Fc fusion proteins

We designed an Ig fusion construct (mouse MOG (1–118, N31Q)-linker-IgG2c (C220S, LALA-PG), shortly MOG-Fc) consisting of mouse IgG2c (215–452) together with mouse MOG. We also designed a fusion construct using mouse IgG2c (215–452) and anti-influenza B llama single-domain antibody heavy chain [20] which we used as a control (shortly SD-Fc). We introduced the following mutations in the IgG2c to abolish complement and FcγR receptor binding: L234A, L235A and P329G (LALA-PG), C220S and L351Q. We also mutated the N-glycosylation site (N31Q) in mouse MOG 1–118. The GGGGSGGGGS linker was used to fuse MOG (1–118) and IgG2c-Fc portions. Igk secretion signal was introduced at the N-terminus and His-Tag and AviTag sequences were introduced at the C-terminus to facilitate the purification and multimerization. DNA fragments (Additional file 4) were synthesized by ThermoFisher Scientific as GeneArt gene fragments and cloned into either pTT5 [21] or PB-T vector [22] using NEBuilder® HiFi DNA Assembly (New England Biolabs). The proteins were expressed and purified the IgG-Fc proteins in HEK cells using either a transient transfection [21] or from a transposase-based stable expression system [22] by the core facility of the Max Planck Institute of Biochemistry. The dimeric nature of the purified proteins was confirmed by reducing SDS-PAGE and by mass spectrometry by the core facility of the Max Planck Institute of Biochemistry. Monomeric MOG protein (without Fc) was produced either in HEK cells or in E. coli as recombinant proteins.

SDS-PAGE

MOG-Fc, SD-Fc, and MOG-specific IgG1 (clone 8.18C5) proteins were denatured in SDS-containing loading buffers (95 °C, 5 min) and loaded (2 μg per lane) onto a gradient (7–15%) polyacrylamide gel under reducing (2 X Laemmli sample buffer; Sigma) or non-reducing (4X Laemmli sample buffer; Bio-Rad) conditions. Two protein markers were used as a protein size reference: Precision Plus Protein standards (Bio-Rad) and PageRuler Plus Prestained Protein Ladder (Thermo Fisher). Gel imaging was performed using a ChemiDoc MP Imaging System (Bio-Rad).

Mice

Wild-type (WT) C57BL/6, WT SJL/J, CD45.1 C57BL/6, CD45.2 SJL/J, IgHMOG SJL/J, IgHMOG C57BL/6 [23], TCR1640 SJL/J (RR) [24] and OSE [23] mice were bred and housed at the animal facilities of the Max Planck Institute of Biochemistry. Mating pairs were fed regular chow ad libitum. Mice were given autoclaved drinking water ad libitum. All animal procedures were performed following the guidelines of the Committee on Animals of the Max Planck Institute of Biochemistry and with approval from the Regierung von Oberbayern (Munich, Germany).

Serum collection

Blood was collected by retro-orbital bleeding into serum gel tubes (Sarstedt), allowed to stand at room temperature (RT) for 1 h, then centrifuged (10,000 rpm, 5 min, 4 °C) to collect serum. For all kinetics experiments, blood was collected at the time points indicated in the respective graphs. For antibody depletion experiments, asymptomatic RR mice (8–16 weeks of age) were pre-selected based on high anti-MOG IgG titers in their sera. On day 0, 200 µg of MOG-Fc or SD-Fc were administered i.p, following which blood was collected at the indicated time points. Sera were frozen at − 20 °C until their use in ELISA.

Cell isolation and flow cytometry

Single-cell suspensions were prepared from the spleen and the lymph nodes by mechanical disruption using 40-µm cell strainers (Corning or Fisher Scientific). Cells were collected in RPMI (RPMI 1640, Sigma) containing 10% heat-inactivated Fetal Bovine Serum (FBS, Sigma). Spleen cells were further resuspended in erythrocyte lysis buffer (0.83% NH4Cl) and incubated for 3 min at room temperature. The lysis buffer was then neutralized with RPMI containing 10% FBS and the cells were washed and collected in flow cytometry buffer for staining.

Leukocytes from the brain and spinal cord were isolated using a Percoll (GE Healthcare) gradient centrifugation. In brief, mice were perfused with PBS, the CNS tissues were collected and forced through 100-µm cell strainers, and washed in RPMI-1640 containing 10% FBS. The pellet was resuspended in 5 ml of serum-free RPMI-1640, mixed with 2.16 ml Percoll (density 1.123 g/ml), and overlaid onto 5 ml of Percoll (density 1.08 g/ml). After centrifugation at 1200 g for 30 min, the cells at the interface were collected and washed with RPMI-1640 containing 10% FBS.

