Extended Data Fig. 3: mITGB1 is a major MHC-II-restricted neoantigen in T3 sarcomas.
From: MHC-II neoantigens shape tumour immunity and response to immunotherapy
a, b, T3 MHC-II neoantigen predictions for all expressed mutations were made using hmMHC (a) and netMHCII-2.3 (b) (netMHCIIpan-3.2 predictions yielded very similar results, data not shown). The predictions are shown as −log10odds predictor value or logIC50 (smaller values indicate higher likelihood of being presented by I-Ab) and expression level (FPKM). Strong binders are defined as mutations residing in the second percentile of I-Ab binding predictions for random natural peptides for each algorithm (−log10odds ≤ 26.21 or logIC50 ≤ 343.8 nM). The N710Y mutation in ITGB1 met the strong binder threshold in the hmMHC predictions but not in the netMHCII-2.3 predictions. Red dots indicate all mutations that were screened for CD4+ T cell reactivity. Green line denotes high-expression cut-off (FPKM = 89.1). Blue line indicates strong binder cut off for each algorithm. c, Two million T3 sarcoma cells were injected subcutaneously into syngeneic mice and CD4+ TILs were isolated on day 12. IFNγ ELISPOT was performed using naive splenocytes pulsed with 2 μg ml−1 of the indicated peptides. Data are shown as mean of three independent experiments ± s.e.m. d, Gating strategy for pI-Ab tetramer staining of whole TILs. e, Quantification of mITGB1–tetramer and CLIP–tetramer staining of CD4+ T cells from whole T3 TILs 12 days after transplantation. Data are shown as mean ± s.e.m. per cent tetramer-positive cells of CD4+ cells from three independent experiments. f, Syngeneic 129S6 mice were injected subcutaneously with 2 × 106 T3 sarcoma cells and TIL-derived CD4+ T cells were collected 12 days after transplantation. CD4+ T cells were stimulated with naive splenocytes pulsed with 2 μg ml−1 OVA323–339 control or mITGB1697–724 peptide for a flow-based multi-cytokine array. Representative data from one of two independent experiments using pools of five tumours each are shown as average of technical triplicate wells from three pooled tumours.
