Immunospot

The MultiScreen filter plates (Millipore) were coated overnight with anti–IFN-γ antibody (BD Pharmingen). A total of 5 × 10⁵ T cells enriched from spleen cell suspensions of the indicated mouse group were placed in each well and were cocultured with intact donor (DBA/2), self (B6), third-party (C3H) splenocytes (direct pathway) or corresponding sonicates (indirect pathway) at 37°C in 5% CO₂. After 72 hours of incubation, biotinylated anti–IFN-γ antibodies were added overnight and then detected with avidin–horseradish peroxidase and 3-amino-9-ethylcarbazole. Resulting spots representing IFN-γ–producing responders were counted and analyzed by ImmunoSpot (Cellular Technology Limited).

PBMCs (2 × 10⁵) were incubated in 96-well flat-bottom plates (MSIPS 4510, Millipore, Millipore, Burlington, MA, USA) coated with anti-IFN-γ mAb (clone 1-D1K, Mabtech, Stockholm, Sweden) with 0.2 mL of complete RPMI containing 10% human AB serum with pools of 12 peptides (2 g/mL, final concentration) or individual peptides (1 g/mL final concentration) for 20 h. Following a 20 h incubation at 37 °C, the wells were washed with PBS/0.05% Tween 20 and then incubated with biotinylated anti-IFN-γ mAb (clone 7-B61-, Mabtech, Stockholm, Sweden) for 1 h 30 min. The spots were developed using streptavidin-alkaline phosphatase (Mabtech, Stockholm, Sweden) and BCIP/NBT substrate (Promega, Madison, WI, USA) and counted using an automated ELISPOT reader (Immunospot, Cellular Technology Limited, Cleveland, OH, USA). The number of IFN-γ-producing cells was expressed as spot-forming cells (SFC) relative to 1 × 10⁶ PBMCs. Values were calculated by subtracting the number of spots detected in the nonstimulated control wells. Values were considered positive if they were equal to or greater than 20 spots and at least three times above the means of the unstimulated control wells. As a positive control, cells were stimulated with CEF peptide pool (Mabtech, Stockholm, Sweden).

The mouse granzyme B and IFN-y ELISpot reagents were purchased from R&D Systems, and the assays were performed following the manufacturer's instructions. Briefly, 96-well filter plates (Millipore) were pre-coated with capture Ab overnight. Isolated CD8⁺ T cells and NK cells were plated with and without LLC stimulator cells and incubated, 37°C for 4 h. The plates were washed three times, the detection Ab was added, and the plates with Ab incubated overnight at 4°C. The plates were then developed using the ELISpot Blue Color Module (R&D Systems) and read on an ImmunoSpot (Cellular Technology) ELISpot reader.

Human peripheral blood mononuclear cells (obtained from Immunospot, Cellular Technology Ltd.) were cultured for 12 days in FBS-free DMEM with regular medium change, and plated at a density of 10⁵ cells/well in Immunospot ELISpot plates. Cells were then exposed to MS-Hu6, CEFT, or DMEM for 48 hr, following which IL-2 and IFN-γ expressing cells were quantitated per the manufacturer’s instructions.