Volume 134, Issue 4, Pages 988-997 (April 2008) Immune Response Against Frameshift-Induced Neopeptides in HNPCC Patients and Healthy HNPCC Mutation Carriers  Yvette Schwitalle, Matthias Kloor, Susanne Eiermann, Michael Linnebacher, Peter Kienle, Hanns Peter Knaebel, Mirjam Tariverdian, Axel Benner, Magnus von Knebel Doeberitz  Gastroenterology  Volume 134, Issue 4, Pages 988-997 (April 2008) DOI: 10.1053/j.gastro.2008.01.015 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Frequency of FSP-specific TiTc from 3 patients with MSI-H CRC expanded with aCD3/aCD28. TiTc (1 × 105 cells/well) were incubated with autologous TiBc (4 × 104 cells/well) pulsed with FSP or with TiBc without FSP (no peptide) as targets. Specific IFN-γ production of TiTc against 13 FSP preparations is shown. The number of IFN-γ releasing activated TiTc (spots) among the total number of Tc analyzed (105) and the standard deviation from 6 replicate wells are given. Gastroenterology 2008 134, 988-997DOI: (10.1053/j.gastro.2008.01.015) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Cytotoxic potential of TiTc expanded with aCD3/aCD28 toward MSI-H HLA-A2+ colon carcinoma cell lines HCT116 and Colo60H compared with MSI-H HLA-A2− LS180 cells and MSS HLA-A2+ SW480 cells. TiTc were stained with CMTMR, colon carcinoma cells with CMFDA, respectively. The upper row shows TiTc and tumor cells from separate cultures, which were mixed before flow cytometry analysis (control). The lower row shows TiTc and tumor cells from common culture after 48 hours (in vitro killing). Before flow cytometry analysis, harvested cells were incubated with propidium iodide (PI). The number of CMFDA+ PI− tumor cells is depicted in lower right quadrants of each blot. The number of CMFDA+ PI+ tumor cells is depicted in upper right quadrants. Gastroenterology 2008 134, 988-997DOI: (10.1053/j.gastro.2008.01.015) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Frequency of FSP-specific pTc from patients with MSI-H CRC (A) and healthy mutation carriers (B) compared with patients with MSS CRC (C) and healthy controls (D). pTc (1 × 105 cells/well) were incubated with autologous pBc (4 × 104 cells/well) pulsed with FSP or with pBc without FSP (no peptide) as antigen-presenting cells (APC). Specific IFN-γ production of pTc against 13 FSP preparations is shown. The number of IFN-γ releasing activated pTc (spots) among the total number of Tc analyzed (105) and the standard deviation from 6 replicate wells are given. Patient numbers are given in the corners of each of the panels. Gastroenterology 2008 134, 988-997DOI: (10.1053/j.gastro.2008.01.015) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Schematic illustration of ELISpot results. Samples are arranged along the x-axis, FSP antigens along the y-axis. Squares represent normalized mean counts of IFN-γ-secreting T cells after subtraction of background and standard errors of peptide-specific counts and no peptide control counts. Open boxes, adjusted T-cell count ≤0; shaded boxes, adjusted T-cell count 0 to <2.5; solid boxes, adjusted T-cell count ≥2.5. Threshold between shaded and solid boxes is derived from highest adjusted counts observed in healthy control individuals. Gastroenterology 2008 134, 988-997DOI: (10.1053/j.gastro.2008.01.015) Copyright © 2008 AGA Institute Terms and Conditions