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. The Kruskal–Wallis test reporter cell suspension in PBS containing 2% FCS by volume was therefore used to assess the effect of antigen condition, and and 1 g/L of sodium azide on ice. The reporter cells include equal Dunn’s Multiple Comparison Test was used to compare each of numbers of unlabeled TE MOG cells and CTV-labeled TE 0 cells. the conditions with the T cells absent condition.

Sanderson et al. www.pnas.org/cgi/content/short/1614472114 2of6 adro tal. et Sanderson www.pnas.org/cgi/content/short/1614472114 Figure 1 A Figure 2 A Figure 2 B, Figure 2C,D, 4D Figure 3 A Figure 4 A,B

4.0M 250K

200K 3.0M Lymphocytes 24.100 150K Lymphocytes 2.0M SSC-A 37.722 100K SSC-A :: SSC-A

1.0M 50K

0 0 0 50K 100K 150K 200K 250K 0 1.0M 2.0M 3.0M 4.0M FSC-A :: FSC-A FSC-A

4.0M 250K Single Cells 200K 3.0M 96.320

Single Cells 150K

2.0M 77.552 FSC-H 100K FSC-H :: FSC-H

1.0M 50K propidium iodide

0 0 0 50K 100K 150K 200K 250K 0 1.0M 2.0M 3.0M 4.0M FSC-A

FSC-A :: FSC-A

5 7 10 10

6 10 4 10 5 10 T cells CD4 4 3 10 10 30.434

APC Cy7 B220 62.440 CD19 - APC CD19 - CD19 - APC CD19 - APC Cy7 B220

0 0

3 4 -10 -10

2 3 4 5 6 7 3 3 4 5 10 10 10 10 10 10 pacific blue B220 -10 0 10 10 10 PerCP Cy5.5 TCR brilliant violet 605 B220 CD4-FITC CD4 - brilliant violet 786

120 7 10 CTV low 6 10 90 54.358

5 10 Count 60

4 10 prolif CD25 - PE-Cy7 30 3 76.435 order of gating 10 2 0 10 4 5 6 3 4 5 10 10 10 0 10 10 10 pacific blue B220 Cell Trace Violet Cell Trace Violet

Fig. S1. Gating strategies for flow cytometry figures. For each labeled figure panel, gating strategies are shown with the largest population at the top and successively reduced populations going down the page. 3of6 Table S1. List of antigen-expressing cell lines and the stocks from which they were derived Antibody used to verify Cell line Transfected antigen Source surface expression

TE 0 No added antigen ATCC CRL-8805 TE MOG MOG—that is, native MOG from Rattus rattus NM_022668.2 PMID: 10428054 8.18C5 TE HA HA—that is, native hemagglutinin from influenza ACF54598.1 FluBI mouse serum A/WSN/33 HEK No added antigen ATCC CRL-1573 HEK MOG-GFP MOG amino acids 1–204 fused to GFP 8.18C5 HEK HA MOG-GFP HA FluBI mouse serum MOG amino acids 1–204 fused to GFP 8.18C5 TE MOG-GFP MOG amino acids 1–204 fused to GFP 8.18C5

TE MOG HA MOG 8.18C5 HA FluBI mouse serum TE mOVA mOVA—a fusion protein comprising the signal pODpCAGGS PMID: 14525595 AB1225 peptide from H-2 Kb, the full sequence of chicken OVA, and the transmembrane domain of H-2 Db, which results in extracellular membrane expression TE MOG mOVA MOG 8.18C5 mOVA AB1225 TE MOG-OVA MOG amino acids 1–204 fused to full-length chicken 8.18C5 OVA TE HA-OVA HA-OVA—that is, A/WSN/33 HA fused to amino acids FluBI mouse serum 312–345 of chicken OVA (this includes the epitope recognized by OT-II T cells) TE MOG HA-OVA MOG 8.18C5 HA-OVA FluBI mouse serum

Table S2. List of primary immune cell types and the mouse lines from which they were derived Name Properties Source

