Patient T cells recognise a SMAD4V370A-containing neoepitope presented by autologous cancer cells. Patient T cells recognise a SMAD4V370A-containing neoepitope.

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Patient T cells recognise a SMAD4V370A-containing neoepitope presented by autologous cancer cells. Patient T cells recognise a SMAD4V370A-containing neoepitope presented by autologous cancer cells. (A) HLA class I and HLA-DR expression in 1247 cancer cells following 48 h IFNγ induction. (B–D) IFNγ ELISPOT of CD8+ T cells from 1247 patient, stimulated with a peptide pool encompassing the SMAD4V370A mutation, assayed for the recognition of: 1247 cancer stem/initiating cells (CSCs) and differentiated colorectal cancer (CRC) cells (B), of SMAD4 mutated peptides presented by HLA-A*0201+ T2 cells (C), of SMAD4m-1 and SMAD41 WT peptides presented by T2 cells (D), ±anti-class I or HLA-DR mAbs. (E) Percentage of GFP expression by sorted HEK293t cells transduced with retroviral vectors encoding the 1247 SMAD4V37A mutated or the corresponding SMAD4V37 WT minigenes; (F) Upper panel. SMAD4V37A-specific CD8+ T cells assayed by IFNγ ELISPOT for the recognition of HEK293t cells transduced with the SMAD4 minigenes, or of three HLA-A*02:01+ CRC cell lines negative for the SMAD4V37A mutation, ±anti-class I mAb. Lower panel. PCR typing for the expression of the SMAD4V37A mutation, or the corresponding SMAD4V37 WT sequence in the CRC cell lines shown in the upper panel, and in untransduced HEK293t (293t) cells. The 1247 and 1869 CRC cell lines are positive and negative controls for the PCR, respectively. (G) SMAD4m-1 peptide-stimulated CD8+ T cells assayed by IFNγ ELISPOT (right panel) for the specific recognition of the peptide epitopes of different lengths (left panel), presented by T2 cells. All IFNγ ELISPOT data are triplicate mean±SD, subtracted of the background spots produced by T cells alone, and are representative of three independent experiments performed with independently induced CD8+ T cell lines. Only the experiment in panel F was performed twice with the same CD8+ T cell line. *p≤0.05; **p≤0.01; ***p≤0.001; ns, non-statistically significant. Daniele Mennonna et al. Gut doi:10.1136/gutjnl-2015-309453 Copyright © BMJ Publishing Group Ltd & British Society of Gastroenterology. All rights reserved.