Figure 2.

Tc17 cells enhance pancreatic tumour growth in vivo. (A) Purified CD8⁺ T cells isolated from CD45.2⁺OT-I mice were stimulated with anti-CD3/CD28 antibodies in the presence of TGFβ+IL-6 (Tc17) or IL-12+IL-2 (CTL) for 4 days. Differentiation was confirmed by intracellular staining for IL-17A and IFNγ and subsequent FACS analysis. Representative FACS plots are shown. (B) Scheme of experimental design. Congenic CD45.2^- mice were subcutaneously (s.c.) injected with 10⁶ Panc^OVA cells. After 5 days, tumour-bearing mice were injected intraperitoneally (i.p.) with 10⁶ Tc17 or CTLs obtained from CD45.2⁺OT-I mice or with PBS (no transfer). The analysis was performed at the indicated end of the experiment. (C) Tumour-growth curve of subcutaneous tumours is shown (tumour volume in mm³ (mean±SE, n=5–7 mice). *p<0.05 indicates the tumour volume comparisons between mice without transfer and mice with Tc17 or CTL transfer. (D) FACS analysis of IL-17A and IFNγ production after restimulation of tumour single-cell suspensions with PMA/Ionomycin in the presence of brefeldin A for 5 hours. Data from endogenous CD45.2^- or transferred CD45.2⁺ CD8⁺ T cells. Left, representative FACS plots are shown. Right, quantification of the frequency of IL-17A⁺ (top) or IFNγ⁺ (bottom) among endogenous (CD45.2^-) or transferred (CD45.2⁺) CD8⁺ T cells with or without Tc17 transfer (n=4–5 tumours). (E) Quantification of cytokines (ng/mL) produced by in vitro differentiated Tc17 cells after restimulation with plate-bound anti-CD3 antibodies for 24 hours (n=5). (F) Top, scheme of the experimental design showing the relative titre of tumour cells cultured for 36 hours with Tc17 cells generated from WT CD8⁺ T cells or with Tc17-conditional media (Tc17-CM). Tc17-CM were obtained after restimulation of differentiated Tc17 cells with plate-bound anti-CD3 for 24 hours. Bottom, the tumour-cell titre was obtained from Panc^OVA or KPC cells tagged with firefly luciferase (Panc^OVA/Luc, KPC^Luc) and assessed as fold of luciferase activity, normalised to the control (0% FCS), which was arbitrarily set to 1. Tumour cells were cultured alone in 0% FCS (control) or 2% FCS (2% FCS), or in 2% FCS containing IL-17A (IL-17A), Tc17-CM (Tc17-CM) or Tc17 cells (Tc17) (n=3). (G) Top, scheme of the experimental design showing treatment of matrigel-embedded 3D organoid cultures with 50 ng/mL IL-17A. Bottom, cell titre assay of mouse (Mm_Bu2548) or human (Hs_ACH0264T) PDAC organoids treated with recombinant murine (rm) or recombinant human (rh)IL-17A (n=3). The relative cell titre without IL-17A treatment (control) was arbitrarily set to 1. (D–G) Bars show mean±SD; biological replicates are plotted. In (C) *p<0.05 determined by mixed-effects model (REML), in (D) **p<0.01, ***p<0.001, ****p<0.0001 by t-test, in (F) statistics evaluated by two-way ANOVA followed by Tukey’s HSD multiple comparison test, ns (non-significant) in (G) statistics evaluated by Mann-Whitney U test. ANOVA, analysis of variance; HSD, honestly significant difference; PBS, phosphate-buffered saline; PDAC, pancreatic ductal adenocarcinoma.