Tumour 2. Tumour 9. Tumour 25 Tumour 26. Tumour 27. t(6;16) doi: /nature06159 SUPPLEMENTARY INFORMATION Supplementary Figure 1

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1 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 1 Tumour Tumour 9 t(6;16) Tumour 5 Tumour 6 Tumour 7 1

2 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 1 Tumour 8 Tumour 9 t(;5) Tumour Tumour 4 Supplementary Figure 1. Spectral karyotype analysis of Pax5 Δ/ progenitor cell tumours. Exponentially growing cells of nine different Pax5 Δ/ tumour cell lines, which were derived from tumours of Cd19-cre Pax5 F/ mice, were treated with colcemid followed by staining of metaphase chromosome spreads as described in Methods. Representative metaphase spreads are shown, indicating that two chromosomal translocations (indicated by red arrows) were detected in tumours 9 [t(6;16)] and 8 [t(;5)]. The fact that the other 7 tumours had a normal diploid karyotype ruled out the possibility that a specific translocation or high genomic instability is required for lymphoma development in Cd19-cre Pax5 F/ mice. Moreover, the re-expression of the Rag1 and Rag genes and thus ongoing V(D)J recombination are unlikely to be involved in development of the progenitor cell lymphomas, as none of the tumours analyzed carried a chromosomal translocation at the Igh locus on mouse chromosome 1.

3 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure a WT pro-b cells Pax5/ pro-b cells Pax5/ Rag/ pro-b cells 1 1 WT pro-b cells Tumour cells Tu-6 Tu- Tu-6 - Igj c Tumour Tu-9 Igh V J558-DJ H H Igk V κ -J κ 5 Igl V λ 1-J λ - Notch1 Tu-1 V H 718-DJ H V κ -J κ 5 V λ 1-J λ 1 - Emb Tu-15 V H J558-DJ H 4 V κ -J κ 5 V λ 1-J λ 1 - Gpr97 Tu-16 V H J558-DJ H 4 V κ -J κ 5 n.d. - Spint Tu-17 V H J558-DJ H V κ -J κ 5 n.d. Tu- V H Q5-DJ H V κ -J κ 5 V λ 1-J λ 1 (oc) - Grap - Blnk Tu-4 Tu-5 V J558-DJ 4 H H V J558-DJ H H n.d. V κ -J κ 5 n.d. V λ 1-J λ 1 - Cd79a Tu-6 V H J558-DJ H n.d. n.d. - Ebf1 Tu-7 V H J558-DJ H 4 V κ -J κ 4 n.d. - Irf4 - Btg1 - Ltb4dh Tu-9 Tu-1 Tu- Tu- V J558-DJ 4 H H V J558-DJ 4 H H V J558-DJ H H V J558-DJ 4 H H V κ -J κ 5 V κ -J κ 4 V κ -J κ 5 V κ -J κ 5 n.d. V λ 1-J λ 1 V λ 1-J λ 1 n.d. 1/6 1 6 in frame out of frame b Pax5 Tumour B cells F Δ Supplementary Figure. Gene expression and V(D)J recombination analyses of Pax5-deficient progenitor cell lymphomas. a, Microarray analysis. A mouse cdna microarray (Delogu et al., Immunity 4, 69-8) was hybridized with a Cy-labelled cdna probe (green colour) prepared from wild-type (WT) pro-b cells in combination with a Cy5-labelled cdna probe (red colour) prepared from the indicated Cd19-cre Pax5 F/ tumour cells or Pax5 / and Pax5 / Rag / pro- B cells. The expression ratios of individual Pax5-repressed and Pax5-activated genes (horizontal bars) are depicted according to the colour scale shown. Selected genes are indicated by their Gene names. b, PCR genotyping. DNA from the lymph node tumours of Cd19-cre Pax5 F/ (9, 1, 16,, 4, 5, 9, 1-) and Cd19-cre Pax5 F/F (15, 17, 6, 7) mice were analyzed by PCR to demonstrate that the floxed (F) Pax5 allele was fully converted to the deleted ( ) Pax5 allele in all tumours. B splenocytes of Pax5 F/ mice were used as control. The Pax5 null () allele is indicated. c, Summary of the V(D)J recombination analysis. The PCR fragments corresponding to the rearrangements of the tumours shown in Fig. 1i were cloned and sequenced to determine the reading frame of the individual rearranged Ig alleles. n.d., not detected; oc, oligoclonal. All microarray data are available at the GEO repository at NCBI ( under the accession numbers GSM198, GSM199, GSM11, GSM111, GSM1574, GSM1575 and GSM176.