IgHMOG Transgenic mouse line whose B cells express a BCR and secrete Guru Krishnamoorthy and Hartmut Wekerle, Max-Planck-Institut antibodies that bind an epitope in the extracellular domain für Neurobiologie, Martinsried, Germany (amino acids 1–125) of MOG FluBI Transgenic mouse line whose B cells express a BCR and secrete Stephanie Dougan and Hidde Ploegh, Whitehead Institute, antibodies that bind an epitope in the extracellular domain of Cambridge, MA HA from influenza A/WSN/33 Wild type “Wild type”—that is, nontransgenic B cells were derived from Jackson 000664 C57BL/6J C57BL/6 mice, which share the same MHC class II allotype I-Ab as the other mice in the study 2D2 Transgenic mouse line whose CD4+, MHC class II-restricted T cells Guru Krishnamoorthy and Hartmut Wekerle, or Jackson, C57BL/ recognize MOG 35–55 in the context of I-Ab 6-Tg(Tcra2D2,Tcrb2D2)1Kuch/J OT-II Transgenic mouse line whose CD4+, MHC class II-restricted T cells Ed Palmer, Basel, or Jackson B6.Cg-Tg(TcraTcrb)425Cbn/J recognize chicken OVA 323–339 in the context of I-Ab

Sanderson et al. www.pnas.org/cgi/content/short/1614472114 4of6 Table S3. List of abbreviations Abbreviation Full name Relevance

ADEM Acute disseminated encephalomyelitis Autoimmune neurological condition characterized by the presence of serum autoantibodies and sometimes preceded by infections BCR B-cell receptor Surface recptor expressed by B cells, whose specificity is identical to the specificity of antibodies produced by that B cell Cherry A variant of red fluorescent protein CNS Central nervous system The brain and spinal cord, the physiological site of MOG expression GFP Green fluorescent protein GMF Geometric mean fluorescence Measure of central tendency in fluorescent flow cytometry HA Hemagglutinin The major envelope glycoprotein of influenza viruses, expressed on the plasma membrane of infected cells, and targeted by the anti-influenza B-cell response IgG IgG The class of Ig (BCR or antibody) expressed by class-switched B cells IgG 2a IgG subclass 2a A subclass of IgG, the production of which is particularly dependent on T-cell help IgM IgM The class of Ig (BCR or antibody) expressed by naive B cells LAMP1 Lysosome-associated membrane protein 1 Protein localized predominantly in lysosomal membranes and used here as a subcellular marker of lysosomes MHCII Major histocompatibility complex, class II Surface receptor expressed by antigen-presenting cells such as B cells and macrophages, which presents antigen fragments to T cells, thereby mediating T-cell antigen recognition MOG Myelin oligodendrocyte glycoprotein CNS-restricted cell-surface protein, autoantibodies against which are found in certain autoimmune conditions OVA Ovalbumin Protein found in chicken egg white, containing the experimental T-cell epitope OVA 323–339 PE Phycoerythrin Red protein–pigment complex fluorochrome used in flow cytometry TCR T-cell receptor Surface receptor expressed by T cells, which recognizes a particular combination of antigen-derived peptide (e.g., MOG amino acids 35–55) and its cognate MHC molecule (I-Ab in all of the experiments in this study)

Movie S1. Live cell imaging of MOG-specific B cells capturing MOG-GFP (green) from stably transfected HEK cells by IgHMOG B cells labeled with Cell Tracker Deep Red (magenta). Frames are separated by approximately 1 min.

Movie S1

Sanderson et al. www.pnas.org/cgi/content/short/1614472114 5of6 Movie S2. 3D reconstruction of confocal z stack of B cell–HEK MOG-GFP interaction fixed at 3 min after contact and immunolabeled for LAMP1 (red) and IgM (magenta) before laser scanning confocal microscopy. Stacks of XY planes were processed digitally to generate 3D reconstructions and then rotated to create successive frames of the movie to show the spatial location of the B and HEK cells and the antigen–IgM complexes being internalized by the B cell.

Movie S2

Movie S3. 3D reconstruction of confocal z stack of B cell–HEK MOG-GFP interaction fixed at 60 min after contact. Other than the time before fixation, the movie is exactly analogous to Movie S2.

Movie S3

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