Automated elispot reader

Spleens from vaccinated animals were collected aseptically, homogenised, and red blood cells were lysed. Splenocytes were resuspended in RPMI medium (Sigma-Aldrich) supplemented with 5% FBS, 2 mM L-Glutamine, 100 U penicillin & 0.1 mg/ml streptomycin, 50 μM 2-mercaptoethanol and 25 mM HEPES solution (Sigma-Aldrich). Splenocytes were assessed for antigen recall response via IFN-γ ELISpot (Mabtech, Sweden), performed as per the manufacturer's instructions. Cells were seeded in PVDF microtitre plates at 2×10⁵ per well and re-stimulated with peptide pools (Mimotopes, Australia). Peptides spanning the tPA-GP-V5 fusion protein sequence were 20 residues long, with an overlap of 12 residues between peptides. They were applied to cells at a final concentration of 2.5 μg/ml per peptide, with 28–32 peptides per pool. Plates were developed after 18 hours at 37°C, 5% CO₂ in a humidified incubator. Spots were counted visually on an automated ELISpot reader (Autoimmun Diagnostika, Germany). Background values from wells containing cells and medium but no peptides, were subtracted and pools summed across the target protein. Results were expressed as spot forming units (SFU) per 10⁶ cells.

To test T cell reactivity against the target cell lines (BR5-FOXL2 and 4T1-FOXL2), cocultures were established in an ELISpot plate using 5 × 10⁵ splenocytes or 1 × 10⁵ T cells from vaccinated mice, respectively. 4T1-FOXL2 and 4T1 WT were pretreated for 24 hours with 5 ng/mL of rIFN-γ used to increase MHCI expression levels. Cells were then washed twice before starting the coculture. Ninety-six–well MAIP plates (MilliporeSigma; N4510) were coated overnight with a 1:400 dilution in sterile PBS of rat anti–mouse IFN-γ (BD Biosciences; clone R4-6A2, 551216). Splenocytes were plated at 0.1 × 10⁶ to 1 × 10⁶ cells/well and incubated overnight at 37°C with 1 μg/mL peptides. After incubation, plates were washed with PBS and 0.05% Tween-20 (Bio-Rad; 170-6531) and incubated with anti–mouse biotin-conjugated anti–IFN-γ antibody (BD Biosciences; clone R4-6A2, 551506). After washing the plate, Streptavidin-alkaline phosphatase conjugate (BD Biosciences; 554065) was then added for 30 minutes. Plates were developed by adding nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Pierce), and spots were then counted using an automated ELISpot reader (Autoimmun Diagnostika GmbH).

IFN-γ production was quantified after 24 h using a human IFN-γ ELISpot kit (Mabtech), according to manufacturer’s instructions. Briefly, Millipore MultiScreen-HA 96-well filter plates (Millipore) were coated with 5 μg/mL IFN-γ mAb in PBS overnight at 4 °C. Cells were seeded at 2 × 10⁵ cells/well in cIMDM and incubated with the relevant compounds overnight. After washing with PBS, plates were incubated with biotin-labeled anti-IFN-γ for 1 h at room temperature. Plates were then washed and treated with 1:1000 dilution of streptavidin-alkaline phosphatase (Sigma) for 30 min at room temperature. Plates were developed with nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate substrate (Mabtech) until all spots were clearly visible. Developed plates were dried and counted on an automated ELISpot reader (Autoimmun Diagnostika, Strassberg, Germany). Cells treated with PHA were used as a positive control.

Mice were immunized s.c. in one hind footpad with 1, 5, or 25 μg of rOprF protein (20 (link)) emulsified with Hunter “TiterMax Gold” adjuvant (Sigma-Aldrich) or with adjuvant emulsified with PBS alone. At 11 d postimmunization, the draining popliteal lymph nodes were harvested and disaggregated into a single-cell suspension. CD4⁺ T cells responding to OprF Ag were quantified by IFN-γ ELISPOT (Diaclone; 2BScientific) performed in HL-1 serum-free medium (BioWhittaker) supplemented with l-glutamine and penicillin–streptomycin. Cells (2 × 10⁵) plus Ag were added to wells of precoated anti–IFN-γ ELISPOT plates and incubated for 72 h at 37°C with 5% CO₂. Plates were developed according to the manufacturer’s instructions, and spots were counted on an automated ELISPOT reader (Autoimmun Diagnostika). Replica stimulations were set up in normal tissue culture plates for the measurement of IFN-γ in culture supernatants by ELISA.

After 8–10 days of culture, the cells were washed and incubated for 18 h with RPMI 1640/FCS at 37°C in 5% CO₂ with one of the Sp17 peptides, HIV control peptide, PHA, or medium only. Peptides were used at 10 μmol and all cultures were carried out in triplicate. Gamma-interferon (γ-IFN) release assays were performed according to manufacturer's instructions (Mabtech, Stockholm, Sweden). Spots were counted using an automated ELISPOT reader (Autoimmun-Diagnostika, Strasberg, Germany). Results were considered positive if the number of spots in the test wells was at least twice those present in the control cultures (media only or containing the irrelevant HIV-1 peptide) and assays were excluded if there were more than 25 spots per well in the absence of peptides. All tests were performed in triplicate.