Endofit ovalbumin

We detected OVA-specific IgG in sera by an indirect ELISA. Briefly, we coated microtiter plate wells (Nunc) with 100 µL OVA solution (2 μg/well; EndoFit Ovalbumin, Invivogen, San Diego, CA, USA) in 0.2 M carbonate buffer for 24 h at 4 °C. We washed the wells three times with PBS containing 0.05% (v/v) Tween 20 (PBST) and then blocked with 5% FCS/PBS (AAJ63160AK; Thermo, San Diego, CA, USA) at 37 °C for two hours. After three washings of PBST, 100 μL of a series of diluted sera samples (initial dilution 1:50) and 0.5% FCS/PBS as a control were added to the wells in triplicate. Then, we incubated the plates for 2 h at 37 °C and washed three times with PBST. We diluted from 100 μL aliquot of rabbit anti-mouse IgG horseradish peroxidase conjugate (AP160P; Sigma) with 1:10,000. Then, we incubated the plates for 2 h at 37 °C. After washing with PBST, we determined the peroxidase activity by adding 100 µL of substrate solution (10 mg of O-phenylenediamine and 37.5 µL of 30% H₂O₂ in 25 mL of 0.1 M citrate–phosphate buffer, pH 5.0) to each well. After incubation for 10 min at 37 °C, we added 50 µL/well of 2 N H₂SO₄ (Merck, NJ, USA) to each well to terminate the enzyme reaction. We used an ELISA reader (TECAN Sunrise^TM, Männedorf, Switzerland) at 450 nm to perform the assays on each of the sera samples after conducting within- and between-group comparisons.

Mouse AAC-Ova was generated as stated above by squeezing RBCs with EndoFit Ovalbumin (InVivoGen) and poly I:C (InVivoGen), while AAC-E7 was generated by squeezing mouse RBCs with mouse-E7 SLP (Biosyntan GmbH) and poly I:C (Dalton Pharma Services). Either were injected RO at 250x10⁶ per C57BL/6J recipient (unless otherwise indicated) on day 0. In cases of multiple immunizations, see Figure S2E and S2F for a timeline. Seven days post last immunization, spleens (or blood in the case of splenectomized mouse studies) were harvested, processed into single cells, and co-cultured with appropriate stimulation, anti-CD28 (8 µg/mL, eBioscience, San Diego, CA, USA) and either Ova_257-264 (1 µg/mL, SIINFEKL, AnaSpec, Freemont, CA, USA) or E7_49-57 (4 µg/mL, AnaSpec) with sequences found in Supplementary Table S1, for one hour and for an additional four hours in the presence of GolgiStop/GolgiPlug (BD Bioscience, Franklin Lakes, NJ, USA). Following restimulation, cells were stained for flow cytometry analysis using BD Bioscience FACS lysis solution and Fixation/Permeabilization kit according to manufacturer’s instructions and with the panel in Supplementary Table S6. For tetramer staining, the panel in Supplementary Table S7 was used.