Np18 ova

For standard NP-OVA immunizations, 100μg NP₁₈-OVA (Biosearch Technologies) in a 1:1 H37RA CFA (DIFCO) emulsion was injected s.c. into the flanks of young or aged mice. The spleen, dLN, and blood were harvested 7-27 days later. PP were harvested from unimmunized mice. Serum was isolated from blood using serum separator tubes (BD vacutainer) and NP-specific IgG measured by ELISA as previously described (Sage et al., 2013 (link)).

For TFH and TFR cell studies mice were immunized with 100μg MOG 35–55 (UCLA biopolymer facility), MOG 1–125 or NP₁₈-OVA (Biosearch Technologies) emulsified 1:1 with H37RA Mtb. CFA (Difco laboratories) in the flanks as previously described(25 (link)). 7 days later draining lymph node or blood was harvested for analysis. For studies of TFH and TFR cell differentiation in PD-L1 conditional knockout mice, all mice received 200μg anti-PD-L2 (clone 3.2) on d2 and d4. For EAE induction, similar emulsions were prepared as above but contained an extra 2mg/ml H37RA Mtb. Mice were additionally given 200ng pertussis toxin i.p. on days 0 and 2. Clinical disease was assessed using the following criteria: 1 = limp tail, 2 = weak gait, 3 = hind limb paralysis, 4 = hind+forelimb paralysis, 5 =moribund as described previously(26 (link)).