Hl 1 serum free medium

Female BALB/c mice were immunized subcutaneously with 10μg of HEL (Sigma) or PBS (Gibco) emulsified in CFA (DIFCO) or IFA. Draining lymph nodes were harvested and single-cell suspensions were prepared in petri dishes containing DME (GIBCO). Large debris was removed by decanting, followed by two washes in DME. LN cells were cultured with 10 μg /ml HEL, 20ng/ml of PPD, 10 μg /ml of Mus musculus lysozyme 2 (ML2 ) peptide RAWVAWRAHCQ, 10 μg /ml of Mus musculus lysozyme 1 (ML1) peptide RAWVAWRTQCQ or PBS. Peptides were purchased from A&A Lab (San Diego).
Cells were suspended at a concentration of 5 × 10⁶ cells per milliliter in chemically defined, HL-1 serum-free medium (Lonza) supplemented with streptomycin and penicillin (Gibco). 24-well plates were used for culture. Incubation conditions maintained at 5% CO2 and 37°C. After 72 hr cell culture supernatants and cells were collected.

Our murine IFN-γ ELISpot kits (R&D Systems) were used according to the manufacturer’s suggested protocol. Freshly isolated splenocytes from naïve or tumor-bearing mice treated or untreated with anti-PD1 were resuspended in HL-1 serum-free medium (BioWhittaker) supplemented with 1% penicillin/streptomycin/l-glutamine, and were plated at 10⁶ per well in triplicate. Splenocytes were restimulated with the following synthetic HY-specific peptides: (i) Uty: the immunodominant Db–restricted HY-derived peptide corresponding to amino acids 245 to 253 (WMHHNMDLI) of the Uty gene product; (ii) Dby: the immunodominant I-Ab–restricted (NAGFNSNRANSSRSS) HY-derived peptide; and as specific controls (iii) for Db binding: the E7 peptide (RAHYNIVTF) derived from HPV and (4 (link)) I-Ab peptide: the T. cruzi peptide (SHNFTLVASVIIEEA). After wash steps and incubation with a biotinylated IFN-γ detection antibody, alkaline-phosphatase conjugated to streptavidin was added to enumerate IFN-γ–positive spots.

Popliteal lymph nodes were removed 10d after immunization in one hind footpad with 25μg rFliC or peptide. Assays were performed in HL-1 serum free medium (BioWhittaker, Lonza, Slough, UK), supplemented with L-glutamine and gentamicin (Life Technologies, Paisely, UK). The frequency of cells producing IFNγ was quantified by ELISpot (Diaclone, 2B Scientific, Oxon, UK). Two ×10⁵ cells and antigen were added to wells and plates incubated for 72h at 37°C in 5% CO₂. Spots were counted using an automated ELISpot reader (Autoimmun Diagnostika, Strasbourg). 50ng/ml Staphylococcal enterotoxin B was used as a positive control and culture medium only as a negative control. Results are expressed as for human ELISpot data above. The presence of an epitope was confirmed when the majority of mice in a group responded, and frequencies as Δsfc/10⁶ cells categorised as low (<25), intermediate (<100), or high (>100).