Volume 138, Issue 3, Pages 1178-1188 (March 2010) Chronic Pancreatitis Is Associated With Disease-Specific Regulatory T-Cell Responses  Hubertus Schmitz–Winnenthal, Dong–Ho Kim Pietsch, Simon Schimmack, Andreas Bonertz, Florian Udonta, Yingzi Ge, Luis Galindo, Sebastian Specht, Christine Volk, Kaspar Zgraggen, Moritz Koch, Markus W. Büchler, Jürgen Weitz, Philipp Beckhove  Gastroenterology  Volume 138, Issue 3, Pages 1178-1188 (March 2010) DOI: 10.1053/j.gastro.2009.11.011 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 TT-specific type-1 TC in CP-patients, PaCa, and HD. (A) TT-specific type-1 and CP-specific regulatory TC in BM of 1 exemplary CP patient. IFN-γ, IL-4, and IL-10 spot numbers secreted after TC stimulation with TT, antigens from the autologous CP-lesion (CP-L), or with negative control antigens (lysate from autologous PBMC; PBL) are shown as mean + SEM of 3 wells/group. *Significant difference (P < .05) between spot numbers in test wells and control wells. (B–D) Numbers of TT-reactive TC in BM and PB of HD (B, n = 21 or 22), CP patients (n = 15–17 (IFN-γ) or 7–13 (IL-4, IL-10); C) or 4 PaCa patients (D). Overall reactivity of TC stimulated with TT or huIgG (control) are shown as mean + SEM spots/105 T cells. N, numbers of tested individuals. (E) Frequencies of TT-specific TC in BM and PB of HD, CP, or PaCa patients analyzed by 40-hour IFN-γ-, IL-4-, or IL-10 ELISPOT assays. TC frequencies in samples containing significantly elevated numbers of cytokine secreting cells in test wells compared with control wells (responders) are shown as individual dots. Nonresponders are depicted at the bottom line. Full lines depict median values of all tested samples per group. P, P values for differences in cytokine reactivity are depicted only in case of statistical trend or significance (P < .1). Gastroenterology 2010 138, 1178-1188DOI: (10.1053/j.gastro.2009.11.011) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 CP-specific TC in CP patients. (A) Lysates from CP lesions do not influence cytokine secretion by TC from HD. Induction of IFN-γ, IL-4-, or IL-10 secretion by PBTC from HD in 40-hour ELISPOT assay upon stimulation with allogeneic CP-L or corresponding PB-L presented by lysate-pulsed autologous DC. Numbers of cytokine-secreting cells/105 purified TC are shown as means + SEM from cumulative data of n = 5 tested HD. (B) Presence and functional phenotype of CP-specific TC in BM and PB of 17 CP patients, stimulated by autologous DC pulsed with lysate from autologous CP-L or autologous PBMC-lysate (PB-L) in short-term IFN-γ (n = 15–17), IL-4, or IL-10 (n = 7–13) ELISPOT assays. Means and SEM of all tested individuals are shown. (C) Frequencies of cytokine-secreting TC specific for antigens derived from CP lesions in BM and PB of 5 healthy donors or 17 CP patients (CP) (BM: n = 14–17; PB: n = 8 or 9), respectively, analyzed by 40-hour IFN-γ, IL-4, or IL-10 ELISPOT assays are shown as individual dots. Nonresponders are depicted at the bottom line. Full lines depict median values. Gastroenterology 2010 138, 1178-1188DOI: (10.1053/j.gastro.2009.11.011) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Type-1 phenotype of PaCa-specific TC in PaCa patients. (A) PaCa lysates do not influence cytokine secretion by TC from HD. Induction of IFN-γ, IL-4, or IL-10 secretion by PBTC from HD in 40-hour ELISPOT assay upon stimulation with allogeneic lysates from PaCa (Ca-L) or PB-L presented by lysate-pulsed autologous DC. Mean + SEM numbers of cytokine-secreting cells/105 purified TC from n = 7 HD are shown. (B) Presence of PaCa-specific TC in BM and PB of CP patients. Reactivity of TC from BM or PB of 25 individuals stimulated by autologous DC pulsed with lysate from autologous PaCa (Ca-L) or autologous PB-L in short-term IFN-γ, IL-4, or IL-10 ELISPOT assays are shown as mean + SEM of all tested patients. (C) Frequencies of tumor-specific TC in BM and PB of HD or PaCa patients analyzed by IFN-γ- IL-4, or IL-10 ELISPOT assays are depicted as individual dots. Nonresponders are depicted at the bottom line. Full lines depict median values. Gastroenterology 2010 138, 1178-1188DOI: (10.1053/j.gastro.2009.11.011) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 TC reactivity against pancreatic antigens in CP patients differs from that of PaCa patients. Proportions of patients with CP or PaCa containing type-1 (IFN-γ), type-2 (IL-4), or regulatory (IL-10) type TC responses against autologous pancreatic lesion-derived antigens in BM or PB. P values indicate significant differences between secreted cytokines (horizontal lines) or between CP and PaCa (asterisks) analyzed by Fisher test. Gastroenterology 2010 138, 1178-1188DOI: (10.1053/j.gastro.2009.11.011) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Pancreas-specific TC in CP and PaCa patients are not cross-reactive. (A–D) Presence of MUC1-reactive TC in PaCa patients but not in CP patients. Cumulative results of IFN-γ- and IL-10 ELISPOT analyses of BMTC and PBTC from 5 PaCa patients (A and C) or 3 CP patients (B and D). Mean + SEM spot numbers after TC stimulation with MUC1p1–100 (MUC-1) or