Enzyme-linked immunospot (ELISpot) assays were performed following the instructions of the manufacturer (Diaclone). Briefly, PVDF membrane plates were coated with capture antibody against mouse IFNγ and blocked with a 2% skimmed milk solution prepared in PBS. 2 × 10 5 Splenocytes per well were cultured in vitro for 36 h in the presence of 10 μg/ml SIINFEKL peptide. Plates were revealed after incubation with anti-mouse IFNγ detection antibody coupled to biotin followed with a streptavidinalkaline phosphatase conjugate and exposure to a ready-to-use solution of nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3 -indolyphosphate (BCIP) for chromogenic development. Plates were analyzed with an ELISpot plate reader and a dedicated ImmunoSpots software (C.T.L.).