Supporting Information Sanderson et al. 10.1073/pnas.1614472114 SI Materials and Methods verified expression at the establishment of the cell line and Mice and Primary Immune Cells. A full list of primary immune cells subsequently periodically checked the expression of the antigen − − used in the study is presented in Table S2. C57BL/6 and Rag / by immunofluorescence flow cytometry (see Table S1). Adherent mice were bred in the University of Basel Mouse Core Facility. cells were washed in PBS, trypsinized, resuspended in complete − − Tg(TcraTcrb)425Cbn (OT-II) mice on a Rag2 / background medium, washed in labeling buffer (PBS containing 2% FCS by were a kind gift from Ed Palmer, University of Basel. FluBI mice volume and 1 g/l of sodium azide), incubated for 30 min on ice in (23) were bred from founder members provided by Stephanie primary antibody, washed again, incubated with fluorescent Dougan and Hidde Ploegh, Whitehead Institute, Cambridge, secondary antibodies, washed, and measured on a BD LSR MA. IgHMOG (ref. 12; also known as Th B cells) and 2D2 mice Fortessa flow cytometer (BD Biosciences). Cells known to be (13) were bred from founder members provided by Guru positive and negative for the target antigen were labeled and Krishnamoorthy and Hartmut Wekerle, Max-Planck-Institut für measured in parallel each time. A typical result from such an Neurobiologie, Martinsried, Germany. Primary immune cells experiment is shown in Fig. 4E. When flow cytometry results are were obtained from spleens by mechanical disruption followed shown as contour plots, the contour lines are spaced at 10%. by brief settlement under gravity to remove tissue fragments. B Gating strategies for each figure are shown in Fig. S1 for each cells and CD4-positive T cells were obtained by negative selec- figure. tion using biotinylated antibodies and magnetic beads from MOG Miltenyi (Pan B Cell Isolation Kit II, mouse 130–104-443, and B-Cell Flow Cytometry. IgH B cells isolated by negative se- – CD4-positive T Cell Isolation Kit, mouse, 130–104-454). When lection were cocultured with TE MOG or TE 0 cells for 8 20 h in higher purity of B cells was required, for example for the T-cell complete medium at 37 °C in 5% carbon dioxide and then re- proliferation assays, the splenocytes were first predepleted with trieved and labeled with the following antibodies before analysis anti-CD43 beads (Miltenyi 130–049-801) before following the as above: APC-Cy7 anti-B220 (clone RA3-6B2), PerCP-Cy5.5 manufacturer’s instructions for the negative selection kit. anti-CD69 (clone H1.2F3), PE-Cy7 anti-CD86 (clone GL1), APC anti–I-A/I-E (clone M5/114.15.2, Biolegend), PE anti- Peptides, Proteins, Antibodies, and Vital Dyes. MOG 35–55 peptide CD25 (clone PC61.5), AlexaFluor488 donkey anti-mouse IgM and MOG 1–125 recombinant protein were obtained from (Jackson Immuoresearch), and FITC R35-95 rat IgG2a kappa AnaSpec. AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgM, μ isotype control. All antibodies were from BD Biosciences unless Chain Specific 115–006-020 was from Jackson Immunoresearch, otherwise specified. and FITC-conjugated anti-mouse IgMa was from BD Pharmin- gen. CTV and Cell Tracker Deep Red were from ThermoFisher. T-Cell Proliferation Assays. B cells were exposed to antigen- expressing adherent cells in 25 cm2 flasks for 3 h and then re- Plasmids and Cell Lines. A full list of antigen-expressing cells used in trieved, counted, and put into the wells of 96-well plates at the study is presented in Table S1. The ORF encoding rat MOG 50,000 per well in complete medium supplemented with 50 μM was amplified with its STOP codon from pRSV-MOG and cloned 2-mercaptoethanol. T cells were labeled with 5 μM CTV in into the blasticidin (Gibco) resistance-conferring pcDNA6.2C- Hanks’ Balanced Salt Solution (Gibco) at 37 °C for 15 min and EmGFP-DEST (Invitrogen) by gateway cloning, following the then washed thrice in complete medium, counted, and added to manufacturer’s instructions. An expression plasmid encoding the B-cell wells at 25,000 per well. After 4 or 5 d, the cocultures A/WSN/33 influenza HA under hygromycin (Sigma Aldrich) se- were retrieved; labeled with anti-CD19, anti-B220, anti-CD4, lection was purchased from Sino Biological. Fusion constructs and anti-CD25 (all from BD Biosciences); and analyzed by flow MOG-GFP, MOG-OVA, and HA-OVA were made by template- cytometry on a BD LSR Fortessa. switching PCR and cloned by Ligation Independent Cloning into the PigLIC vector, which confers puromycin (Gibco) resistance. Fixed and Live Cell Microscopy. For high-resolution immunolabel- The mOVA construct was amplified from pODpCAGGS (24) (a ing, B cells were added to HEK MOG-GFP cells growing in kind gift from Marc Jenkins, University of Minnesota, Minneapolis) chambered coverslips (Ibidi) at 37 °C and at various time points and cloned into