysis of

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1 Table S1. Summary of IgV gene sequence analysis of lymphoproliferations from CDR-deleted and wild-type mice Animal # Diagnosis V H D H J H V H mut. CDR +/ 4 DLBCL V286 P7inv 3 41 CDR +/+ 51 CLL/SLL V265 DFL CDR +/ 67 CLL/SLL V332 DFL CDR +/+ 114 MBL V235 DSP2.1 1 CDR +/ 117 DLBCL V188 IGHD CDR +/ 147 CLL/SLL V54 DFL * CDR +/ 174 CLL/SLL V313 DSP2.9 1 CDR +/ 179 CLL/SLL V19 DSP2.x 1 CLL/SLL V251 DQ52a.2 4 CDR +/ 181 CLL/SLL V235 DSP2.1 1 CDR fl/fl CD19-Cre 14 CLL/SLL V563 DQ52.a1 2 1 CDR fl/fl CD19-Cre 27 CLL/SLL V12 DFL CDR fl/+ CD19-Cre 47 CLL/SLL V186 DSP2.9 2 CDR fl/fl CD19-Cre 178 CLL/SLL V126 DFL CDR fl/fl CD19-Cre 22 CLL/SLL V332 DFL CDR +/+ CD19-Cre 3 CLL/SLL V364 n.i. 1 6* CDR fl/ CD19-Cre 39 CLL/SLL V54 DSP CDR fl/ CD19-Cre 184 CLL/SLL V163 DFL CLL/SLL V27 DSP * CDR fl/ CD19-Cre 224 CLL/SLL V122 DSP2.9 2 CDR +/ CD19-Cre 225 CLL/SLL V235 DSP2.x 1 CDR fl/ CD19-Cre 34 CLL/SLL V34 DSP2.2 1 CLL/SLL V346 DSP2.x 2 CDR fl/ CD19-Cre 38 CLL/SLL V328 P5 1 CLL/SLL V126 DSP2.2 2 CDR +/ CD19-Cre 328 CLL/SLL V332 P8inv 4 CDR fl/ CD19-Cre 33 CLL/SLL V286 DFL * n.i.: not identifiable *likely to be polymorphisms since BLAST search reveals 1% homology to rearranged IgV genes

2 Table S2. HCDR3 rearrangements of lymphoproliferations from CDR, MDR and mir-15a/16-1-deleted and wild-type mice Animal V H V H N D H N J H J H CDR3 Length Diagnosis CDR +/ #4 V286 SR GVE IL N W 3 9 DLBCL CDR +/ #147 V54 AR C TTVVAT KE NAMDY 4 16 CLL/SLL MDR +/ #29 V175 AR GV TTVVA P YFDY 2 14 MBL CDR +/+ #51 V265 TR R GSSY YYAMDY 4 13 CLL/SLL CDR fl/ CD19-Cre #39 V54 AR S DYR SQY YYAMDY 4 15 CLL/SLL MDR / #232 V249 VK AV YYGNY YYAMDY 4 15 LPL MDR / #143 V183 AR K NYYGSSY EG YYAMDY 4 18 CLL/SLL CDR fl/fl CD19-Cre #27 V12 ARD HYGSSY AWFAY 3 14 CLL/SLL CDR fl/ CD19-Cre #33 V286 AR ERL DYGY E AWFAY 3 15 CLL/SLL CDR +/ #67 V332 AR R DYGSSY WYFDV 1 14 CLL/SLL CDR fl/fl CD19-Cre #22 V332 AR I YYGSSY WYFDV 1 14 CLL/SLL MDR fl/ CD19-Cre #219 V128 YYGSSY WYFDV 1 11 MBL CDR +/ #179-J1 V19 AK E PYYSNY D YWYFDV 1 16 CLL/SLL CDR fl/fl CD19-Cre #178 V126 AR DP YYYGSS L YYFDY 2 16 CLL/SLL MDR fl/+ CD19-Cre #211 V126 AR YYYGSSY YFDY 2 13 MBL MDR +/ #176 V244 TR KI FNDFN NWYFDV 1 15 LPL mir / #189 V227 AR I YYDY YWYFDV 1 13 CLL/SLL CDR +/+ #114 V235 MRY GNY WYFDV 1 11 MBL CDR +/ #181 V235 MRY GNY WYFDV 1 11 CLL/SLL MDR / #138 V153 MR YGNY WYFDV 1 11 CLL/SLL CDR +/ CD19-Cre #225 V235 MRY SNY WYFDV 1 11 CLL/SLL MDR +/ #27 V235 MR YSNY WYFDV 1 11 CLL/SLL mir fl/ CD19-Cre #1 V328 A IYYGNY WYFDV 1 12 CLL/SLL CDR fl/ CD19-Cre #184-J1 V163 AS YYGY WYFDV 1 11 CLL/SLL MDR fl/ CD19Cre #99 V328 AR YYSNY WYFDV 1 12 CLL/SLL mir fl/ CD19Cre #234 V328 AR GEK YSNY WYFDV 1 14 CLL/SLL MDR +/ #175-PB V261 AG DR YGY WYFDV 1 12 CLL/SLL CDR fl/ CD19-Cre #38-J1 V328 AR PS LPY WYFDV 1 12 CLL/SLL CDR +/ #174 V313 AR GVY YDGYY PP FDV 1 15 CLL/SLL MDR fl/ Cd19-Cre #45 V313 AR GGI YDGYY DYFDY 2 16 CLL/SLL mir / #59 V218 AR EGEGD DGYY WYFDV 1 16 CLL/SLL CDR fl/ CD19-Cre #38-J2 V126 AR G YDYD DYFDY 2 12 CLL/SLL CDR +/ #179-J4 V251 ARD P NWD Y YYAMDY 4 14 CLL/SLL MDR +/ #269 V211 AR G YGN S YAMDY 4 12 DLBCL CDR fl/+ CD19-Cre #47 V186 AR IYDGYY YFDY 2 12 CLL/SLL CDR fl/ CD19-Cre #224 V122 ARD DGYY YFDY 2 11 CLL/SLL CDR +/ CD19-Cre #328 V332 AR E EA G YYAMDY 4 12 CLL/SLL mir +/ #4 V332 AR EE TG PY YFDY 2 12 CLL/SLL CDR fl/ CD19-Cre #34-J1 V34 AS HDYD WYFDV 1 11 CLL/SLL MDR fl/ CD19-Cre #197 V227 A SP NWD WYFDV 1 11 MBL