4 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure Tumour Controls B T GL Igh J H 4 probe EcoRI GL Igk Jκ5 probe BamHI Supplementary Figure. Monoclonal origin of the Cd19-cre Pax5 F/ progenitor cell lymphomas. DNA of the indicated tumours was digested with EcoRI and analyzed for V(D)J rearrangements at the Igh locus by Southern blotting using a J H 4 probe (1.6-kb HindIII-EcoRI fragment of plasmid JH4.). V κ -J κ rearrangements at the Igk locus were determined by Southern blot analysis of BamHI-digested tumour DNA using a J κ 5 probe (1-kb XbaI-EcoRV fragment of plasmid pbs- JκMAR). Thymocytes (T) served as negative control, whereas B splenocytes (B) were used as a positive control to detect the multiple Igh and Igk rearrangements (indicated by arrowheads) present in the mature B cell population. GL denotes the position of the germline DNA fragment. Each tumour is characterized by the presence of two prominent Igh DNA bands and one or two Igk DNA fragments, demonstrating that the Cd19-cre Pax5 F/ progenitor cell lymphomas are monoclonal in origin. 4

5 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 4 Pax5 Δ/ tumour cell lines Pro-B B / / IgM V H J558-DJCµ V κ -JC κ V λ 1-JC λ Hprt Supplementary Figure 4. Expression of rearranged Ig transcripts in Pax5 Δ/ tumour cell lines. Expression of the rearranged Igh (V H J558-DJC µ ), Igk (V κ -JC κ ) and Igl (V λ 1-JC λ ) transcripts was analyzed by RT-PCR in six in vitro grown Pax5 Δ/ tumour cell lines, which were established from lymphomas of Cd19-cre Pax5 F/ mice. Pax5 / and Pax5 / pro-b cells as well as sorted splenic IgM B cells served as controls. The functionally rearranged Igh allele was efficiently expressed in all tumour cell lines, whereas the rearranged Igk or Igl alleles were either weakly or not at all expressed in 4 of 5 tumours analyzed. 5

6 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 5 a Control 1 5 Bone marrow 1 pro-b pre-b pre-b Anti-IL-7Rα Ab treatment pro-b pre-b pre-b B b 1 5 Spleen Control 1 pro-b imm B T1 1 5 Anti-IL-7Rα Ab treatment 1 pro-b imm B B IgM T Supplementary Figure 5. B cell developmental block upon anti-il-7rα antibody treatment. A 6-week-old CreED- Pax5 F/F Eµ-bcl mouse was intravenously injected every second day with 1 mg of anti-il-7rα antibody during 8 days (5 injections), followed by flow cytometric analysis two days after the last injection. Pro-B, pre-b and immature B cells (highlighted by boxes) were absent in the bone marrow (a) and spleen (b) of the anti-il-7rα Ab-treated mouse in contrast to a non-injected control littermate. T1, transitional 1 B cells. 6

