Tumor diameters were measured by calipers every 2 to 3 3 days

Tumor diameters were measured by calipers every 2 to 3 3 days. peptide epitopes within an inquiry polypeptide sequence according to an embedded interchangeable scoring matrix. For an inquiry polypeptide with amino acids, the Optimizer breaks the sequence into N-8 nine-mer peptides and calculates the scores one by one, as well as the maximum increase and maximum score obtainable with one amino acid substitution. ELISPOT Assays The ELISPOT assay has been previously described (24). Briefly, MultiScreen-IP plates (Millipore, Billerica, MA) were coated with 100 l anti-mouse IFN- antibody at a concentration of 100 g/ml (10 mg/ml; clone AN18; Mabtech, Inc. Cincinnati, OH). Purified CD4+ or CD8+ T cells (>95% enriched) from tumor, spleen or pooled inguinal lymph nodes, isolated using anti-CD4 or anti-CD8 MACS magnetic beads (Miltenyi Biotec, Inc. Auburn, CA), were plated at a concentration of 1 1 105 cells/well or as indicated. To assess CD8+ T cell responses, CD8+ cells were incubated at 37C for 20 hours with irradiated (10,000 rads) APCs, using either 1 104 EL-4 lymphoma cells pulsed with 1 g/ml peptide or B16 cells. For CD4+ T cell assays, CD4+ cells were incubated for 40 hours with 105 irradiated (3,000 rads) na?ve syngeneic splenocyte APCs pulsed with 10 g/ml peptide. Wells were then incubated with 100 l/well biotinylated antibody against mouse IFN- at a concentration of 4 g/ml (2 mg/ml; clone R4-6A2; Mabtech, Inc. Cincinnati, OH). Spots were developed with streptavidin-conjugated horseradish peroxidase and counted with an automated ELISPOT reader system with KS 4.3 software (Carl Zeiss MicroImaging, Inc. Thornwood, NY). Tumor challenge and measurement For tumor challenge, indicated numbers of B16 or LiHa cells with a viability >95% were used. Tumor cells were challenged intradermally into the lower right flank of mice. Tumor diameters were measured by calipers every 2 to 3 3 days. Mice were sacrificed when tumors ulcerated or reached a maximum diameter of 10 mm or mice showed any sign of discomfort. Flow cytometry Fluorochrome-labeled anti-mouse mAbs recognizing mouse CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), Ly-6G and Ly-6C (Gr-1) (RB6-11 8C5), NK1.1 (PK136), CD11b (M1/70) CD25 (3C7) and IFN- (XMG1.2) 5-BrdU were used from BD Pharmingen (San Diego, CA). Phycoerythrin-labeled anti-mouse Foxp3 (FJK-16s) mAb from eBiosciences was used in our experiments (San Diego, CA). Cells were assessed on a FACSCalibur or FACScan flow cytometer (Becton Dickinson, San Jose, CA) and results analyzed using FlowJo (TreeStar, San Carlos, CA). For tetramer assays, phycoerythrin-conjugated Db/WM455C463 tetramer, containing the H2-Db-restricted mutant Tyrp1 epitope WM455C463 (19), was made by Beckman Coulter (Fullerton, CA). One million cells were incubated with 1 l tetramer for 30 minutes at 37C and then incubated with fluorochrome-labeled anti-CD3, anti-CD4 and anti-CD8 antibodies (BD Pharmingen, San Diego, CA) for 20 minutes at 4C. Percentage of 5-BrdU tetramer positive cells was calculated within the CD3+CD4?CD8+ gate or 5-BrdU as indicated. For intracellular IFN- assays, lymphocytes were stimulated either with or without 0.1C1 g/ml peptide. Brefeldin A (Sigma-Aldrich, St. Louis, MO) was added after one hour at a concentration of 10 g/ml. Following incubation for 12 to 16 hours at 37C, cells were stained for surface markers, fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA), and stained for intracellular IFN- using phycoerythrin-conjugated anti-mouse IFN- (BD Pharmingen, San Diego, CA). Statistical analysis Differences in tumor-free survival were analyzed by log-rank analysis of Kaplan-Meier curves (comparisons pooled over strata using SPSS 10.0 software for Windows). Statistical differences and T cell responses between groups of mice were determined by two-tailed, non-parametric Mann Whitney test (GraphPad Prism 4.0, San Diego, CA). Results Generation of tumor cells expressing wild-type and mutant Tyrp1 To investigate natural tumor immunity elicited by immunogenic point mutations in mutant self-proteins, mutant self-genes encoding defined immunogenic point mutations were expressed in inherently immunogenic LiHa tumor cells and poorly immunogenic B16 tumor cells (6). Native and a mutated version of mouse tyrosinase-related protein-1 (Tyrp1) were selected for this study based on extensive characterization of the 5-BrdU biology and immunogenicity of these proteins (19, 22, 25C30). We have previously reported the mutant form of mouse Tyrp1, Tyrp1-WM, which Ets2 contains point mutations to create heteroclitic.