Fig 2: Blood and pancreatic tissue samples of CP patients contain increased numbers of Treg cells.T cells were isolated from EDTA blood samples of patients (CP) and blood doners (BD) and were analyzed by flow cytometry. a The T cell activation marker CD25 (p = 0.0317, BD n = 10/CP n = 7) was significantly elevated in CP patients compared to BD, whereas the elevation of the surface expression of CD69 (BD n = 10/CP n = 7) did not reach significance. T cell differentiation was analyzed by intracellular transcription factor staining of FOXP3, GATA3 and TBET. b FOXP3+CD25+ Treg cells were significantly elevated in the blood of CP patients (p = 0.0345, BD n = 10/CP n = 7), c in the same manner as GATA3+ Th2 cells (p = 0.0026, BD n = 10/CP n = 7). d Interestingly also TBET+ Th1 cells were significantly increased (p = 0.0478, BD n = 10/CP n = 7). e Representative immunofluorescence labeling images of CD4 and FOXP3 illustrate a high number of CD4+FOXP3+ Treg cells in human CP tissue. f Summary illustration showing how regulatory T cells control organ fibrosis during chronic pancreatitis by suppressing Th2/ILC2-mediated alternative activation of macrophages via IL-4/IL-13. Alternatively activated macrophages in turn induce activation of pancreatic stellate cells (PSC) which contribute to extracellular matrix production and fibrosis. All data were presented as means ± SEM, statistically significant differences were tested by unpaired two-tailed students t-test for independent samples and significance levels of p < 0.05 are marked by an asterisk (a–d). Source data are provided as a Source Data file.

Fig 3: Systemic immune response during chronic pancreatitis in mice.Splenocytes were analyzed from control (con) and chronic pancreatitis (CP) mice. a T cell (CD4+) differentiation is shown by intracellular transcription factor staining of GATA3 for Th2 cells (p = 0.0016, con n = 4/CP n = 8), b TBET for Th1 cells, c ROR?t for Th17 cells and d FOXP3 in combination with CD25 to discriminate Treg cells (p = 0.0085, con n = 4/CP n = 8). e, f In contrast to GATA3+ Th2 cells an increase of CD206+ or CD163+ alternatively activated macrophages could not be observed. g Serum concentration of IL-4 was significantly increased (p = 0.0422, con n = 7/CP n = 15), whereas the rise of IL-10, IFN?, TNF, and IL-17A concentrations were not significant (con n = 7/CP n = 15). h The intracellular cytokine production of CD4+ T cells was analyzed by labeling for IL-4, IL-13, IFN?, TNF, and IL-10 after brefeldin a/monensin treatment. Th2 cytokines IL-4 (p = 0.0443, con n = 7/CP n = 15), IL-13 (p = 0.0455, con n = 7/CP n = 15) and IL-10 (p = 0.0294, con n = 7/CP n = 15) were elevated in CD4+ T cells, whereas of Th1 cytokines only IFN? (p = 0.0072, con n = 7/CP n = 15) was elevated but not TNF (con n = 7/CP n = 14). i Gene expression in splenocytes was also analyzed by RT-qPCR for Il4 (p = 0.0250, con n = 7/CP n = 15), Il13 (con n = 3/CP n = 13), Il10 (con n = 6/CP n = 15), Tnf (con n = 7/CP n = 15) and Ifng (con n = 6/CP n = 15) and confirmed the Th2 differentiation. Transcript levels as determined by RT-qPCR were normalized using Rn5s as internal calibrator gene and were related to the corresponding mRNA amounts in control mice. All data were presented as means ± SEM, statistically significant differences were tested by unpaired two-tailed students t-test for independent samples and significance levels of p < 0.05 are marked by an asterisk (a, d, g, h, i). Source data are provided as a Source Data file.