huIgG as negative control antigen (IgG) are shown. (E) Frequencies of MUC1-specific TC in BM or PB samples of PaCa patients (PaCa, n = 11) and in CP patients (CP, n = 6) as detected by IFN-γ (PaCa) or IL-10 (CP) ELISPOT assays. (F and G) Tissue-specific TC of CP patients and PaCa patients recognize different antigens. (F) Cumulative results of increased TC reactivity in the blood of PaCa patients against pooled lysates from 5 allogeneic pancreatic carcinomas (PaCa-L) but not against pooled lysates from 5 allogeneic pancreatitis lesions (CP-L) compared with pooled allogeneic PBMC lysates (PB-L) tested by IFN-γ ELISPOT assay. Bars show mean + SEM of spot numbers in triplicate wells. (G) Exemplary result of increased TC-reactivity in a CP patient against pooled CP-L but not against pooled PaCa lysates (PaCa-L) compared with pooled allogeneic PB-L tested by IFN-γ ELISPOT assay. Bars show mean + SEM of spot numbers in triplicate wells. P values for differences between test and control groups are depicted only in case of statistical trend or significance (P < .1), N, numbers of individuals tested. Gastroenterology 2010 138, 1178-1188DOI: (10.1053/j.gastro.2009.11.011) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Accumulation of Treg in CP patients compared with HD. (A) Exemplary flow cytometric detection of Treg (percent CD3+CD4+EMA−CD25+FoxP3+ Treg gated on CD3+CD4+ cells) in BM, PB, and CP lesions. (B) Flow cytometry showing one representative histogram out of n = 3 CP patients of CD127 expression in Treg (CD3+CD4+EMA−FoxP3+CD25high) and Tcon (CD3+CD4+EMA−FoxP3-CD25low). (C) Mean + SEM proportions of Treg in freshly isolated BM, PB, and CP lesions from 4 CP patients and 10 HD. (D) Increased proportion of IL-10+ among ex vivo isolated Treg compared with Tcon (CD3+CD4+CD25−FoxP3−) in tissue-infiltrating lymphocytes in CP patients but not in PaCa patients. Columns show mean values from 2–5 patients measured in duplicates ± SEM. Numbers depict P values of significant differences between indicated groups. Gastroenterology 2010 138, 1178-1188DOI: (10.1053/j.gastro.2009.11.011) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Treg, but not Tcon, secrete IL-10 in response to stimulation with CP antigens. (A) Eighty percent of CD3+CD4+CD25high Treg coexpress FoxP3 as shown as representative histogram (left) and as the mean proportion from 6 different patients (right). (B) Exemplary results show the dose-dependent suppressive effect of isolated Treg (CD3+CD4+CD25+) polyclonally activated and cocultured with Tcon at varying ratios. Bars show mean ± SEM. (C) Antigen-unspecific polyclonal stimulation with Staphylococcus aureus enterotoxin B resulted in IL-10 secretion of Treg but not Tcon as shown by mean + SEM IL-10 spot numbers in an exemplary ELISPOT assay. Treg or corresponding Tcon stimulated with PB-L were used as negative control groups. (D) Treg isolated from BM, PB, or CP lesions were stimulated with CP antigens (+) or control antigens (−) and analyzed for intracellular coexpression of IL-10 and IFN-γ by flow cytometry. Proportions of IFN-γ+ IL-10+ (open columns) and of IFN-γ− IL-10+ (solid columns) Treg subsets among total IL-10+ Treg are shown (n = 3; 4–9 data points per group). Columns show mean ± SEM. (E and F) Treg but not Tcon from PB or CP lesions stimulated with autologous CP antigens but not with Tu-L secreted IL-10 determined by IL-10 ELISPOT as shown by mean + SEM spot numbers of triplicate wells/group compared with TC stimulated with negative control antigens (PB-L). Gastroenterology 2010 138, 1178-1188DOI: (10.1053/j.gastro.2009.11.011) Copyright © 2010 AGA Institute Terms and Conditions

Figure 8 Treg in CP patients recognize CP antigens. (A and B) Exemplary Treg-specificity assays with Treg/Tcon isolated from BM (A) or from a CP lesion (B). Columns show mean counts per minute (c.p.m.) of 3 wells per antigen ± SEM; P < .05, significant difference in counts in test wells (auCP-L) compared with negative control antigens (PB-L). Cumulative data from 8–10 test samples derived from 4 CP patients show that Treg stimulated with CP-L suppressed TC proliferation stronger than Treg stimulated with PB-L (C) and an increased extent of TC suppression when Treg were prestimulated with CP antigens compared with PaCa antigens (D). Columns show mean ± SEM. (2-tailed Student t test: unpaired, except for C, which is paired t test). (E) The ratio of tissue-specific concentrations of IL-10: IFN-γ in tissue lysates of 11 CP lesions (CP) or of 10 individual carcinomas PaCa as determined by ELISA reflect the dominant phenotype of tissue-specific PB- and BM-derived TC in both diseases. (F) Increased IL-10-positive areas (left) and increased relative expression of IL-10 compared with IFN-γ (right) in PaCa tissues compared with CP lesions as determinded by 2-color fluorescence immunohistochemistry. Columns show mean values from 3 CP and 3 PaCa patients ± SEM. Numbers depict P values of significant differences between indicated groups. Gastroenterology 2010 138, 1178-1188DOI: (10.1053/j.gastro.2009.11.011) Copyright © 2010 AGA Institute Terms and Conditions