3 MDR +/+ CD19-Cre #212 V186 AR NWD WYFDV 1 1 MBL MDR / #58 V235 M SH NWD G WYFDV 1 12 CLL/SLL mir +/+ #136 V357 AR HEEG NWD M YFDV 1 14 MBL CDR fl/fl CD19-Cre #14 V563 A G NW DFDY 2 8 CLL/SLL CDR +/+ CD19-Cre #3 V364 AR WDFDV 1 7 CLL/SLL CDR +/ #117 V188 LR EDK YDG FDF 2 11 DLBCL MDR / #241 V315 LK KDP YD E YFDV 1 12 DLBCL CDR fl/ CD19-Cre #184-J4 V27 VR D DYD AMDY 4 1 CLL/SLL MDR fl/+ CD19-Cre #215 V386 AR RT NWD EG DY 2 11 CLL/SLL MDR +/ #175-SP V219 AR DF YGDVDV 1 1 B-NHL CDR fl/ CD19-Cre #34-J2 V346 AR DYSN YDY 2 9 CLL/SLL mir / #46 V231 VT FDS DFN Y 2 9 DLBCL MDR fl/ CD19-Cre #246 V262 T EG YYGSS FAY 3 11 CLL/SLL mir +/ CD19-Cre #163 V139 AR SDE G FSH 3 9 DLBCL MDR / #313 V126 AR ME LLW SP FDY 2 12 CLL/SLL mir +/ CD19-Cre #144 V281 AR SEG LR P FAY 3 11 B-NHL MDR +/ #236 V11 AI RP FTY 3 7 DLBCL Order of cases is according to the alignment obtained after hierarchical clustering of all HCDR3 sequences. Cases that showed 8% amino acid sequence homology in the CDR3 to the next neighbor/s are grouped. Of altogether 57 sequences, 46 are of CLL or MBL origin. MDR and mir cases were derived from ref. 22.