7 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 6 a Spleen After Lin depletion After FACS sorting % 99.4% 5.9% % 1x Lin IgM IgM Lin b Lymph node 1 After MACS FACS sorting 1 1x 96.1% IgM IgM IgM Supplementary Figure 6. FACS purification of mature B cells. a, Isolation of mature IgM IgD high B cells for in vitro deletion of Pax5. Splenocytes of anti-il-7rα Ab-treated CreED- Pax5 F/F Eµ-bcl Ly5. mice were depleted of Lin non-b cells by magnetic cell sorting (MACS) after staining with lineage marker antibodies (CDε, CD4, a, CD11c, CD49b, CD9, Gr1, c-kit, Mac1, TCRβ, Ter119 and Thy1.). Mature B cells were FACS-sorted as Lin IgM IgD high cells that revealed a purity of more than 99% upon flow cytometric reanalysis. b, FACS sorting of in vivo Pax5-deleted mature B cells from the lymph nodes of anti-il-7rα Ab-treated Cd19-cre Pax5 F/- Eµ-bcl Ly5. mice. Lymph node cells were depleted of Lin non-b cells by MACS sorting after staining with lineage marker antibodies (CDε, CD4, a, CD11c, CD49b, Gr1, c-kit, Mac1 and Ter119). As in vivo Pax5 inactivation results in IgD loss and CD5 induction (Horcher et al., Immunity 14, ), Pax5-deficient mature B cells were FACS-sorted as Lin CD5 IgM IgD cells with a purity of more than 95%. The Pax5-deficient CD5 IgM B cells do not express IgD (gate 1) in contrast to the non-deleted CD5 IgM B cells (gate ). 7

8 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 7 Spleen 1% B 6 1 SSC-A IgM Supplementary Figure 7. Homing of injected Pax5-deleted B cells in the spleen. In vitro Pax5- deleted mature B cells were stained with carboxyfluoroscein succinimidyl ester (CFSE) prior to injection of -4x1 6 CFSE-labelled cells into Rag / Ly5.1 mice. One week later, the spleen contained 1% of CFSE cells, which retained the CD19 IgM IgD c-kit cell surface phenotype of mature B cells, suggesting that the conversion of Pax5-deleted mature B cells to the Pax5 mutant phenotype is an inefficient process. For CFSE labelling, the B cells were resuspended at 1 7 cells/ml in PBS,.1% BSA followed by addition of CFSE (5 mm in DMSO) to a final concentration of.5 µm. The cells were incubated at 7ºC for 1 min and washed in PBS prior to intravenous injection into Rag / Ly5.1 mice. 8

9 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 8 a In vitro cultured Pax5 Δ/Δ pro-b cells d In vivo Spleen B B Gr1 7% Ly5. Ly5.1 Ly5. b In vitro macrophage differentiation c Mac1 F4/8 Gr1 B Supplementary Figure 8. Myeloid differentiation of Pax5-deleted mature B cells. a, Surface phenotype of a representative Pax5 Δ/Δ pro-b cell line. Ly5. c-kit B CD19 pro-b cells, which were sorted from the bone marrow of a Rag / Ly5.1 mouse transplanted with in vitro Pax5-deleted mature Ly5. B cells, were cultured on stromal ST cells in the presence of IL-7, SCF and FltL for two weeks prior to flow cytometric analysis. b, c, Macrophage differentiation of Pax5 Δ/Δ Ly5. pro-b cells. Pro-B cells were grown on the M-CSF-producing ST cells in the absence of IL-7, SCF and FltL for 1 days, followed by terminal macrophage differentiation in the presence of recombinant mouse M-CSF (5 ng/ml; R&D) for one week. May-Grünwald-Giemsa staining of cytospin preparations (b) identified macrophages with their characteristic vacuolar morphology, which are shown at lower (top) and higher (bottom) magnification. Flow cytometric analysis (c) of the same in vitro differentiated cells revealed expression of the macrophage markers Mac1 and F4/8 in the absence of Gr1 and B expression (blue lines). The same cells were also stained with an isotype control antibody (red lines). d, Presence of myeloid Ly5. Mac1 Gr1 low/ cells in the spleen of Rag / Ly5.1 mice, which were transplanted with in vitro Pax5-deleted mature B cells 8 weeks before flow cytometric analysis. The expression of Ly5. is shown for the gated Mac1 Gr1 low/ cell population. 9