PigLIC. Cells transfected with this construct express were fixed in ice-cold 4% phosphate-buffered paraformaldehyde OVA at the cell surface, which can be detected by flow cytometry for 20 min and then washed in PBS, blocked in PBS containing on living cells using a polyclonal rabbit anti-OVA antibody 5% normal goat serum and 0.02% TritonX, and and immuno- (Ab1225, Millipore). HEK 293 embryonic kidney cells and TE671 labeled with rat anti-mouse LAMP1 (Southern Biotech), fol- rhabdomyosarcoma cells (referred to as “TE cells” throughout the lowed by Alexa 647-conjugated goat anti-mouse IgM and text) were obtained from ATCC (LGC). Cells were transfected Rhodamine RedX-conjugated goat anti-rat antibodies, both by exposure to 2 μg/mL of plasmid DNA and 5 μg/mL of poly- from Jackson Immunoresearch. Images were aquired with a ethylenimine in RPMI medium without serum for 4 h. Cells were Nikon a1r in line-scanning mode with a 60×, 1.40 NA oil im- cultured in complete medium [RPMI medium containing 10% mersion objective using voxel dimensions set at approximately (vol/vol) FCS, 100 units/mL of penicillin, and 100 μg/mL of Nyquist sampling. Acquisition was controlled by Nikon Ele- streptomycin, all from Gibco] at 37 °C in 5% carbon dioxide. ments software, and the same software was used to create three- Cells resistant to the appropriate selective antibiotic were sorted dimensional reconstructions and maximum intensity projections by the Department of Biomedicine Flow Cytometry Core after from the stacks of XY planes. Regions of interest were sub- labeling, as described below. Cells were tested for mycoplasma squently cropped, and when necessary to enhance visibility, infection on arrival and subsequently (LookOut Mycoplasma brightness and contrast were increased using the Fiji distribution PCR Detection Kit, Sigma). of ImageJ (25). For live cell imaging, a similar setup was used, but the B cells were prelabeled with Cell Tracker Deep Red Cell-Surface Antigen Confirmation and General Flow Cytometry. For (Invitrogen) following the manufacturer’s instructions, and each cell line designed to express a cell membrane antigen, we without fixing the cells, the chambered coverslips with the cells Sanderson et al. www.pnas.org/cgi/content/short/1614472114 1of6 growing in complete medium were placed into a humidified After 30 min on ice, the cells are washed, and antibodies adhering chamber on the microscope stage held at 37 °C and 5% carbon to the TE MOG cells were detected with Alexa 647 anti-mouse dioxide by an INU-TIZ-F1 controller (Tokai Hit). The pinhole IgG or PerCP-conjugated anti-IgG2a secondary antibodies (both was opened to 5.0 Airy units and other parameters (PMT volt- from Jackson Immunoresearch) and measured by flow cytometry. ages, voxel dimensions, etc.) optimized to minimize laser light TE 0 and TE MOG populations were then separated by CTV exposure. Approximately one frame per minute was captured, label, and the ratio of geometric mean fluorescence intensities and frames were assembled into movies with Nikon Elements between TE MOG and TE 0 indicated the abundance of antibody software. in the supernatant. Adoptive Transfer. CD4-positive OT-II cells were separated from Statistics. Geometric mean fluorescence intensities of activation mouse spleens as above, washed in PBS, resuspended in cold PBS, −/− markers on B cells were analyzed by two-way analysis of variance and injected intraperitoneally into Rag mice at 1 million per (time versus condition), and because an effect of condition but not mouse. One day later, IgHMOG B cells were isolated as above, 2 of time was found, the effect of condition was examined by one-way exposed to antigen-expressing adherent cells in 75 cm flasks for analysis of variance followed by Dunnett’s Multiple Comparison 3 h, and then retrieved, washed in cold PBS, counted and Test to compare each of the conditions against “no stimulation.” resuspended in cold PBS, and injected intraperitoneally at T-cell proliferation data (proportions of cells in the CTV-low 4 million cells per mouse. Two weeks later, mice were killed and gate) and anti-MOG Ig levels in sera were analyzed by one-way blood was taken from the right atrium, refrigerated overnight, ’ and then cetrifuged at 5,000 × g for 10 min. Sera were stored at analysis of variance followed by Dunnett s test. Anti-MOG IgG –20 °C until antibody assay. levels in culture supernantants, expressed as ratios of geometric mean immunofluorescence TE MOG:TE 0, were analyzed by two- Anti-MOG Antibody Assay. Anti-MOG antibodies were measured in way analysis of variance (presence of T cells versus antigen con- sera and in culture supernatants by flow cytometry as previously dition), revealing a strong interaction, with an effect of condition described (3). Supernatants were mixed with two volumes of limited to the T cells present condition.