Fig 1.
Experimental pipeline for identifying and predicting immunogenic mutations in the ID8-G7 tumor line.
Mutations were identified by whole exome sequencing, and expression was assessed using RNA-seq. Epitope prediction was performed using NetMHCpan2.4[52]. Starting with 92 mutations in genomic DNA, 17 mutations within peptides predicted to bind MHCI (IC50 < 1500 nM) were advanced to subsequent experiments.
Table 1.
Predicted epitopes and 29mer peptides selected for vaccination study.
Each mutation that was found in a peptide predicted to bind to H-2Kb or H-2Db with an IC50 < 1500 nM was annotated. Shown for each mutation are the amino acid substitution, predicted highest binding epitope, binding score, binding H-2 allele, the RNA-seq read count supporting the mutated and wild type allele, and the 29mer peptide used for vaccination.
Fig 2.
Immunogenicity of mutant peptides.
Mice (n = 3 per group) were vaccinated daily on days 0–3 and 21–24 with mutant 29mer peptides (50 μg, one peptide per group) and poly(I:C) (10 μg). On day 28, mice were euthanized, and splenocytes (106 cells/well) were stimulated in duplicate with mutant 29mer peptides (20 μg/ml) or media, and assessed by IFN-γ ELISPOT. Positive control mice were vaccinated with a 29mer peptide (long-SIINFEKL) encompassing the known OVA257-264 epitope. A. Eleven of 17 mutant peptides elicited robust T cell responses. Dots represent responses in individual mice, and bars represent the mean for three mice. The dashed line shows the threshold for positivity (3 x maximum background for splenocytes in media alone determined post-hoc). B. Representative ELISPOT wells from 3 mice vaccinated with mutant Cul2 peptide and stimulated in duplicate with mutant Cul2 peptide or media alone.
Fig 3.
T cell specificity for mutant versus wild type peptides.
Mice (n = 2 per group) were vaccinated as in Fig 2. On day 28, mice were euthanized, and splenocytes (5 x 105 cells/well) were stimulated in duplicate with titrated concentrations of mutant or wild type 29mer peptides in IFN-γ ELISPOT wells. Dots and bars represent the mean and range of responses, respectively.
Fig 4.
Determination of CD4 versus CD8 T cell responses to mutant peptide vaccines.
Mice (n = 2 per group) were vaccinated as in Fig 2. On day 28, splenocytes were harvested from vaccinated mice, incubated overnight in media containing the indicated mutant peptides or media alone, and analyzed by flow cytometry. Cells were gated on viability and lack of MHCII expression (activated murine T cells are MHCII negative). The percentage of IFN-γ-secreting CD8 and CD4 T cells was determined. A. Example of a mixed T cell response (CD8 top and CD4 bottom) to mutant Dync1h1 29mer peptide versus media alone. B. Summary of data for 7 mutant peptides and the positive control peptide long-SIINFEKL. Bars and dots represent mean and individual responses, respectively.
Fig 5.
Therapeutic and prophylactic vaccination with mutant peptides.
A. For therapeutic vaccination, mice (n = 5 per group) were inoculated with ID8-G7 tumor cells (106 cells/mouse) on day 0 and vaccinated on days 3–6 with individual mutant peptides (50μg) and poly(I:C) (10μg). As a positive control, one group of mice received adoptive transfer of OT-I splenocytes on day 2, followed by vaccination on days 3–6 with OVA protein (100 μg) and poly(I:C) (10 μg). Non-vaccinated mice served as negative controls. Each graph represents one group of 5 mice vaccinated with a single mutant 29mer peptide. The same group of positive and negative control mice were used for each graph. Mice were euthanized once they displayed abdominal distension due to ascites. B. For prophylactic vaccination, mice (n = 5 per group) were vaccinated on days -28 to -25 and -7 to -4 with mutant 29mer peptides (25 μg, 1 peptide per group) and poly(I:C) (10 μg). As a positive control, one group of mice received adoptive transfer of OT-I splenocytes on day -8 followed by vaccination on days -7 to -4 with OVA protein (100 μg) and poly(I:C) (10 μg). As negative controls, one group of mice was vaccinated with an irrelevant peptide, and a second group of mice received no vaccination. On day 0, mice received intraperitoneal inoculation of 106 ID8-G7 tumor cells. Mice were euthanized once abdominal distension due to ascites was observed.
Fig 6.
In vitro assessment of mutation-reactive T cells for recognition of ID8-G7 tumor cells.
Mice were vaccinated as in Fig 2, and splenocytes were harvested on day 28. OT-I splenocytes were used as positive controls. Splenocytes (5 x 105 per well) were stimulated in duplicate with cognate mutant peptide (20 μg/ml), media alone, or ID8-G7 tumor cells (105 cells per well) and assessed by A. IFN-γ ELISPOT, or B. IL-2 ELISPOT. C. To assess tumor recognition by mutation-reactive CD4 T cells, 4-1BB+OX40+ CD4 T cells from the above mice were FACS purified and cultured in recombinant murine IL-2 (100 U/ml). ID8-G7 tumor cells were incubated in IFN-γ (100 U/ml) for 72 hours to cause upregulation of MHCI and MHCII. D. Sorted and expanded CD4 T cells (104 per well) were stimulated in duplicate with either 20 μg/ml of cognate mutant peptide, media, ID8-G7 tumor cells or ID8-G7 tumor cells incubated in IFN-γ for 72 hours. OT-I T cells were used as a positive control for tumor cell recognition. Bars and lines represent mean and standard deviation of duplicate wells, respectively.
Fig 7.
Mutation and neoantigen loads in the human HGSC and lung cancer TCGA datasets.
Individual cases are aligned on the X axes in rank order according to the total number of mutations identified by whole exome sequencing (panel A); the same order of cases was used in panels B and C. Y axes indicate the number of mutations meeting the indicated criteria for each panel. Lung cancer cases (n = 603) are shown as solid black triangles, whereas HGSC cases (n = 274) are shown as hollow circles. A. Somatic, non-synonymous mutations identified by whole exome sequencing (total mutations). B. Somatic, non-synonymous mutations with at least one corresponding read in the RNA-seq data (transcribed mutations). C. Peptides predicted to bind autologous HLA-A with an IC50 < 100nM and containing somatic, non-synonymous, transcribed mutations. Only samples with unambiguous HLA-A allele calls are shown (lung n = 158, HGSC n = 220).