4.1. Mice

Experiments were conducted under the approval of the Austrian animal ethics committee (BMWF 66.012/0009-II/3b/2012 and TGV/52/11-2012, BMBWF-66.012/0002-V/3b/2018). CLL tumors were originally derived from the Tcl1 mouse model and further adoptively transferred into WT mice (C57BL/6 N/J), OT1 (C57BL/6-Tg(TcraTcrb) [15], OT2 (C.Cg-Tg(DO11.10)10Dlo/J) [28] mice, and OT1/OT2 mice harboring a deletion of the third exome of the Rag2 gene (OT1/OT2 Rag) (OT1 Rag: B6.Cg-Rag2tm1Fwa Tg(TcraTcrb)1100Mjb/Tac, MGI:4838647, OT2 Rag: B6.Cg-Rag2tm1Fwa Tg(TcraTcrb)425Cbn/Tac, MGI:4838759). All mouse models possess the same genetic background of C57BL/6 N/J. After sacrificing mice, spleen was removed and homogenized for flow cytometry analysis, BCR analysis or frozen for subsequent FACS sorting or transplantation. CLL tumors were transplanted by intraperitoneal injection of splenocytes including CLL cells. Injected cell numbers varied according to tumor and transplantation experiment: in the preceding two transplantation experiments 4 × 10⁶ splenocytes of tumor 658, 5 × 10⁶ splenocytes of tumor C755 and 1.425 × 106 splenocytes of tumor 703 were injected separately in WT, OT1, and OT2 mice (Figure 1A). For the subsequent transplantation experiment, CLL cells derived from the spleen of the WT mice 370 and 371 (transplanted in the previous experiment using tumor 703) were untouched. MACS purified (Pan B cell isolation Kit, 130-104-443, Miltenyi Biotech, Bergisch Gladbach, Germany) via an antibody cocktail (CD3ε, CD4, CD8a, CD49b, Gr-1, and terr119) targeting non-B/non-CLL cells and transplanted individually into WT, OT1, OT2, OT1 Rag, and OT2 Rag mice (tumor 370 1.725 × 10⁶ cells; tumor 371 1.6 × 10⁶ cells). Additionally, healthy B cells of the untransplanted WT mouse 483 were purified in the same manner and transplanted into OT1 Rag mice as a control (1.48 × 10⁶ cells) (Figure 3A). Tumor load was checked weekly via flow cytometry measurements of venous blood samples and mice were sacrificed by CO₂ suffocation when showing signs of weakness or a lymphocytic tumor content of >80%.