10 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 9 a In vitro deletion 6 weeks 8 weeks 8 weeks Ly5. Ly5. Ly5. 8 weeks 8 weeks 8 weeks Ly5. Ly5. Ly5. 8 weeks 8 weeks (LN) 1 weeks Ly5. Ly5. Ly5. 1 weeks 14 weeks weeks Ly5. Ly5. Ly5. b In vivo deletion 9 weeks (LN) weeks (LN) 6 6 Ly5. Ly5. Supplementary Figure 9. Reconstitution of T lymphopoiesis by Pax5-deleted mature B cells. Rag / Ly5.1 recipient mice were transplanted with in vitro Pax5-deleted (a) or in vivo Pax5- deleted (b) mature Ly5. B cells. T cell development of all transplanted mice (in addition to the two mice shown in Fig. ) was analyzed by flow cytometry. The expression of CD4 and is shown for the donor-derived Ly5. thymocytes together with the time of analysis after transplantation. All mice were transplanted with Pax5-deleted splenic B cells except for three mice, which were injected with Pax5-deleted B cells prepared from lymph nodes (LN). 1

11 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 1 Mice D β -J β In vitro deletion Ly5. DP cells B T Control In vivo deletion ES Ly5. DP cells 1 GL V β -DJ β V β 4-DJ β V β 5-DJ β V β 8-DJ β V β 14-DJ β Supplementary Figure 1. Polyclonal Tcrb rearrangements in B cell-derived DP thymocytes. Ly5. CD4 (DP) thymocytes were sorted 8-1 weeks after transplantation of Rag / Ly5.1 recipient mice with in vitro or in vivo Pax5-deleted mature B cells. The same sorted Ly5. DP thymocytes, which were analyzed for Ig gene recombination in Fig., were used for PCR amplification and subsequent Southern blot detection of the indicated Tcrb gene rearrangements. Wild-type B splenocytes (B), DP thymocytes (T) and ES cells were used as controls. GL denotes the position of the germline PCR product, and numbers indicate the different rearrangements involving the J β.1 to J β.7 gene segments. These data demonstrate that the B cell-derived DP thymocytes contain polyclonal Tcrb rearrangements like wild-type DP thymocytes. 11

12 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 11 a Ex vivo sorted Ly5. pro-b cells In vitro cultured Ly5. pro-b Mice B cells V H J V κ -J κ 4 5 V λ 1-J λ Pax5 F X Δ b Mice Ly5. Pax5 Δ/Δ pro-b cell lines Pro-B B / / IgM V H J558-DJCµ V H 718-DJCµ V H 69-DJCµ Hprt Supplementary Figure 11. Ig gene rearrangements in dedifferentiated Pax5 Δ/Δ pro-b cells. a, PCR analysis. V H -DJ H, V κ -J κ and V λ 1-J λ rearrangements as well as the Pax5 genotype were determined by PCR analysis of in vitro cultured Pax5 Δ/Δ Ly5. pro-b cells and ex vivo sorted Ly5. B c-kit CD19 cells from the bone marrow of Rag / Ly5.1 mice transplanted with in vitro Pax5-deleted mature B cells. B splenocytes from a Pax5 F/F mouse were used as a control to detect all Igh and Igk rearrangements to the J H 1 to J H 4 or J κ 1 to J κ 5 gene segments (indicated by numbers), respectively. F, floxed;, deleted; X, PCR artefact. b, RT-PCR analysis. The expression of rearranged Igh gene transcripts was analyzed by RT-PCR in six in vitro cultured Pax5 Δ/Δ Ly5. pro-b cells cell lines. Pax5 / and Pax5 / pro-b cells as well as sorted splenic IgM B cells served as controls. 1

