Gr 1 rb6 8c5

For FCM staining spleens of mice after adoptive transfer were isolated in RPMI supplemented with 2% FCS and antibiotics and a single cell suspension was prepared. Red blood cells were lysed using ACK buffer prior to staining. Fc receptors were blocked using an anti-CD16/32 antibody (2.4G2, BD). To discriminate Qβ-specific plasma cells (PCs) from Qβ-specific activated and CS B cells, surface immunoglobulins (Ig) of specific cells were blocked using unlabelled Qβ VLPs. PCs were further stained with and characterized as IgM (polyclonal, Jackson ImmunoResearch), IgD (11-26c (11-26), eBioscience), CD4 (H129.19, BD), CD8 (53-6.7, BD), GR1 (RB6-8C5, BD), CD11b (M1/70, BD), CD11c (HL3, BD) negative (all antibodies labeled with PE), and B220-PE-Cy7 (RA3-6B2, BD) low. To detect Qβ specific PCs by intracellular staining of specific Ig, splenocytes were permeabilized using FACS lysing solution (BD, 349202) containing 0.04% Tween20 and stained with Alexa Flour 488 labeled Qβ VLPs. The congenic marker Ly5.1 (antibody labeled with APC, A20, eBioscience) identified all transfer derived B cells.
Qβ VLPs were labeled with the Alexa Flour 488 protein labeling kit (Thermo Fisher Scientific, A10235) according to the manufacturer's instructions.

Lymphoma immunophenotype was assessed as previously described [22 (link)]. The following antibodies were used for staining: CD19-APC, B220 (CD45R)-FITC, IgM biotin, IgD-PE, and Streptavidin-APC/Cy7 (BD Biosciences, USA). Data were collected by a flow cytometer (BriCyteE6, Mindray Bio- Medical Electronics Co. Ltd., Shenzhen, China) and analyzed using FlowJo Version 10.1 software. For the analysis of tumor-associated macrophages, cell suspensions of lymph nodes were obtained by grinding and filtering tissues through 0.4-μm cell strainers (BD Biosciences, USA) in PBS. Cells were then transferred to a fresh tube for centrifugation at 1000× g for 5 min. The cell pellet was incubated in red blood cell lysis buffer (Lonza) for 1 min at room temperature, diluted in PBS, and centrifuged for 1000× g for 5 min. The cell pellet was incubated with fluorescent-conjugated antibodies (dilution 1:50 in PBS) for 15 min at 4 °C in the dark, washed, and analyzed by flow cytometry. The following antibodies were used: F4/80 (6F12) from Miltenyi; CD11b (M1/70.15.5) and Gr1 (RB6–8C5) from BD Pharmingen.

Hematopoietic stem and progenitor cell and lineage-positive cell profiling was perform on BM, spleen, and thymus of mice from each genotype. Antibodies (clones) used: CD3 (145-2C11) [BD: #553064], CD4 (H129.19) [BD: #553653], CD8 (53-6.7) [BD: #553033], Gr-1 (RB6-8C5) [BD: #553128], B220 (RA3-6B2) [BD: #553090], Ter119 (Ter119) [BD: #553673], Mac1 (M1/70) [BD: #553311], IL7Rα (A7R34) [eBiosceinces: #12-1271-82], cKit (2B8) [BD: #558163], Sca-1 (E13-161.7) [eBiosceinces: #17-5981-83], CD25 (7D4) [eBiosciences: 13-0252-82], CD44 (IM7) [BD: 559250], CD43 (S7) [BD: 561856], HSA (M1/69) [BD: #553262] and BP-1 (6C3) [BD: #553159], CD4 (GK1.5) [BD: #561830], CD8 (7D4) [eBiosciences: #12-0081-82], B220 (RA3-6B2) [BD: #561880].
Flow cytometric gating strategy is described in Supplementary Fig. 6.

Bone marrow cells were harvested from femurs and tibiae by crushing the bones with a mortar and pestle. The cell suspension was collected after filtering through 40 μ membrane filter (BD). These cells were used for surface staining after red blood cell (RBC) lysis treatment with Red Blood Cell Lysing Buffer Hybri-Max™ (Sigma). Spleens were crushed between two glass slides and isolated cells were used for surface staining with respective antibodies after RBC lysis. Surface staining was done with B220 (RA3-6132, BD), CD3 (145-2C11, BD), Gr-1 (RB6-8C5, BD), Mac1 (M170, BD), CD45.1 (A20, BD), and CD45.2 (104, BD). After surface staining with respective fluorescent antibodies, polychromatic flow acquisition was performed using a LSR II instrument (BD) at the University of Rochester Flow Cytometry Core. Flow data was analyzed using FlowJo software (version 10.0.7 Treestar, Ashland, OR).