Gamma-delta T cells stimulate IL-6 production by pancreatic stellate cells in pancreatic ductal adenocarcinoma

Abstract

Introduction: The immunosuppressive tumor microenvironment promotes progression of pancreatic ductal adenocarcinoma (PDAC). γδ T cells infiltrate the pancreatic tumor stroma and support tumorigenesis through αβ T cell inhibition. Pancreatic stellate cell (PSC) activation contributes to pancreatic fibrosis in PDAC, limiting the delivery and efficacy of therapeutic agents. Whether γδ T cells have direct effects on PSC activation is unknown.

Methods: In this study, we analyzed tumor tissue from 68 patients with PDAC and determined the frequency and location of γδ T cells using immunohistochemistry and immunofluorescence. PDAC samples from the TCGA database with low and high TRGC2 expression were correlated with the expression of extracellular matrix genes. Further, PSCs were isolated from pancreatic tumor tissue and co-cultured with γδ T cells for 48 hours and cytokine production was measured using a cytometric bead array.

Results: γδ T cells infiltrated the pancreatic tumor stroma and were located in proximity to PSCs. A high infiltration of γδ T cells was associated with increased expression of several extracellular matrix genes in human PDAC. In vitro, γδ T cells stimulated IL-6 production by PDAC-derived PSCs.

Conclusion: γδ T cells activated PSCs and modulation of this interaction may enhance the efficacy of combinational therapies in human PDAC.

Keywords: Gamma-delta T cells; IL-6; Pancreatic cancer; Pancreatic stellate cells.

Fig. 1

γδ T cells infiltrate human PDAC. a Frozen sections of human PDAC (n = 68) and adjacent normal pancreas (n = 8) were tested for the expression of the γδ T-cell receptor (γδTCR). Representative images are shown. Scale bar, 100 μm. b Quantification of γδTCR⁺ cells per high-power field (HPF). c Number of γδTCR⁺ cells per HPF correlated with T (left), N (middle), and UICC stages (right). Each point represents data from one patient. Data, median, unpaired t test, or one-way ANOVA. *P < 0.05

Fig. 2

High γδ T-cell infiltration is associated with fibrosis in PDAC. a Frozen sections of human PDAC (n = 68) were tested for the expression of the γδ T-cell receptor (γδTCR) and analyzed by their intratumoral location (duct vs. stroma). Representative images are shown. Scale bar, 100 μm. b Quantification of γδTCR⁺ cells per high-power field (HPF). Each point represents data from one patient. Data, median, unpaired t test. ***P < 0.001 (c) Heatmap showing low and high tertiles of TRGC2 expression and indicated extracellular matrix genes in human PDAC samples from the TCGA database. Row, indicated gene; columns; color key indicates row Z-score

Fig. 3

γδ T cells are located in proximity to PSCs in the pancreatic tumor stroma. (a) Frozen human PDAC specimens were stained for alpha-smooth muscle actin (αSMA), γδTCR and CK19 by immunofluorescence. Representative image is shown. Scale bar, 25 μm. b Correlation between the high and low tertiles of TRGC2 expression and ACTA2 expression was tested in human PDAC samples from the TCGA database. Each point represents data from one patient. Data, median, unpaired t test. *P < 0.05

Fig. 4

γδ T cells activate PSCs and stimulate their IL-6 production. a PSCs were tested for alpha-smooth muscle actin (αSMA) expression by immunofluorescence. Representative image is shown. Scale bar, 25 μm. b PSCs (7.5 × 10⁴) were plated alone or together with expanded γδ T cells (1:2 ratio) in 12-well plates and IL-6 expression was measured in the supernatant by cytometric bead array (CBA). c γδ T cells and PSCs were tested for IL-6 expression. Representative histograms and quantification are shown. d Correlation between the high and low tertiles of TRGC2 expression and IL6 expression was tested in human PDAC samples from the TCGA database. Each point represents data from one patient. Data, mean or median, unpaired t test, or one-way ANOVA. **P < 0.01, ****P < 0.0001