Mice

C57BL/6 wild type and B6.129S7-Rag1tm1Mom/J (Rag1−/−) mice were obtained from the Jackson Laboratory (Bar Harbor, MA, USA) and bred in-house. The animals were housed in the Comparative Medicine Center, University of Utah Health, UT, USA at constant temperature (22–24 °C), relative humidity ~55% and maintained under 12 h light/dark cycle with access to standard chow pellets and tap water ad libitum. The research has been approved by the University of Utah Institutional Animal Care and Use Committee. Every effort was taken to minimize animal suffering and to reduce the number of animals used.

Isolation and polarization of naive CD4+ T cells

Spleens were harvested from C57BL/6 wild type mice and naive CD4+ T cells were isolated by magnetic separation using Naive CD4+ T-Cell Isolation Kit (cat. 130-104-453, Miltenyi Biotec., Auburn, CA, USA). The naive CD4+ T-cell polarization was performed according to the manufacturer’s instructions using the CytoBox Th2 (cat. 130-107-760, Miltenyi Biotec., Auburn, CA, USA). The isolated naive CD4+ T cells were resuspended in TexMACS™ media (cat. 130-097-196, Miltenyi Biotec., Auburn, CA, USA), supplemented with 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Corning, Tewksbury, MA, USA), 50 U/ml mouse IL-2 (CytoBox Th2, Miltenyi Biotec., Auburn, CA, USA), 200 U/ml mouse IL-4 (CytoBox Th2, Miltenyi Biotec., Auburn, CA, USA) and 10 μl/ml anti-IFN-γ antibody (CytoBox Th2, Miltenyi Biotec., Auburn, CA, USA). 2 × 105 of naive CD4+ T cells were transferred to 96-well round-bottom plate, activated using T-cell activation beads coupled with anti-CD3 and anti-CD28 antibodies (cat. 11456D, ThermoFisher Scientific, Waltham, MA, USA) and incubated at 37 °C and 5% CO2. After 6 days of differentiation of naive CD4+ T cells, Th2 cells were collected and used for in vitro or in vivo analyses.

Isolation of eosinophils

Eosinophils were isolated from spleens of C57BL/6 wild type mice by magnetic separation using Anti-Siglec-F MicroBeads (cat. 130-118-513, Miltenyi Biotec., Auburn, CA, USA) according to manufacturerʼs protocol. After separation, eosinophils in RPMI media (Corning, Tewksbury, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Corning, Tewksbury, MA, USA) and 1% l-glutamine (ThermoFisher Scientific, Waltham, MA, USA) were resuspended and used for in vitro studies.

Murine allograft models and treatments

BRAF (BRAFV600EΔTRZI) cells were derived from a mouse tumor developed from organoids kindly shared by Dr. Daniel Worthley from the South Australian Medical and Health Institute, Australia [15]. The tumor was dissociated using the gentleMACS™ instrument (Miltenyi Biotec., Auburn, CA, USA) and cultured in RPMI media (Corning, Tewksbury, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Corning, Tewksbury, MA, USA) and 1% l-glutamine (ThermoFisher Scientific, Waltham, MA, USA). After several passages, the resulting cell line was utilized to induce tumors for this study. PK5L1940 cells were provided by Dr. Michael Gough from the Earle A. Chiles Research Institute, Portland, OR, USA [16]. Cells were cultured in RPMI media (Corning, Tewksbury, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Corning, Tewksbury, MA, USA) and 1% l-glutamine (ThermoFisher Scientific, Waltham, MA, USA). In all studies, cells testing negative for mycoplasma were used. BRAF cells (2 × 106) or PK5L1940 cells (1 × 106) resuspend in PBS and mixed with Matrigel® (Corning, Tewksbury, MA, USA) were injected into the flank of 6–10-week-old male or female randomized Rag1−/− mice, investigators were not blinded. Ten animals per cell line were used in tumor experiments. Tumors were manually measured using caliper starting from day 1. Some mice with BRAF and PK5L1940 cell-derived allografts were administered intratumorally with 5 × 105 of Th2 cells or 200 ng of IL-5 (cat. 200–20, Shenandoah Biotechnology, Inc., Warwick, PA, USA) resuspended in PBS. Tumor size was calculated according to the following formula: tumor size = (length × length × width)/2. Mice reached the endpoint when tumor volumes were approximately 2000 mm3.

