Clone 37

The WT, PTEN^flox, PTEN^M−KO, β-catenin^flox, and β-catenin^M−KO mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.), and then Bio-Gel elicited peritoneal macrophages were isolated as described previously (22 (link)). The macrophages were cultured in medium (Invitrogen) supplemented with 10% FBS, 100 μg/ml of penicillin/streptomycin (Life Technologies; Grand Island, NY). The peripheral blood or spleen T cells were purified using the EasySep™ mouse T cell isolation kit (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. T cells were then stimulated with anti-CD3 (1 μg/ml, Clone 145-2C11) and anti-CD28 (2 μg/ml, Clone 37.51) (eBioscience).

Ninety-six-well plates (Nunc) were coated overnight with monoclonal antibodies (mAb) anti-CD3 (1 µg/ml, clone 17A2, unlabeled, in-house produced by rat hybridoma cell lines) and CD28 (1 µg/ml, clone 37.51, unlabeled, purchased from eBioscience) to stimulate γδ T cells. Single-cell suspensions of PPs were obtained like previously described. Then 4×10⁶ cells/ml were resuspended in RMPI 1640 (10% FCS, 1% Pen/Strep, 1% L-Glutamine) and incubated at 37°C for 2 h. After stimulation, antibodies for surface staining were added for 30 min on ice together with anti-FcR antibodies (clone 2.4 G2) and live/dead cell discrimination agent (Zombie Aqua Fixable Viability Kit, BioLegend). Next, IL-4 secretion assay (Miltenyi Biotec) was performed according to the manufacturer’s instructions. Briefly, cells were incubated for 5 min with IL-4 catch antibodies on ice and afterwards for further 45 min at 37°C to secrete IL-4. The secreted IL-4 was then detected with PE-labeled IL-4 detection antibodies for 10 min on ice. After washing, cells were acquired and data analysis was performed using Flowjo software (Version: 10.1, Three Star). Gating strategy and FMO control of IL-4 are shown in Supplementary Figure 1A.

Splenic naïve CD8⁺ T cells were purified by using Naive CD8a⁺ T Cell Isolation Kit, mouse (Miltenyi Biotec) following the manufacturers protocol, and polarized in vitro toward differentiated Tc1 and Tc2. In brief, naive cells were seeded into anti-CD3e (2 μg/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 μg/ml, clone 37.51, eBioscience) coated plates. The medium contained the following cytokines and/or antibodies:
Tc1 subtype: recombinant murine IL2 (10 ng/ml, R&D Systems), recombinant murine IL12 (10 ng/ml, R&D Systems) and neutralizing anti-IL4 (10 μg/ml, clone 11B11, eBioscience). Tc2 subtype: recombinant murine IL2 (10 ng/ml, R&D Systems), recombinant murine IL-4 (10 ng/ml, R&D Systems) and neutralizing anti-IFNg (10 μg/ml, clone XMG1.2, eBioscience).