Intracellular ifnγ

1x10⁵ T cells were stained with CFSE (Thermo) and cultured in 200μl RPMI (Gibco) + 10% HI-FCS (Gibco) + 1% Peniciliin/Streptomycin (100x, Gibco) + 1% L –glutamine (100x, Gibco) with 2x104 Human T-Activator CD3/CD28 Dynabeads (Thermo) alone or with 2x105 LDNs or NDNs isolated form the same donor for 96 hours at 37°C 5% CO2. Activation cocktail containing protein transport inhibitors (500x eBioscience) was added for 4 hours then stained with Live/Dead Yellow (Thermo), followed by antibodies for CD4, CD8, CD66b (Biolegend) and intracellular IFNγ (Biolegend) following fixation and permeabilisation with 1x Fix/Perm solution (BD). Cells were then run on an LSR Fortessa flow cytometer (BD) and analysed with FlowJo Software (BD). Co-culture conditions were adapted from previously described methods (34 (link)).

CD4⁺ T cells were harvested from the lymph nodes and spleens of naive NR1 mice and enriched with a mouse naïve CD4-negative isolation kit (BioLegend) following the manufacturer’s protocol. CD4⁺ T cells were cultured in media consisting of RPMI 1640 (Invitrogen), 10 % FCS, l-glutamine, HEPES, 50 μM 2-ME, 50 U/ml penicillin, and 50 mg/ml streptomycin. NR1 cells were activated by coculture with mitomycin-treated splenocytes pulsed with 5 μM Cta1_133–152 peptide at a stimulator/T cell ratio of 4:1. Th1 polarization was achieved by supplying cultures with 10 ng/ml IL-12 (PeproTech, Rocky Hill, NJ) and 10 μg/ml anti–IL-4 (Biolegend) One week after initial activation resting NR1 cells were co-incubated with untreated or IFNγ-treated macrophages of different genotypes, that were or were not pulsed with Cta1 peptide. Six hours following co-incubation NR1 cells were harvested and stained for intracellular IFNγ (BioLegend) using an intracellular cytokine staining kit (BioLegend) as done previously. Analyzed T cells were identified as live, CD90.1⁺ CD4⁺ cells.

After 4 hours co-culture, the activations of CD8+ T-cells were determined by the expressions of tomato fluorescent protein, intracellular IFN-γ and IL-2 (Biolegend) using flow cytometry. For in vivo study, the tumor draining lymph nodes (TDLNs) and bone marrow from the vehicle, Dox- and Tax-treated mice were harvested and analyzed on day 3 post Dox and Tax administration to trace the percentages of Tregs and MDSCs (n = 6 per group). The single cell suspensions isolated from the spleen, TDLNs and bone marrow were stained with anti-FOXP3-Alexa Fluor 488/CD4-APC/CD25-PE antibodies using a Mouse Treg Flow Kit (Biolegend) according to manufacturer's protocol, and CD11b-FITC and Gr-1-PE antibodies (eBioscience) for detecting Tregs and MDSCs, respectively. The percentages of these cell types were acquired using FACSCalibur flow cytometry (BD Bioscience), and data were analyzed by FlowJo software (Tree Star).