In samples where cytokine expression was not analyzed, cells were directly stained after isolation. In samples that were analyzed for cytokine expression, cells were activated with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of 5 µg/ml brefeldin A for 4 h at 37 °C before staining. For detection of cell surface markers, cells were washed twice with flow cytometry buffer (PBS containing 1% bovine serum albumin (BSA; Carl-Roth) and 0.1% NaN3) and stained with the following antibodies: anti-CD4 (RM4-5), anti-CD45 (30-F11), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD19 (1D3), anti-CD11c (N418), anti-CD5 (53–7.3), anti-CD21 (7G6), anti-PD-L1 (MIH5), anti-PD-L2 (122), anti-VISTA (MH5A), anti-CD86 (GL1), anti-CD83 (Michel-19), anti-IA/IE (M5/114.15.2), anti-LAG3 (C9B7W), anti-Vα3.2 (RR3-16), anti-Vβ11 (RR3-15), anti-IgMa (DS-1), anti-PD1 (J43) and anti-CD25 (PC61). Fixable viability dye eFluor-780 (Thermo Fisher Scientific) was used at a 1:1000 concentration. Cells were then washed twice in flow cytometry buffer and either resuspended in flow cytometry buffer for acquisition or used for intracellular staining.

For intracellular/intranuclear staining, surface-stained cells were fixed and permeabilized by incubation with 100 µl of Fixation/Permeabilization Buffer (Transcription factor staining set, eBioscience). Cells were then stained with the following antibodies: anti-IFNγ (XMG1.2), anti-IL-17A (TC11-18H10.1), and anti-FoxP3 (FJK-16s). Finally, cells were washed twice in Permeabilization Buffer and resuspended in flow cytometry buffer for acquisition.

Antibodies were purchased from BD Biosciences, Biolegend, or Pharmingen. Antibodies were used in conjugation with one of the following fluorophores: FITC, PE, PerCP-Cy5.5, PeCy7, APC, APC-Cy7, BV421, eFluor450, eFluor780, BV605, BV711, BV785, or APC-R700. Stained samples were acquired on FACS Canto (BD Biosciences). Analysis was performed using FlowJo (TreeStar) software.

In vivo B cell frequency analysis

Leukocytes from the spleen and mesenteric lymph nodes of IgHMOG SJL/J mice were transferred intravenously (i.v.) at a concentration of 10 million cells/mouse, into CD45.2 SJL/J mice. 200 µg of MOG-Fc or SD-Fc was administered intraperitoneally (i.p.) one day later. The percentage of transferred CD45.1+ CD19+ B cells was measured by flow cytometry from the spleen and lymph nodes 3 days later.

Immune cell functionality analysis

To characterize MOG-specific B cell functionality, IgHMOG C57BL/6 mice were injected i.p. with 200 µg of MOG-Fc or SD-Fc. Lymph node cells were isolated 3 days later for flow cytometry.

To characterize CD4 T cell functionality and non-specific APC functionality, splenocytes from OSE mice were transferred i.v. at a concentration of 3–4 million cells/mouse, into WT C57BL/6 mice, along with an i.p. injection of 200 µg of MOG-Fc or SD-Fc. Lymph node cells were isolated 3 days later for flow cytometry.

EAE

WT C57BL/6 were injected subcutaneously with 200 μl of emulsion containing 100 μg of MOG 35–55 peptide and 500 μg M. tuberculosis strain H37 Ra (Difco) in incomplete Freund’s adjuvant (Difco). Mice additionally received 400 ng pertussis toxin (Sigma) i.p. on days 0 and 2 after immunization. 100 µg MOG-Fc or SD-Fc were administered i.p. on days 10, 12, 14, and 16 post EAE induction. Clinical signs of EAE were assessed according to the standard scoring scheme [23]: score 0, healthy; 1, flaccid tail; 1.5, flaccid tail and impaired righting reflex; 2, impaired righting reflex and hind limb weakness; 2.5, one hind leg paralyzed; 3, both hind legs paralyzed with residual mobility in both legs; 3.5, both hind legs completely paralyzed; 4, both hind legs completely paralyzed and beginning front limb paralysis; and 5, moribund or death of the animal after preceding clinical disease. In some cases, the animals were killed before reaching the maximal disease scores in accordance with the animal license regulations.

In vitro proliferation assay

Single-cell suspensions of splenocytes from OSE mice were washed once with PBS and then subjected to B cell isolation (B cell isolation kit, Biolegend). Purified B cells were loaded with MOG-Fc or SD-Fc (10 µg protein per million cells) for 1 h at 37 °C, washed in RPMI containing 10% FBS, and plated in 96-well U-bottom plates (5 × 105 cells/well) and left overnight at 37 °C. The next day, CD4+ T cells were isolated from OSE mice using a CD4 T cell isolation kit (Biolegend). The isolated cells were labeled with 5 µM cell proliferation dye eFluor450 in PBS for 10 min at RT, washed in RPMI containing 10% FBS, and added to the cultured B cells at the concentration of 5 × 105 cells per well. The plates were incubated at 37 °C for 60 h, then stained and analyzed by flow cytometry.