13 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION 1

14 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 1. Dedifferentiated Pax5 Δ/Δ pro-b cells restore T cell development in Rag / cγ / mice. a, Schematic diagram of the serial reconstitution experiment. Sublethally irradiated Rag / Ly5.1 recipient mice were transplanted with in vitro Pax5-deleted mature B cells isolated from Ly5. HK b donor mice of the Cd19-cre Pax5 F/ Eµ-bcl genotype. After 6-8 weeks, pro-b cells of donor origin were FACS-sorted as Ly5. c-kit B CD19 cells from the bone marrow of transplanted mice and were cultured for only two weeks in the presence of ST cells and the cytokines IL-7, FltL and SCF followed by defining the Pax5 genotype and Ig recombination status as shown in Supplementary Fig. 11. In vitro cultured, dedifferentiated Ly5. HK b pro-b cells of the reconstituted mice 1 or 5 were subsequently injected into sublethally irradiated Ly5. HK d Rag / cγ / mice followed by FACS analysis of the bone marrow, thymus and spleen of the transplanted mice after 4 weeks. b-d, Flow cytometric analysis of reconstituted mice. After the last transplantation, HK b c-kit B CD19 cells of donor origin were detected in and therefore homed to the bone marrow (b), from where they were seeding the thymus and restored T cell development in the Rag / cγ / mice (c). The pro-b cell-derived CD4 and SP T cells subsequently emigrated from the thymus and were detected as peripheral T cells in the spleen of reconstituted Rag / cγ / mice (d). The experiment shown in panels b-d was obtained with dedifferentiated Pax5 Δ/Δ pro-b cells of the reconstituted mouse 5 (see also Supplementary Fig. 11). Moreover, the FACS data shown are representative of 5 different mice reconstituted with dedifferentiated Pax5 Δ/Δ pro-b cells of the transplanted mice 1 or 5. In summary, the data of these serial transplantation experiments demonstrate (i) that mature B cells following Pax5 deletion dedifferentiate to Pax5 Δ/Δ pro-b cells and (ii) that these dedifferentiated Pax5-deficient pro-b cells reconstitute thymopoiesis in a T cell-deficient mouse. Hence, these experimental results confirm our conclusion that Pax5 inactivation allows mature B cells to develop in vivo into functional T cells via dedifferentiation to uncommitted progenitor cells. 14

15 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 1 a b Rag / 4 1 Rag / Reconst. Rag / Reconst. Rag / TCRβ CD4 Supplementary Figure 1. Normal proliferation, cytokine production and Foxp expression of B cell-derived T cells. a, Normal in vitro proliferation of CD4 Ly5. DP thymocytes, which were reconstituted by transplantation of Rag / Ly5.1 mice with in vitro Pax5-deleted mature B cells. Wild-type and reconstituted (reconst.) DP thymocytes were stimulated in vitro with anti-cdε and anti-cd8 antibodies for six days, and H-thymidine was added during the last 4 hours to measure its incorporation into DNA. The data represent the average ( s.d.) incorporated radioactivity (cpm) determined in four independent experiments. b, Normal TNFα, IL- and Foxp expression of reconstituted thymocytes. TNFα and IL- expression was measured by intracellular staining after a 6-h treatment with PMA and ionomycin in the presence of brefeldin A. As shown by Foxp expression, normal numbers of regulatory T cells (Foxp CD4 ) were generated in the reconstituted mice. 15

