Keyhole limpet hemocyanin (klh)

Serum was collected by cardiac puncture from B6 mice three weeks after immunization with rhMOG(200 μg/mouse) /CFA or KLH(200 μg/mouse) /CFA (LGC biosearchtechnologies, N-5060) as described above. Mice from indicated groups were injected i.p. with 400 μl serum starting at the time of EAE induction and continuing every 2 days for the following weeks. IgG was purified from serum using Protein G sepharose 4 Fast Flow resin (Cytiva 17-0618-05) and Pierce IgG Elution Buffer (ThermoFisher Scientific 21009). Purified IgG was buffer-exchanged to PBS, sterile filtered and antibody integrity confirmed by SDS-PAGE analysis. Purified IgG was injected in the same amount as contained in the 400 μl serum. This protein G purification removed >95% of IgG from serum as determined by ELISA, and the IgG-depleted serum was also used in transfer experiments.

To induce GC formation, age- and sex-matched mice were immunized intraperitoneally at 8 to 12 weeks of age with either 0.5ml of a 2% sheep SRBC suspension in PBS (Cocalico Biologicals; Reamstown, PA, USA), or 100 μg of the highly substituted hapten NP (NP₁₆ to NP₃₂) conjugated to the carrier protein ovalbumin (OVA), or CGG (Chicken Gamma Globulin), or KLH (Keyhole Limpet Hemocyanin; all from Biosearch Technologies; Novato, CA, USA) absorbed to Imject™ Alum Adjuvant (77161; ThermoFisher Scientific; Waltham, MA, USA) at a 1:1 ratio. In the experiments where T_FH interactions were blocked in vivo, mice received and i.v. injection of 100μg anti-CD40L antibody (clone MR-1, BE0017; BioXCell; West Lebanon, NH, USA) or control IgG antibody (BE0091; BioXCell) 4 days after SRBC immunization, and a second dose of antibody 2 days later. In the experiments where IL-9 was blocked in vivo, mice received a single i.v. injection of 200μg anti-IL-9 antibody (clone 9C1, BE0181; BioXCell) or control IgG2a antibody (BE0085; BioXCell) 7 days after SRBC immunization. In these experiments, littermates of the same sex were randomly assigned to experimental groups. In experiments where GC cell cycle distribution was assessed, animals received an i.v. injection of 1mg EdU (E10187; ThermoFisher Scientific), 1h before euthanasia.