Fig 4: ILC2 are suppressed by regulatory T cells.Splenocytes of CP animals, additionally treated with DT or PBS were isolated and analyzed by flow cytometry. a The fraction of ILCs was discriminated as lin-CD45+CD127+CD90+ cells (PBS n = 6/DT n = 7). b The presence of the transcription factors GATA3 and TBET was used to differentiate ILC1 and ILC2. c, d Treg depletion increases the number of GATA3+ ILC2 (p = 0.0064, PBS n = 6/DT n = 7) but not of TBET+ ILC1 (PBS n = 6/DT n = 7) in spleen. e Isolated leukocytes from CP tissue of mice were investigated for pancreatic ILCs. The total number of lin-CD45+CD127+CD90+ cells was dramatically increased in the absence of Treg cells (p < 0.0001, con PBS/DT n = 5/ CP PBS/DT n = 11), f and the number of GATA3+ ILC2 was significantly increased (p = 0.0155, PBS n = 7/DT n = 11). g Expression analysis of Areg encoding amphiregulin in pancreatic tissue by RT-qPCR confirms an increase in Treg-depleted animals during CP (p = 0.0253, PBS n = 7/DT n = 6). Transcript levels as determined by RT-qPCR were normalized using Rn5s as internal calibrator gene and were related to the corresponding mRNA amounts in control mice. h, i Representative immunofluorescence labeling of CD90+ AREG+ detected AREG in pancreatic ILC2 from CP animals as well as in human tissue sections, scale bars represent 20 µm. All data were presented as means ± SEM, statistically significant differences were tested by unpaired two-tailed students t-test for independent samples and significance levels of p < 0.05 are marked by an asterisk (d–g). Source data are provided as a Source Data file.

Fig 5: Chronic pancreatitis leads to pancreatic tissue remodeling.a Flow cytometry analysis of isolated leukocytes from pancreatic tissue showed a significant increase of CCR2+ cells (p = 0.0032, con n = 7/CP n = 15). b CD11b+ labeling identified that also cells of the innate immune system infiltrate into the damaged pancreas (p < 0.0001, con n = 7/CP n = 15). c Most of these cells were positive for CD206 (p < 0.0001, con n = 7/CP n = 15) or CD163 (p < 0.0001, con n = 7/CP n = 15), which characterized them as alternatively activated macrophages. d Labeling of CP tissue for CD206 illustrates their increase in pancreatic tissue of mice. A quantative analysis showed that ~9% of all cells were positive for CD206 in CP tissue (p < 0.0001, con n = 5/CP n = 9), scale bars represent 20 µm. e IL-33 expression of CD206+ macrophages was demonstrated by immunofluorescent labeling, scale bars represent 20 µm. f This cytokine is a strong inducer of ILC2s (lin-CD45+CD127+CD90+GATA3+) which were also significantly elevated in the pancreas of CP mice (p = 0.0002, con n = 7/CP n = 15). g The replacement of exocrine pancreas by fibrotic tissue in CP was illustrated by azan blue staining of the pancreas, scale bars represent 50 µm. h Flow cytometry analysis of isolated pancreatic cells for PSC marker proteins CD271 (p < 0.0001, con n = 7/CP n = 15) and GFAP (p < 0.0001, con n = 7/CP n = 15) identified a significant increase. i Serum collagen levels were significantly elevated in CP (p = 0.0433, con n = 7/CP n = 13) and collagen deposition and tissue fibrosis were observed in histology. j Masson Goldner trichrome staining was utilized to evaluate the tissue remodeling during CP, scale bars represent 50 µm. k Area quantification (in%) by pattern quant software illustrates the loss of exocrine and endocrine tissue (red) and the increase of fibrotic area (green) and of free space, which reflects fatty tissue and edema (yellow). l Area quantification of exocrine and endocrine active tissue (p < 0.0001, con n = 6/CP n = 10) versus fibrosis (p < 0.0001, con n = 6/CP n = 10) demonstrated the significant changes in organ architecture. All data were presented as means ± SEM, statistically significant differences were tested by unpaired two-tailed students t-test for independent samples and significance levels of p < 0.05 are marked by an asterisk (a–f, h, i, l). Source data are provided as a Source Data file.