16 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Figure 14 a Tail DNA FACS-sorted splenocytes from reconstituted Tcra / mice Sorted B cells for injection into Tcra / mice WT Tcra /Reconst. Tcra CD4 SP SP OHT Pax5 - Floxed - WT - Deleted Tcra - Mutant - WT b Reconstituted Tcra / mice FACS-sorted splenocytes CD4 SP SP FACS-sorted thymocytes CD4 SP SP Sorted B cells for injection into Tcra / mice OHT T GL D β -J β V β 5-DJ β V β 8-DJ β V β 14-DJ β Supplementary Figure 14. B cell-mediated reconstitution of peripheral T cells in Tcra / mice. a, Genotyping. Tcra / mice were transplanted with in vitro Pax5-deleted mature B cells as indicated in Fig. 4a. The wild-type (WT) and mutant alleles of the Pax5 and Tcra genes were identified by PCR in the sorted in vitro Pax5-deleted mature B cells used for transplantation, in tail DNA from wild-type, Tcra / and reconstituted (reconst.) Tcra / mice as well as in FACS-sorted splenic SP T cells from reconstituted Tcra / mice. The PCR genotyping data unequivocally demonstrate that the reconstituted splenic T cells were derived from in vitro Pax5-deleted mature B cells, as they carried only deleted Pax5 and wild-type Tcra alleles. b, Tcrb recombination. FACS-sorted SP T cells from the spleen and thymus of reconstituted Tcra / mice were analyzed by PCR for the presence of the indicated D β -J β and V β -DJ β rearrangements. Wild-type CD4 thymocytes (T) were used as positive control. GL denotes the position of the germline PCR product, while the numbers indicate rearrangements involving the J β.1 to J β.7 gene segments. The presence of multiple rearrangements demonstrates that the B cell-derived SP T cells were characterized by a polyclonal TCR repertoire like wild-type T cells. 16

17 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION a Supplementary Table 1: Histological analysis of Cd19-cre Pax5 F/ tumour mice Mouse Lymph nodes Spleen Thymus Liver Heart Lung Kidney Uterus Testis Intestine Salivary gl. Muscle Tu-1 Tu- Tu- Tu-4 Tu-5 Tu-6 Tu-7 Tu-8 Tu-9 Tu-1 Tu-11 Tu-1 Tu-1 Tu--15 Tu-16 Tu-17 Tu-18 Tu-19 Tu- Tu-1 Tu- Tu- Tu-4 Tu-4 Tu-4 b Transplantation experiments Mouse Lymph nodes Spleen Thymus Liver Heart Lung Kidney Uterus Testis Intestine Salivary gl. Muscle Tu-5- Tu-5- Tu-5-4 Tu-5-5 Tu-5-9 Tu-5-1 Tu-5- Tu-5-7 Tu-5-8 Tu-5-1 Tu-5-11 Tu-5-1 Tu-6- Tu-6-4 Tu-6-5 Tu-6-6 Tu

18 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Table 1. Histological analysis of Cd19-cre Pax5 F/ tumour mice. a, The indicated organs were dissected from Cd19-cre Pax5 F/ tumour mice and analyzed for lymphoma cell infiltration by histological analysis. The presence () or absence () of infiltrating tumour cells in the different organs is indicated. No entry indicates that the corresponding organ of the tumour mouse was not analyzed. b, Transplantation experiments. Cells of the primary tumours Tu-5, Tu- 5 and Tu-6 were culture in vitro for 1- days prior to intravenous injection into syngeneic wild-type mice (1 7 cells/mouse). Secondary tumours with multiorgan infiltration developed in the recipient mice within 4 weeks after transplantation and were designated according to the primary tumour and injected recipient mouse (i.e. Tu-5- corresponding to tumour Tu-5 injected into recipient mouse ). Supplementary Table Surface protein B cells (Δ/) B cells (Δ/) Tumor (Δ/) Surface protein B cells (Δ/) B cells (Δ/) Tumor (Δ/) CD19 B CD1 Igµ CD Igκ CD Igλ CD4 CD5 CD7 CD9 IgD pre-bcr MHCII c-kit IL-7Rα Flt Supplementary Table. Immunophenotype of Cd19-cre Pax5 F/ lymphomas. The expression of the indicated cell surface proteins on mature B cells and tumour cells was examined by flow cytometric analysis of lymph node cells. The expression pattern is summarized for control mature B cells (Δ/) of Cd19-cre Pax5 F/ mice, for Pax5-deficient mature B cells (Δ/-) of young Cd19- cre Pax5 F/ mice before lymphoma development and for tumour cells (Δ/-) of older Cd19-cre Pax5 F/ mice. The expression () or absence () of the different cell surface proteins is indicated, while an arrow denotes the down-regulation of protein expression in Pax5-deficient (Δ/-) mature B cells (Horcher et al., Immunity 14, ). 18

