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-cell expansion and lymphomagenesis induced by chronic CD4-signaling is strictly dependent on CD19 Caroline Hojer 1*, Samantha Frankenberger 1*, Lothar J. Strobl 1, Samantha Feicht 1, Kristina Djermanovic 1, Franziska Jagdhuber 1, Cornelia Hömig-Hölzel 1, Uta Ferch 2, Jürgen Ruland 2, Klaus Rajewsky 3, Ursula Zimber-Strobl 1,4 Supplementary Figures Suppl. Figure 1. M cell development is comparable in LMP1/CD4//CD19 +/- and LMP1/CD4//CD19 -/- mice. Suppl. Figure 2. CD4 employs CD19 to mediate survival signals. Suppl. Figure 3. Loss of CD19 abrogates lymphoma development in LMP1/CD4 mice. Suppl. Figure 4. Stimulation of cells with αigm in the presence of Dasatinib. Suppl. Figure 4. Titration of the PI3K inhibitor LY2944 Suppl. Figure 5. Sorafenib does not affect Erk phosphorylation in LMP1/CD4 + cells. Suppl. Figure 5. CD19 expression on different DLCL and HL cell lines. Suppl. Figure 6. CD4 expression in different DLCL cell lines. Suppl. Figure 6. CD19 phosphorylation after stimulation with a CD4 ligand.

* 5 cells 15.9 3 M 2 15.7 6.2 6.7 22 CD19+/- CD19-/- LMP1/CD4// CD19+/- LMP1/CD4// CD19-/- 14±7 13±6 4±2 4±2 46±8 5±4 42±7 6±5 33±4 5±5 33±6 6± CD43 IgM 2±4 19±5 2±3 17±3 17±8 21±8 6±3 2±1 CD19+/- CD19-/- LMP1/CD4// CD19+/- LMP1/CD4// CD19-/- IgD Suppl. Figure 1. M cell development is comparable in LMP1/CD4//CD19 +/- and LMP1/CD4//CD19 -/- mice. () In the bone marrow (M) the total cell numbers were slightly decreased in LMP1/CD4 mice independent of the absence and presence of CD19 in comparison to the controls. Total cell numbers in the different genotypes: cell numbers were calculated by offsetting the total cell number of the bone marrow that was determined by cell counting against the percentage of TO-PRO-3 - cells gated on lymphocytes and 22. Symbols represent data from individual mice and horizontal bars mark the mean values. The numbers indicate the mean value of each data set. () nalysis of the different cell subsets in the bone marrow revealed that mainly the mature recirculating cells (IgM + /IgD + ) and not the developing cells were decreased in LMP1/CD4 mice, whereupon the reduction was stronger in LMP1/CD4//CD19 -/- than in LMP1/CD4//CD19 +/- mice. one marrow preparations were analyzed by flow cytometry for the expression of 22 and CD43 to determine the percentages of pro/early pre- (22 pos CD43 pos ), late pre/immature (22 pos CD43 neg/low ) as well as recirculating cells (CD43 neg, 22 high ). To determine the percentage of recirculating cells (22 high, IgM med, IgD pos ) more precisely an IgM and IgD analysis was performed. Numbers indicate mean percentages and SDs of lymphocyte-gated populations and were calculated from at least five independent experiments.

(%) cells viable 8 7 * 6 * * * 5 4 3 2 1 2 3 4 5 days CD19 +/- - CD19 /- LMP1/CD4//CD19 +/- -/- LMP1/CD4//CD19 nnexin-pe 1 1 1 2 1 3 1 4 CD19 +/+ CD19 +/- CD19 -/-.8 nnexin-pe 1 1 1 2 1 3 1 4.8 nnexin-pe 1 1 1 2 1 3 1 4 9.5 1 2 3 4 7-D 1 2 3 4 7-D 1 2 3 4 7-D Suppl. Figure 2. CD4 employs CD19 to mediate survival signals. () Splenic cells were cultured for five days. Each day the percentages of viable cells were determined by FCS after staining with TO-PRO-3. sterisks indicate significant differences between the CD19-proficient and - deficient LMP1/CD4-expressing cells. () Splenic cells of the indicated genotypes were cultured for four days and percentages of 7-D +, nnexin + cells were determined by using an nnexin staining Kit purchased from D iosciences for FCS analysis.

LMP1CD4//CD19 +/- LMP1CD4//CD19 -/- #492 #684 #848 #445 #685 #384 #494 #687 #49 #738 #626 #794 #795 #942 #627 #943 * + + + + + + + Suppl. Figure 3. Loss of CD19 abrogates lymphoma development in LMP1/CD4 mice. nimals were kept under observation and palpated regularly to detect lymphoma development. Lymphomas were scored by investigating mono-/ oligoclonality by Southern-lot analysis using an IgH specific probe spanning the J H3 and J H4 region. Genomic DN was prepared and digested with EcoRI from splenic cells of LMP1/CD4//CD19 +/- mice that developed neoplasias and of LMP1/CD4//CD19 -/- mice that, if they did not get sick, were analysed with an age of 19 month. The band corresponding to the unrearranged Ig locus (indicated by an asterisk) is detected in each sample since DN was prepared from total splenic cells. dditional bands are indicative for lymphoma development. In the Southern-lot some representative examples of samples displaying lymphoma development are shown. Samples with a mono- or oligoclonal cell population are indicated with an (+).

