OVA Peptide s.c. Immunization

A mixture of OVA peptides was administrated in a s.c. injection in mice anesthetized by i.p. injection of xylazine (10 mg/kg) and ketamine (100 mg/kg). OVA_257–264 (SIINFEKL) (InvivoGen, San Diego, CA, USA) is a class I (Kb)-restricted peptide epitope of OVA presented by the class I MHC molecule (CMH) H-2Kb. OVA_323–339 (ISQAVHAAHAEINEAGR) (AnaSpec, San Jose, CA, USA) is an H-2b-restricted OVA class II epitope. 100 μg of each peptide emulsified with the same volume of IFA (Sigma-Aldrich) was administered. Controls were injected with PBS-IFA complexes. OVA peptides and PBS-IFA complexes were injected 2 h or 21 days post-first i.p. injection of MnTBAP (or PBS). Eight days post-OVA peptide injection, mice were sacrificed, and spleens were harvested to analyze OVA-specific T cell response.