19 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Table reconstituted mice V H J558-DJ H 7 V H 718-DJ H V κ -J κ 8 V λ 1-J λ 8 cloned fragments different sequences sequences / mouse reconstituted mice cloned fragments different sequences sequences / mouse Supplementary Table. Oligoclonality of Ig rearrangements in B cell-derived T cells. The different PCR fragments of the Ig gene rearrangements detected in DP thymocytes of reconstituted Rag / mice (Fig. a, b) were cloned and sequenced to determine how many different rearrangements were present in these PCR fragments. As the number of B cells giving rise to T cell development is best approximated by the sum of the different Igk and Igl rearrangements, we estimated that on average 14 in vitro Pax5-deleted and in vivo Pax5-deleted B cells contributed to T cell reconstitution in Rag / mice. In this context, it is worth noting that 6-fold less in vivo Pax5- deleted B cells relative to in vitro Pax5-deleted B cells were used on average for transplantation of Rag / Ly5.1 mice. 19

20 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Table 4: Primers used for PCR analysis of Ig and Tcrb rearrangements Sense oligonucleotides V H J558 CGAGCTCTCCARCACAGCCTWCATGCARCTCARC V H 718 CGGTACCAAGAASAMCCTGTWCCTGCAAATGASC Igh locus V H Q5 CGGTACCAGACTGARCATCASCAAGGACAAYTCC V H Gam.8 CAAGGGACGGTTTGCCTTCTCTTTGGAA V H 69 KCYYTGAAGAGCCRRCTCACAATCTCC IgL loci Tcrb locus V κ V λ 1 D β V β V β 4 V β 5.1 V β 8 V β 14 GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC GCCATTTCCCCAGGCTGTTGTGACTCAGG GTAGGCACCTGTGGGGAAGAAACT GGGTCACTGATACGGAGCTG GGACAATCAGACTGCCTCAAGT GTCCAACAGTTTGATGACTATCAC GATGACATCATCAGGTTTTGTC CTTCTACCTCTGTGCCTGGAGT Antisense oligonucleotides Igh locus J H 4 TCTCAGCCGGCTCCCTCAGGG IgL loci J κ 5 ATGCGACGTCAACTGATAATGAGCCCTCTCC J λ 1, ACTCACCTAGGACAGTCAGCTTGGTTCC Tcrb locus J β TGAGAGCTGTCTCCTACTATCGATT Southern probe PCR product using D β and Jβ primers K: G or T M: A or C S: C or G R: A or G W: A or T Y: C or T

21 doi: 1.18/nature6159 SUPPLEMENTARY INFORMATION Supplementary Table 5: Primer used for RT-PCR analysis of rearranged Ig transcripts 5 Oligonucleotides Oligonucleotides V H J558-DJC µ CGAGCTCTCCARCACAGCCTWCATGCARCTCARC ATGCAGATCTCTGTTTTTGCCTCC V H 718-DJC µ CGGTACCAAGAASAMCCTGTWCCTGCAAATGASC ATGCAGATCTCTGTTTTTGCCTCC V H 69-DJC µ KCYYTGAAGAGCCRRCTCACAATCTCC ATGCAGATCTCTGTTTTTGCCTCC V κ -JC κ GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC TTGGTCAACGTGAGGGTGCTG V λ 1-JC λ CGCGAATTCTCAGGCTCCCTGATTGGAGACAAGG GACCTAGGAACAGTCAGCACGGG Hprt GGGGGCTATAAGTTCTTTGC TCCAACACTTCGAGAGGTCC K: G or T M: A or C S: C or G R: A or G W: A or T Y: C or T 1