6 kda 6 kda 44 kda 42 kda 44 kda 42 kda 53/56 kda 56 kda 36 kda nm αigm nm nm nm 5 nm nm 2 nm Dasatinib pkt kt perk1/2 Erk1/2 plyn Lyn GPDH Suppl. Figure 4. Stimulation of cells with a IgM in the presence of Dasatinib. CD19 +/- cells were stimulated for 2min with αigm (15μg/ml; Jackson ImmunoResearch) in the presence of different concentrations of Dasatinib as indicated. s controls unstimulated cells in the absence (nm) and presence of Dasatinib (nm) were used. Phosphorylation of Erk, kt and Lyn in the absence and presence of different inhibitor concentrations was investigated by Western-blotting. GPDH served as loading control. 9 % To-Pro-3 negative 8 7 6 5 4 3 2 1 2 3 4 5 days L/C L/C DMSO CD19 +/- L/C LY 5μM L/C LY μm L/C LY 2μM Suppl. Figure 4. Titration of the PI3K inhibitor LY2944 () Splenic cells from LMP1/CD4 (L/C) mice were cultured for up to five days with different concentrations of the PI3K inhibitor LY2942 (5-2μM). s control, cells from LMP1/CD4 and control mice (CD19 +/- ) were used. dditionally, LMP1/CD4 + cells were cultured in the presence of DMSO, which was used to solve the PI3K inhibitor. Percentages of living cells (TO-PRO-3 - ) were determined by flow cytometry at day, 1, 3 and 5.

ctrl LMP1/CD4 42/44 kda 42/44 kda DMSO LY2942 UO126 Sorafenib 3 μm Sorafenib μm perk1/2 Erk1/2 55 kda Tubulin Suppl. Figure 5. Sorafenib does not affect Erk phosphorylation in LMP1/CD4 + cells. Protein extracts of cells from control (ctrl) and LMP1/CD4/CD19 +/- mice (LMP1/CD4) were prepared after incubation with the depicted inhibitors in concentrations as described in Materials and Methods 1h prior to extract preparation. Western-lots were performed and stained with antibodies against Erk and perk. Tubulin was used as loading control.

5 SU-DHL-6 SU-DHL-4 J 1 2 3 4 5 5 1 2 3 4 1 2 3 4 5 U2932 1 2 3 4 % of max 5 HL1 1 2 3 4 5 TMD8 1 2 3 4 5 RIV 1 2 3 4 5 Oci-Ly3 1 2 3 4 Oci-Ly KM-H2 L428 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 CD19-PC Suppl. Figure 5. CD19 expression on different DLCL and HL cell lines. Histograms showing an overlay of the CD19 expression on the surface of different DLCL cell lines in comparison to an isotype control. The plots are gated on living cells. Description of the cell lines: SU-DHL-6, SU-DHL-4, J: DLCL of the GC subtype. U2932, HL1, TMD8, RIV, Oci-Ly3, Oci-Ly: DLCL cell lines of the C subtype KM-H2 and L428: Hodgkin-Lymphoma cell lines. Origin and confirmation of the cell lines: The cell lines SU-DHL-4 and SU-DHL-6, J, U2932, HL1 were kindly provided by Dr. Gerhard Laux. The identity of these cell lines has been confirmed by DSMZ (raunschweig) in the year 28 by DN-Fingerprinting. fter confirmation of the identity, cell lines were frozen in several vials. One original vial was thawed for the experiment. Cell lines were cultured not longer than 5 months. The cell lines TMD8, RIV Oci-Ly3 and Oci-Ly were kindly provided by Dr. Daniel Krappmann. These cell lines are from the same batch as recently used in the publications by Kloo et al., PNS, 211 and Nagel et al., Cancer Cell, 212. The cell lines are routinely checked by gene expression analysis. ll DLCL were cultured as described by Kloo et al., PNS, 211. The HL cell lines KM- H2 and L428 were purchased from DSMZ in the year 213, expanded, and frozen in several vials. One of these vials was thawed for the experiments.

5 SU-DHL-6 SU-DHL-4 J 1 2 3 4 5 5 1 2 3 4 1 2 3 4 5 U2932 1 2 3 4 % of max 5 HL1 1 2 3 4 5 TMD8 1 2 3 4 5 RIV 1 2 3 4 5 Oci-Ly3 1 2 3 4 Oci-Ly KM-H2 L428 5 1 2 3 4 CD4 ligand h.5 h 1 h 24 h 48 h 5 1 2 3 4 5 1 2 3 4 CD4-PE Suppl. Figure 6. CD4 expression in different DLCL cell lines. Histograms show an overlay of the CD4 expression on the surface of different DLCL cell lines in comparison to an isotype control. The plots are gated on living cells. pcd19 plyn pkt kt perk1/2 Erk1/2 Suppl. Figure 6. CD19 phosphorylation after stimulation with a CD4 ligand. Protein extracts derived from J cells that were stimulated for the indicated time points in the presence of a CD4 ligand (.2μg/ml; Cell Signaling) were investigated by Western-lot and probed with the indicated antibodies. GPDH was used as loading control. GPDH

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