Pancreatic ductal adenocarcinoma (PDAC) responds poorly to checkpoint blockade, such as anti-CTLA-4 and anti-PD-1. Galectin-9, a β-galactoside-binding lectin, promotes immune suppression through T-cell inhibition, and programming of tolerogenic macrophages. Of all cancers tested, PDAC showed the highest expression of LGALS9 (galectin-9) mRNA. We analyzed formalin-fixed and paraffin-embedded specimens from 83 patients with PDAC stained for galectin-9. Using flow cytometry, we determined galectin-9 expression on immune cells from tumor and matched blood samples from 12 patients with resectable PDAC. Furthermore, we analyzed galectin-9 serum levels by enzyme-linked immunosorbent assay using serum samples from 70 patients with PDAC, from 36 individuals with benign pancreatic disease, and from 28 healthy controls. Galectin-9 was highly expressed in human PDAC compared with normal pancreas and present on both tumor and immune cells. Tumor-infiltrating immune cells, especially CD3 + T cells, showed upregulation of galectin-9 compared with immune cells from matched blood. Blood γδ T cells from PDAC patients had higher galectin-9 expression than γδ T cells from healthy individuals. Galectin-9 polarized macrophages toward a protumoral M2 phenotype leading to suppressed T-cell cytokine secretion. Furthermore, serum concentration of galectin-9 was able to discriminate PDAC from benign pancreatic disease and healthy individuals, and was prognostic for stage IV patients. Galectin-9 is a new biomarker for the detection of PDAC.

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Immunotherapy has shown promising results in multiple solid tumors and hematological malignancies. However, pancreatic ductal adenocarcinoma (PDAC) has been largely refractory to current clinical immunotherapies. The V-domain Ig suppressor of T-cell activation (VISTA) inhibits T-cell effector function and maintains peripheral tolerance. Here, we determine VISTA expression in nontumorous pancreatic (n = 5) and PDAC tissue using immunohistochemistry (n = 76) and multiplex immunofluorescence staining (n = 67). Additionally, VISTA expression on tumor-infiltrating immune cells and matched blood samples (n = 13) was measured with multicolor flow cytometry. Further, the effect of recombinant VISTA on T-cell activation was investigated in vitro, and VISTA blockade was tested in an orthotopic PDAC mouse model in vivo. PDAC showed significantly higher VISTA expression compared to that of a nontumorous pancreas. Patients with a high density of VISTA-expressing tumor cells had reduced overall survival. The VISTA expression of CD4+ and CD8+ T cells was increased after stimulation and particularly after a coculture with tumor cells. We detected a higher level of proinflammatory cytokine (TNFα and IFNγ) expression by CD4+ and CD8+ T cells, which was reversed with the addition of recombinant VISTA. A VISTA blockade reduced tumor weights in vivo. The VISTA expression of tumor cells has clinical relevance, and its blockade may be a promising immunotherapeutic strategy for PDAC.

Purpose: Immunotherapy has led to a fundamental shift in the treatment of several cancers. However, its efficacy in pancreatic ductal adenocarcinoma (PDAC) is limited. Understanding the expression of inhibitory immune checkpoint receptors (ICR) by intratumoral T cells may help to unravel their involvement in insufficient T cell-mediated antitumor immunity. Experimental Design: Using multicolor flow cytometry, we analyzed circulating and intratumoral T cells from blood (n = 144) and matched tumor samples (n = 107) of PDAC patients. We determined the expression of PD-1 and TIGIT by CD8+ T cells, conventional CD4+ T cells (Tconv) and regulatory T cells (Treg) and their association with T cell differentiation, tumor reactivity, and cytokine expression. A comprehensive follow-up was used to determine their prognostic value. Results: Intratumoral T cells were characterized by increased PD-1 and TIGIT expression. Both markers delineated distinct T cell subpopulations. PD-1+TIGIT- T cells highly expressed proinflammatory cytokines and markers of tumor reactivity (CD39, CD103), whereas TIGIT expression was linked to antiinflammatory and exhausted phenotypes. In addition, the enhanced presence of intratumoral PD-1+TIGIT- Tconv was associated with improved clinical outcomes, while high ICR expression on blood T cells was a significant hazard for overall survival. Conclusions: Our results uncover the association between ICR expression and T cell functionality. PD-1 and TIGIT characterized intratumoral T cells with highly divergent phenotypes linked to clinical outcomes, further underscoring the relvance of TIGT for immunotherapeutic approaches in PDAC. The prognostic value of ICR expression in patient blood may be a valuable tool for patient stratification.