Immunohistochemistry analysis

Tumors were fixed in 4% paraformaldehyde for up to 24 h, incubated in 15% and then 30% sucrose-PBS solutions for up to 12 h, each. Tumor pieces were embedded in the Tissue-Plus™ O.C.T. Compound Tissue-Plus™ (cat. 23-730-571, ThermoFisher Scientific, Waltham, MA, USA). Sections (5 μm) were blocked with 2% normal serum and incubated with commercially available antibodies against F4/80-PE (cat. 11-4801-82), GATA3-PE (cat. 46-9966-41), MBP (cat. MA1-24990), MPO (cat. PA5-16672), NOS2-APC780 (cat. 47-5920-82), SIGLEC-F-PE (cat. 552125). The above-mentioned antibodies conjugated and unconjugated with fluorochrome were used at 1:200 dilution and were purchased from ThermoFisher Scientific (Waltham, MA, USA) or BD Bioscience (San Diego, CA, USA). The sections where antibodies conjugated with fluorochrome were incubated for 1 h at room temperature. The sections where unconjugated with fluorochrome antibodies were used were incubated overnight at 4°C. Next, the sections were washed and incubated with donkey anti-rat secondary antibodies (cat. A-21209, ThermoFisher Scientific, Waltham, MA, USA) for 1 h. Subsequently, the sections were washed with PBS and mounted in SlowFade™ Gold Antifade Mountant with DAPI (cat. S36938, ThermoFisher Scientific, Waltham, MA, USA). The sections were analyzed using EVOS™ M7000 Imaging System (ThermoFisher Scientific, Waltham, MA, USA) featuring 10x and 20x objectives.

Tumor-killing assay and flow cytometry analysis

BRAF and PK5L1940 cells were plated and Th2 cells or eosinophils in a ratio 1:2 were added. Cells were incubated for up to 24 h and co-cultures stained with CellEvent™ Caspase-3/7 Green Detection Reagent (cat. C10423, ThermoFisher Scientific, Waltham, MA, USA). Supernatants from co-cultures were collected for multiplex analysis. Flow cytometry analysis was performed using an Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Waltham, MA, USA) and analyzed with Attune™ NxT Software (ThermoFisher Scientific, Waltham, MA, USA).

RNA extraction, reverse transcription and real-time PCR

Tumor pieces were homogenized in TRIzol™ reagent (cat. 15596026, ThermoFisher Scientific, Waltham, MA, USA) and RNA extraction was performed according to the manufacturer’s instructions. The quality and quantity of RNA were measured with a NanoDrop™ Lite Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Total RNA (100 ng/µl) was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (cat. 4368814, ThermoFisher Scientific, Waltham, MA, USA) with the following PCR settings: 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 min. Quantitation of mRNA was performed using real-time PCR with validated FAM dye-labelled TaqMan® probes (Applied Biosystems, Foster City, CA, USA) for Actb—Mm02619580_g1, Adgre1—Mm00802529_m1, Fas—Mm01204974_m1, Fasl—Mm00438864_m1, Gsr—Mm00439154_m1, Gzmb—Mm00442837_m1, Mbp—Mm01266402_m1, Mpo—Mm01298424_m1, Nos2—Mm00440502_m1, Prf1—Mm00812512_m1, Siglef—Mm00523987_m1. The reaction mixture consisted of cDNA, TaqMan® Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA), TaqMan® Assays, and RNase-free water in a total volume of 10 μl. Cycle parameters for TaqMan® assays were as follows: initial denaturation at 95 °C for 3 min, followed by 40 cycles of sequential incubations at 95 °C for 15 s and 60 °C for 1 min. Results were normalized to the expression of housekeeping gene, i.e., Actb. All experiments were performed at least as duplicates on QuantStudio™ 5 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). The endpoint used in real-time PCR quantification—CT—was defined as the PCR cycle number that crossed the signal threshold. Quantification of gene expression was performed using the comparative CT method (Sequence Detector User Bulletin 2; Applied Biosystems) and reported as the fold-change relative to the mRNA of the mouse housekeeping gene.

Multiplex analysis

BRAF and PK5L1940 cell-derived allograft tumors were divided into 8 mg pieces (± 0.5 mg) and incubated in RPMI media (Corning, Tewksbury, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Corning, Tewksbury, MA, USA) and 1% l-glutamine (ThermoFisher Scientific, Waltham, MA, USA) up to 18 h. Tumor culture supernatants were analyzed for cytokine and chemokine levels by multiplex arrays (MilliporeSigma, Burlington, MA, USA) and Luminex® in accordance with the manufacturer’s instructions. Mouse cytokine panel 1 and a custom panel consisting of GZMB and FAS were used for this study (MilliporeSigma, Burlington, MA, USA).

Statistical analyses

Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Results are presented as means ± standard error of mean (SEM). Non-parametric Mann–Whitney U-test and two-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for comparison of studied groups. P-values < 0.05 was considered statistically significant.