Galectin-3 Released by Pancreatic Ductal Adenocarcinoma Suppresses γδ T Cell Proliferation but Not Their Cytotoxicity

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor microenvironment with a dense desmoplastic stroma. The expression of β-galactoside-binding protein galectin-3 is regarded as an intrinsic tumor escape mechanism for inhibition of tumor-infiltrating T cell function. In this study, we demonstrated that galectin-3 is expressed by PDAC and by γδ or αβ T cells but is only released in small amounts by either cell population. Interestingly, large amounts of galectin-3 were released during the co-culture of allogeneic in vitro expanded or allogeneic or autologous resting T cells with PDAC cells. By focusing on the co-culture of tumor cells and γδ T cells, we observed that knockdown of galectin-3 in tumor cells identified these cells as the source of secreted galectin-3. Galectin-3 released by tumor cells or addition of physiological concentrations of recombinant galectin-3 did neither further inhibit the impaired γδ T cell cytotoxicity against PDAC cells nor did it induce cell death of in vitro expanded γδ T cells. Initial proliferation of resting peripheral blood and tumor-infiltrating Vδ2-expressing γδ T cells was impaired by galectin-3 in a cell-cell-contact dependent manner. The interaction of galectin-3 with α3β1 integrin expressed by Vδ2 γδ T cells was involved in the inhibition of γδ T cell proliferation. The addition of bispecific antibodies targeting γδ T cells to PDAC cells enhanced their cytotoxic activity independent of the galectin-3 release. These results are of high relevance in the context of an in vivo application of bispecific antibodies which can enhance cytotoxic activity of γδ T cells against tumor cells but probably not their proliferation when galectin-3 is present. In contrast, adoptive transfer of in vitro expanded γδ T cells together with bispecific antibodies will enhance γδ T cell cytotoxicity and overcomes the immunosuppressive function of galectin-3.

Keywords: T cells; autologous; bispecific antibodies; galectin-3; gammadelta T cells; pancreatic cancer; proliferation; α3β1 integrin.

Figure 1

Intracellular galectin-3 expression in PDAC cells, and effect of recombinant galectin-3 on γδ T cell proliferation. (A) Histograms depicted are representative results of indicated PDAC cells. Thin and bold lines represent isotype control and gal-3 expression (clone M3/38), respectively. (B) Median fluorescence intensity (MFI) ± SD (n = 3, duplicates) of gal-3 expression corrected by the MFI of the isotype control is shown for the indicated PDAC cell lines measured by FACS Calibur. (C) PancTu-I cells were labeled with anti-gal-3 mAb (clone Gal397) and analyzed on the ImageStream® X Mark II. Five representative cells out of 5 × 10³ recorded cells are shown with bright field image (left side) and fluorescence image of gal-3 (right side). Scale bars represent 10 μm. (D) 1.25 × 10⁵ PBMC of paired healthy donors (n = 4, different closed symbols), additional healthy donors (n = 7, two conc. of gal-3, closed circles) and paired PDAC patients (n = 4, open symbols) were stimulated with 300 nM BrHPP with the different indicated concentrations of rgal-3 and 50 IU/mL rIL-2. After 6–7 days, the absolute cell number was determined as a percentage of the medium control of the Vγ9 γδ T cells using SCDA. The mean ± SD of duplicates is shown. Statistical comparison of non-matched samples was carried out parametrically by using one-way ANOVA followed by Tukey's multiple comparison test. Significances are shown as P-value; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

Coculture of PDAC cells with PBMC induces galectin-3 release and inhibition of γδ T cell proliferation. (A–C) In total, 5 × 10³ of the indicated PDAC cells were plated in complete medium. After 24 h (A,B) 2.5 × 10⁵ PBMC (n = 4–7) or (C) 2 × 10⁵ freshly isolated CD4 or CD8 αβ T cells or γδ T cells (Tc) (n = 6) were added or cultured alone (medium). Cells were stimulated (A) with 2.5 μM zoledronic acid in the presence of 50 IU/mL rIL-2, (B) with Activation/Expander Beads or (C) with Activation/Expander Beads (TCR), 300 nM BrHPP (PAg) plus rIL-2 or 1 μg/mL bsAb [HER2xCD3] for αβ T cells and [(HER2)₂xVγ9] plus rIL-2 for γδ T cells. (A,B) Vγ9 γδ T cell proliferation was determined after 6–7 days and αβ T cell proliferation after 3–4 days by SCDA. The means ± SD of duplicates are shown. (C) Cell culture supernatants were collected after 72 h and released gal-3 was determined by ELISA. The bars represent the mean ± SD (n = 6), determined in duplicates. Statistical comparison of (A) non-matched samples was carried out parametrically by using one-way ANOVA followed by Tukey's multiple comparison test, and of (B,C) matched samples also parametrically by using paired, two-tailed t-test. P-value; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns, non-significant.