4 Figure S1. Generation of conditional and null alleles of the mouse equivalent of human 13q14-CDR (14qC3-CDR) (A1) Generation of the CDR and MDR 5 Tag. Shown from top to bottom: i.) centromeric region of the 14qC3 locus with the presumptive exon 1B of dleu5 indicated; ii.) the targeting vector comprising a TK gene, an egfp, a triple SV4 polya site (tpa), a PGK neo r pa cassette, and loxp and frt sites; arrows denote transcriptional orientation; iii.) the targeted locus. Restriction sites, probes used for detection, and the expected fragments detected by Southern blot analysis are indicated. Correctly targeted ES cell lines were identified by Southern blot analysis using 5 and 3 probes (bottom); in the SpeI-digest, the 8.9 kb and 19.5 kb fragments appear at unequal intensities most likely due to the size differences that can affect DNA transfer during blotting. (A2) Generation of the CDR 3 Tag. 5 Tag-targeted ES cell lines were used for targeting of the 14qC3-telomeric region. Shown from top to bottom: i.) telomeric region of the 14qC3 locus with the last exon of Gucy1B2 indicated; ii.) the targeting vector comprising a TK gene, a PGK hygro r pa cassette, a PGK promoter; and loxp and frt sites; arrows denote transcriptional orientation; iii.) the targeted locus. Restriction sites, probes used for detection, and the expected fragments detected by Southern blot analysis are indicated. Correctly targeted ES cell lines were identified by Southern blot analysis using 5 and 3 probes (bottom). ES cell lines with both centromeric and telomeric targetings on the same chromosome were identified by infecting the lines with Adeno-Cre and monitoring for egfp-expression by flow cytometry. (A3) Generation of a 14qC3-CDR null allele. Shown from top to bottom: i.) the double-targeted locus; ii.) the locus after recombination of the frt-sites; arrows denote transcriptional orientation. Restriction sites, probes used for detection, and the expected fragments detected by Southern blot analysis are indicated. Correct Flp/frt-mediated recombination of the locus obtained by crossing CDR fl/+ mice with transgenic mice that express Flp-recombinase in germ cells was verified by Southern blot analysis (bottom). (A4) Testing the conditional 14qC3-CDR allele for its ability to undergo Cremediated loxp-recombination. Shown from top to bottom: i.) the double-targeted locus; ii.) the locus after recombination of the loxp-sites that activates egfp expression through juxtaposing a PGK promoter; arrows denote transcriptional orientation, the generation of an egfp mrna is indicated. Restriction sites, probes used for detection, and the expected fragments detected by Southern blot analysis are indicated. Correct Cre/loxP-mediated recombination of the locus by infecting ES cells with Adeno-Cre was verified by Southern blot analysis (bottom); in the Spe I- digest, the 7.7 or 8.9 kb on the one hand and the 19.5 kb fragments on the other appear at unequal intensities most likely due to the size differences that can affect DNA transfer during blotting. Figure S2. Identification of double-targeted CDR clones with loxp-sites in cis and characterization of deletion at molecular and functional levels (A) Approach of identifying double-targeted CDR clones with loxp-sites in cis. (B) Deletion strategy with egfp-activation after Cre-mediated recombination PCR detection strategy. Shown from top to bottom: i.) the double-targeted locus; ii.) the locus after recombination of the loxp-sites that activates egfp expression through juxtaposing a PGK promoter; arrows denote transcriptional orientation, the generation of an egfp mrna is indicated; primers used for the detection of the correct Cre/loxP-mediated recombination of the locus are indicated; iii.) identification of double-targeted CDR clones with loxp-sites in cis by PCR; iv.) FACS analysis of an Adeno-Cre treated ES clone with loxp-sites in cis demonstrating that Cre/loxP-mediated recombination leads to egfp expression. (C) PCR and FACS results of subcloned Adeno-Cre

5 treated bulk ES cells of clone CDR-1F9/2B1 demonstrating that egfp expression can be used as readout for the identification of deletion of the CDR allele in cis. Subclones PCR-positive for the deleted but not for the conditional allele expressed egfp, whereas subclones that were PCRpositive for the conditional but not the deleted allele were egfp-negative. A small number of subclones showed a mixture of deleted and non-deleted ES cells, most likely since the Adeno- Cre mediated deletion occurred after subcloning. (D) Southern blot analysis verifying at the DNA-level that subcloned Adeno-Cre treated CDR-1F9/2B1 bulk ES cells that are PCRpositive for the deletion and express egfp show correct Cre/loxP-mediated recombination of the CDR allele. Restriction sites, probes used for detection, and the expected fragments detected by Southern blot analysis are indicated. Subclones identified as carrying a deleted CDR allele show a 7.7 kb band next to the 19.5 kb wild-type band, while subclones identified as non-deleted display the expected 8.9 kb band. Figure S3. Mice with CDR-deletion show normal lymphoid development at a young age (A) No difference in the fractions of splenic B- or T-cell subpopulations in 3-months-old CDR fl/ CD19-Cre, CDR fl/+ CD19-Cre and CD19-Cre mice. Splenic mononuclear cells were stained against surface antigens that define B-cell subpopulations, including naïve B cells (IgM + IgD + ), marginal zone B cells (IgM-only), CD5 + B cells, germinal center (GC) B cells (PNA + ), plasma cells (CD138 + ), and memory B cells (IgG1 + ), as well as CD4 + and CD8 + T cells. Each three to four mice per genotype were analyzed, data are shown as mean ± SD. None of the subpopulations showed significant differences among the genotypes. Figure S4. Mice with heterozygous deletion of the CDR show a trend towards a more aggressive disease course compared to MDR-heterozygous mice Percent event-free survival in the MDR and the CDR +/ cohorts. Mice were followed for duration of 2 months. Events comprised mice that succumbed to illness or those identified as moribund or sick (palpable tumor or visible ascietis) and thus sacrificed. P values between the CDR +/ and wild-type (WT) littermates and the CDR /, CDR +/, and WT littermates are indicated. The number of mice of each genotype is indicated in brackets. Mice shown in Fig.5 A&B on the one hand and C on the other corresponded to different cohorts. CDR +/ mice developed lymphoproliferations with a similar time-point of disease onset and a trend towards a more aggressive disease course compared to that observed in MDR +/ mice (7% of CDR +/ vs. 45% of MDR +/ mice have died at 2 months).

6 Figure S1

7 Figure S2

8 Spleen (* indicates % of B22 + cells) 8 %B *%IgM + IgD + 9 *%IgM-only (from ref. 22) 6 *%PNA + 2 %CD *%IgG1 + B 1 1 *%CD5 + P=.13 6 %CD4 + (of B22 ) 6 %CD8 + (of B22 ) Supplementary Fig. 4 Figure S3 Figure S4