In vivo proliferation assay

Protocol 1 Single-cell suspensions of splenocytes from OSE mice were washed once with PBS and then subjected to B cell isolation (B cell isolation kit, Biolegend). The B cells were loaded with MOG-Fc or SD-Fc (10 µg protein per million cells) for 1 h at 37 °C, after which they were washed in PBS and injected i.v. into WT C57BL/6 mice (5–10 million cells per mouse). After 24 h, single-cell suspensions of splenocytes from OSE mice were washed once with PBS and then subjected to CD4+ T cell isolation (CD4 T cell isolation kit, Biolegend). The isolated cells were labeled with 5 µM Cell Proliferation Dye eFluor450 in PBS for 10 min at RT, washed in PBS, and injected i.v. into WT C57BL/6 mice (6–8 million cells per mouse). The spleen and lymph node cells were stained and analyzed by flow cytometry on day 6 after T cell transfer.

Protocol 2 Single-cell splenocyte suspension from OSE mice was washed in PBS and stained in 5 µM cell proliferation dye eFluor450 in PBS for 10 min at RT. The cells were washed in PBS and injected i.v. into CD45.1 C57BL/6 mice (20–30 million cells per mouse). 2 days after cell transfer, mice were injected i.p. with 200 μg of MOG-Fc or SD-Fc in 200 μl PBS. Six days after cell transfer, the spleen, and lymph node cells were stained and analyzed by flow cytometry.

Enzyme-linked immunosorbent assay (ELISA)

ELISAs were performed with the following general considerations. 96-well Maxisorp Nunc-immuno plates (Thermo Scientific) were coated overnight at 4 °C, PBS-T (0.1% Tween 20 in PBS) was used for washing the plates, and blocking was done with 10% FCS in PBS for one hour in room temperature. Incubations with serum samples, antibodies, and streptavidin were all performed in the blocking buffer at room temperature. TMB (3,3ʹ,5,5ʹ-tetramethylbenzidine) or ABTS (2,2ʹ-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) substrate solution (activated with 0.1% H2O2) (Biolegend) was used for detection. TMB reaction was stopped with 1N H2SO4. Plates were measured in a spectrophotometer (Perkin Elmer) at 450 nm (for TMB) or 405 nm (for ABTS) after 10 min.

MOG-specific antibody detection

Plates were coated with 20 µg/ml of MOG, MOG-Fc, or SD-Fc in PBS (100 µl per well), washed, blocked for one hour, washed again, and incubated with serum samples at different dilutions for 2 h at RT. The MOG-specific antibody 8.18C5 and mouse IgG1 (Biolegend) were used in a dilution series as standards. After washing, biotin-labeled anti-mouse IgG (5 ng/ml; SouthernBiotech) was added for 1 h, then washed and incubated with HRP-labeled streptavidin (0.5 µg/ml; Biolegend) for another hour. After final washing, the signal was developed by adding a substrate solution and measured in a spectrophotometer.

Residual MOG-Fc ELISA

Plates were coated with 1 µg/ml of anti-His-tag antibody (Biolegend, 100 µl per well), washed, blocked for one hour, washed again, and incubated with serum samples for 2 h at RT. MOG-Fc and MOG were used as standards. After washing, a biotin-labeled 8.18C5 antibody (1 µg/ml; produced in-house) was added for 1 h, then washed and incubated with HRP-labeled streptavidin (0.5 µg/ml; Biolegend) for another hour. After final washing, the signal was developed by adding a substrate solution and measured in a spectrophotometer.

MOG binding assay

AviTag-containing MOG-Fc and monomeric MOG proteins were biotinylated using BirA biotin-protein ligase kit (BirA-500, Avidity LLC) according to the manufacturer’s instructions and conjugated with APC-labeled streptavidin to form tetramers. Splenocytes from wild-type and IgHMOG C57BL/6 mice were mixed in different ratios (100, 50, 25, 12.5, 6.2, and 0 percent of IgHMOG cells) and stained with equimolar amounts of MOG-Fc or MOG tetramers, as well as with anti-CD19 (6D5, Biolegend) and Fixable viability dye eFluor 780 (Thermo Fisher Scientific). Stained samples were acquired on FACS Canto (BD Biosciences). Analysis was performed using FlowJo (TreeStar) software.

Statistical analysis

GraphPad Prism 9 (GraphPad Software, Inc.) was used for all statistical analyses. Information on statistical tests used for analysis is mentioned in figure legends. P values below 0.05 were considered significant. Bars depict the mean ± standard error of the mean.