Distinct immune characteristics distinguish hereditary and idiopathic chronic pancreatitis

Abstract

Chronic pancreatitis (CP) is considered an irreversible fibroinflammatory pancreatic disease. Despite numerous animal model studies, questions remain about local immune characteristics in human CP. We profiled pancreatic immune cell characteristics in control organ donors and CP patients including those with hereditary and idiopathic CP undergoing total pancreatectomy with islet autotransplantation. Flow cytometric analysis revealed a significant increase in the frequency of CD68+ macrophages in idiopathic CP. In contrast, hereditary CP samples showed a significant increase in CD3+ T cell frequency, which prompted us to investigate the T cell receptor β (TCRβ) repertoire in the CP and control groups. TCRβ sequencing revealed a significant increase in TCRβ repertoire diversity and reduced clonality in both CP groups versus controls. Interestingly, we observed differences in Vβ-Jβ gene family usage between hereditary and idiopathic CP and a positive correlation of TCRβ rearrangements with disease severity scores. Immunophenotyping analyses in hereditary and idiopathic CP pancreases indicate differences in innate and adaptive immune responses, which highlights differences in immunopathogenic mechanisms of disease among subtypes of CP. TCR repertoire analysis further suggests a role for specific T cell responses in hereditary versus idiopathic CP pathogenesis, providing insights into immune responses associated with human CP.

Keywords: Cellular immune response; Gastroenterology; Immunology; Macrophages; T-cell receptor.

Figure 1. CD68⁺ macrophages are predominant in idiopathic CP compared with hereditary CP.

(A) Immunohistochemistry staining using the pan-leukocyte marker CD45 (original magnification, ×400). Scale bars: 100 μm. The percentage of CD45⁺ cells among total pancreatic cells is presented as a dot plot. Mean ± SD; unpaired 2-tailed t test. (B) The frequency of CD3⁺ T cells and CD68⁺ cells in live CD45⁺ cells from control (n = 8) and CP (n = 24) samples. Mean ± SD; unpaired 2-tailed t test was used. (C) Graphs show frequencies of CD68⁺ cells and their subsets in live CD45⁺ cells from control (n = 8), hereditary CP (n = 15), and idiopathic CP (n = 9) tissues. Mean ± SD; 1-way ANOVA with Tukey’s multiple-comparisons test. (D) Heatmap represents expression levels of analytes with MFI values by the human 62 multiplex Luminex assay (t test, P < 0.05, FDR < 0.25). Fold change in the average expression of each analyte in idiopathic versus hereditary CP. (E) Comparison of MFI values of the most differentially regulated chemokine (CCL7) between hereditary (n = 17) and idiopathic (n = 8) CP. Mean ± SD; unpaired 2-tailed t test was used. *P < 0.05, **P < 0.01, ***P < 0.001. HPF, high-power field.

Figure 2. CD3⁺ T cells are more frequent in hereditary CP compared with idiopathic CP.

(A) Representative plots of flow cytometry analyses of CD3⁺ T cells based on CD4 and CD8 expression in control (n = 8) and CP (n = 24) samples (mean ± SD). Graphs show frequency of CD4⁺ or CD8⁺ T cells in control and CP samples (mean ± SD; unpaired 2-tailed t test). (B) Frequencies of total CD3⁺, CD4⁺, and CD8⁺ T cells among live CD45⁺ cells from control (n = 8), hereditary CP (n = 15), and idiopathic CP (n = 9) tissues. Mean ± SD; 1-way ANOVA with Tukey’s multiple-comparisons test. (C) Pie charts represent the average frequencies of T-bet⁺, GATA3⁺, RORγt⁺, and FOXP3/CD25⁺ T cell subsets in CD4⁺, CD8⁺, or double-negative (DN) T cells from hereditary (n = 15) and idiopathic (n = 9) CP tissues. *P < 0.05, ***P < 0.001.

Figure 3. TCRβ repertoire of control and CP pancreatic T cells.

(A) Waterfall and dot plots show the ratio of CD3⁺ T cell to CD68⁺ macrophage frequency in control, hereditary CP, and idiopathic CP tissues. Identified gene mutations are indicated in an individual hereditary CP patient. Mean ± SD; 1-way ANOVA with Tukey’s multiple-comparisons test. Her, hereditary. (B) One hundred most frequent rearrangements in each sample ranked from bottom (most frequent clone) to top (100th most frequent clone), and samples are listed by their clonality order from left to right. Number of productive rearrangements (C), TCR clonotype diversity (mean normalized Shannon-Wiener diversity index) (D), and productive clonality are shown (E). (C–E) Comparison between control (n = 5) and CP (n = 13). Nonparametric Mann-Whitney U test. *P < 0.05, **P < 0.01.

Figure 4. Differences in TCRβ repertoire between hereditary and idiopathic CP pancreatic T cells.

(A) Circos plots indicate frequencies of Vβ-Jβ productive gene usage in control (n = 5), hereditary CP (n = 7), and idiopathic CP (n = 6) cells. The width of the Vβ-Jβ pair band is proportional to the frequency in each group. (B) Comparison of Vβ-Jβ2 gene family usage among groups, shown as mean frequencies. Mean ± SEM; 1-way ANOVA with Kruskal-Wallis test, comparison between hereditary and idiopathic CP; *P < 0.05. (C) Heatmap representing frequencies of Vβ-Jβ gene pairs that are significantly different (significance analysis of microarray t test, 90th percentile FDR = 0) between control and CP cells. (D) Full-length CDR3 amino acid sequences shared among at least 4 subjects. Numbers in squares represent the count of unique clonotypes in a subject’s repertoire, with the CDR3β sequence indicated. (E) Correlation of the number of functional TCR rearrangements with CP disease severity score (n = 13, nonparametric Spearman’s correlation r = 0.8361, P < 0.001).