Figure 3

Galectin-3 knockdown in PancTu-I cells partially restores γδ T cell proliferation within PBMC, and gal-3 release in coculture is cell contact-dependent. (A) In total, 5 × 10³ PancTu-I cells left non-transfected or were transfected with either control siRNA or gal-3 siRNA and cultured in complete medium 72 h after transfection. After 24 h culture of PDAC cells, 2.5 × 10⁵ PBMC were added (E/T 50:1) or cultured alone. After addition of 50 IU/mL rIL-2, cells were left unstimulated (med) or stimulated with 2.5 μM zoledronic acid (Z). After 6 days, the absolute cell number of the Vγ9 γδ T cells was determined using SCDA. The mean ± SD of duplicates from 5 donors are shown. (B) Short-term activated Vγ9Vδ2 T cells were cultured with 50 IU/mL rIL-2 alone and directly cocultured with 2 x 10⁴ PancTu-I cells or indirectly separated by a semipermeable membrane with 0.4 μm pores of a transwell insert in 24-well plates at an E/T ratio of 40:1. Cells were left untreated or stimulated with PAg. After 24 h, gal-3 was measured in the cell culture supernatant using ELISA. The means ± SD of 3 donors are shown. (A,B) Statistical comparison of matched samples was carried out parametrically by using paired, two-tailed t-test. P-value; *P < 0.05, **P < 0.01, ns, not significant.

Figure 4

CD49c/CD29 play a role in the galectin-3 mediated inhibition of γδ T cell proliferation within PBMC. (A,B) In total, 5 × 10³ PancTu-I cells were seeded for 24 h. 2.5 × 10⁵ PBMC/well were pre-incubated with (A) the indicated concentrations of the displayed antibodies or appropriate isotype controls or (B) with 10 μg/mL anti-CD29 mAb together with 10 μg/mL anti-CD49c mAb or appropriated isotype controls for 2 h. (A,B) Thereafter, PancTu-I cells were transferred and stimulated with 2.5 μM zoledronic acid and 50 IU/mL rIL- 2. After 7 days, the absolute cell number of Vγ9 γδ T cells was determined using SCDA. (A) The means ± SD of duplicates of 2 donors are shown. (B) Each point represents an individual donor. Statistical comparison of matched samples was carried out parametrically by using paired, two-tailed t-test. P-value; *P < 0.05.

Figure 5

Coculture of PancTu-I cells with activated γδ T cells increases galectin-3 release and its localization in the tumor cell periphery. (A) In total, 5 × 10³ indicated PDAC cells were plated in complete medium. After 24 h, 2 × 10⁵ short-term activated CD4 or CD8 αβ T cells or γδ T cells (Tc) (n = 6) were added or cultured alone (cells alone). Cells were stimulated with Activation/Expander Beads (TCR), 300 nM BrHPP (PAg) plus 12.5 IU/mL rIL-2 or 1 μg/mL bsAb [HER2xCD3] for αβ T cells and [(HER2)₂xVγ9] plus 12.5 IU/mL rIL-2 for γδ T cells. Cell culture supernatants were collected after 72 h and gal-3 was determined by ELISA. The means ± SD of duplicates are shown. Statistical comparison was carried out parametrically by using paired, two-tailed t-test. P-value; *P < 0.05. As samples of CD8 and γδ T cells cultured in medium in comparison to bsAb did not follow a normal distribution, a Wilcoxon non-parametric, matched-pairs signed rank test was applied, which revealed no significance (ns, non-significant). (B) PancTu-I cells alone or cocultured with short-term activated Vγ9 γδ T cells for the indicated time. Thereafter, cells were stained with anti-gal-3 mAb (clone Gal397) followed by secondary goat-anti-mouse Ab and anti-EpCAM mAb (clone REA-125) for tumor cells, anti-CD3 (clone UCHT-1) for T cells and phalloidin for actin-filament staining and analyzed on the ImageStream® X Mark II. The fluorescence image of gal-3 plus overlays with transmitted light image and peripheral mask of a representative PancTu-I cell with gal-3 expression predominately in the center or periphery is shown. In addition, the mean ± SD of the intensity of gal-3 expression in the periphery of PancTu-I cells after addition of γδ T cells (n =3) are presented.

Figure 6

Galectin-3 knockdown does not influence γδ T cell-mediated cytotoxicity against PDAC cells, and does not induce cell death in γδ T cells. (A) In total, 5 × 10³ control (gray bars) or gal-3 siRNA transfected (striped bars) PancTu-I cells were labeled with ⁵¹Cr and used as targets in a standard ⁵¹Cr release assay. Short-term activated γδ T effector cells were titrated at the indicated E/T ratio and cultured in medium or stimulated with the bsAb [(HER2)₂xVγ9]. Mean values of triplicates (SD < 10%) are calculated. Mean ± SD of 3 independent experiments with 3 different donors are shown. (B) 10⁵ short-term activated γδ T cells were treated with the indicated concentrations of gal-3 or gal-9 for 24 h. The cells were then labeled with annexin-V and propidium iodide (PI) and analyzed by flow cytometry. The proportion of alive (black bars, annexin-V⁻ PI⁻), early apoptotic (light gray bars, annexin-V⁺ PI⁻) and late apoptotic/necrotic (dark gray bars, annexin-V⁺ PI⁺) cells are shown as the mean value of 3 donors in 3 independent experiments. SD was <10%.

Figure 7

Monitoring of T lymphocyte subsets within blood and PDAC tissue, and galectin-3 serum levels. (A) The relative percentage of different T cell subsets within ex vivo isolated PBMC and TIL of PDAC patients (n = 6) was determined by staining the cells with the indicated mAbs and analyzed by LSR Fortessa. A gate was set on lymphocytes based on the side scatter properties and CD45 leukocytes to analyze γδ T cells within leukocytes. To distinguish between Vδ1 and Vδ2 within the CD3/pan γδ TCR, a gate was set on CD3/pan γδ TCR-expressing T cells. For discrimination between CD4 and CD8 αβ T cells a gate was set on CD3 T cells excluding CD3/pan γδ TCR⁺ cells. The gating strategy is shown with PBMC of one patient. Each symbol presents the data of one donor, and the lines in the boxes represent the median of different independent experiments. Statistical comparison of matched samples was carried out parametrically by using paired, two-tailed t-test. P-value; *P < 0.05, **P < 0.01, ***P < 0.001. As γδ T cell samples did not follow a normal distribution, Wilcoxon non-parametric, matched-pairs signed rank test was applied. P-value n.s., non-significant. (B) Gal-3 concentrations in serum samples from PDAC patients (n = 22), Ovarian cancer patients (OvCa, n = 9) and age-matched healthy donors (HD, n =13) measured by ELISA are presented as dot plots. The filled dots are the patients presented also in (A). Samples present no significant differences.

Figure 8

Coculture of ex vivo isolated PDAC cells with autologous PBMC or TIL inhibits γδ T cell proliferation and enhances release of galectin-3. (A) In total, 5 × 10³ or (B) 1.5 × 10⁵ ex vivo isolated tumor cells (TuC) and (A) 2.5 × 10⁵ autologous PBMC or (B) 1.5 × 10³ autologous TIL were cultured alone or together in complete medium or were stimulated with 2.5 μM zoledronic acid with 50 IU/mL rIL-2 (Z). At day 0 and 11 days after culture, the absolute cell number of the Vγ9 γδ T cells was determined using SCDA. Cell culture supernatants were collected after 96 h and galectin-3 was determined by ELISA. Each symbol presents the data of one donor, and the lines represent the median of 4 different independent experiments. (A) Statistical comparison of matched samples was carried out parametrically by using paired, two-tailed t-test. P-value; *P < 0.05, ***P < 0.001. (B) Wilcoxon non-parametric, matched-pairs signed rank test (left panel) or parametric, matched-pairs, two-tailed t-test (right panel) was carried out. Samples present no significant differences. (C) Histograms are showing intracellular gal-3 expression stained with anti-gal-3 Ab (gray) compared to the appropriate isotype-control (unfilled) in pan-Cytokeratin⁺ tumor cells and CD45⁺ leukocytes of 2 representative donors (PDAC #1 and #2) out of 4.