Anti cd3

CD4⁺ T cells for T-cell activation were extracted from the spleens of experimental and control mice. We used a Magnisort^TM Mouse CD4 Naïve T cell Enrichment Kit (Invitrogen, Waltham, MA, USA) to isolate purified naïve T cells. The purity of the enriched CD3⁺CD4⁺CD44^lo T cells was 90–95%. Naïve CD4⁺ T cells were stimulated with 1 μg/mL anti-CD3 (Invitrogen, Waltham, MA, USA) and 1 μg/mL anti-CD28 antibodies (BioLegend, San Diego, CA, USA). The phosphorylation of AKT1, mTOR and RPS6KB1 was assessed by flow cytometry. For TSG101-deficient CD4⁺ T cell analysis, whole CD4⁺ T cells from the spleens of experimental or control mice were enriched using a Magnisort™ Mouse CD4 T cell Enrichment Kit (Invitrogen, Waltham, MA, USA) and stimulated with 1 μg/mL anti-CD3 and 1 μg/mL anti-CD28 antibodies for 8 h. Activated CD4⁺ T cells were harvested and analyzed by flow cytometry. The antibodies used for flow cytometry are listed in Supplementary Table 1.

8 × 10⁵ cells/well in 200 µl culture medium (RPMI 1640, 10% FBS, pen/strep, β-mercaptoethanol) in 96-wells U-bottom plates (Greiner, Frickenhausen, Germany) were stimulated with either culture medium, anti-CD3 (1 µg/ml, eBioscience) or anti-CD3/anti-CD28 (10 µg/ml anti-CD3 and 1 µg/ml anti-CD28, eBioscience, transfer experiment) or whey (50 µg/ml). Polyclonal stimulation (48 h) and whey stimulation (96 h) were conducted at 37°C and 5% CO₂. Culture supernatant was collected and stored at −20°C until measurements of IL-5, IL-10, IL-13, and IFNγ production by means of ELISA according to the protocol described earlier for galectin-9. Purified rat anti-mouse coating antibodies (1 µg/ml for IL-5 and IFNγ and 2 µg/ml for IL-10 and IL-13), recombinant mouse cytokines for the standard curve, and biotinylated detection antibodies (1 µg/ml for IL-5, IL-10 and IFNγ and 400 ng/ml for IL-13) were purchased at BD Biosciences.

To detect IL-17 produced by Th17 cells, cells were stimulated with 1 μg/mL anti-CD3 (eBioscience) and 1 μg/mL anti-CD28 (eBioscience) for 24 h. To detect other cytokines produced by the CD4⁺ T cells, cells were cultured with 1 μg/mL anti-CD3 and 1 μg/mL anti-CD28 for 8 h or 24 h. Culture supernatants were collected at the relevant times. To measure the concentration of cytokines in culture supernatants we used ELISA kits for IFN-γ (eBioscience) and IL-17 (R&D Systems) according to the manufacturers’ instructions. We analyzed the cytokine concentrations using microplate reader software (Thermo Fisher Scientific).

Mouse splenic CD4+ T cells were purified with a CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and stimulated with plate-bound anti-CD3 (5 µm/ml, eBioscience) and anti-CD28 (10 µm/ml, eBioscience) antibodies together with IL-12 (10 ng/ml) for Th1 differentiation, IL-4 (10 ng/ml) for Th2 differentiation, TGF-β (5 ng/ml) and IL-6 (20 ng/ml) for Th17 differentiation, and TGF-β (5 ng/ml) alone for Treg differentiation. All cytokines were obtained from eBioscience. Monocytes and Treg were cocultured at a ratio of 2∶1 according to a previously reported method [21] (link). Treg and monocytes were isolated from spleen using a CD4+CD25+ isolation kit or CD11b+ isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD11b+ isolated monocytes is greater than 88% by flow cytometry. Cells were cocultured with RPMI 1640 and stimulated by anti-CD3 (5 µm/ml, eBioscience) and LPS (50 ng/ml, Sigma, St Louis, USA) for 36 h.

PBMC (2×10⁶ cells/ml) were cultured for 18 h or 6 d in Falcon polystyrene tubes (BD Bioscience, San Jose, CA, USA) at 37°C in a humidified 5% CO₂ atmosphere, in complete medium with or without strains M, 410, or H37Rv (2∶1 ratio of Mtb to PBMC). In some experiments, recombinant Interleukin-2 (Biolegend Inc., San Diego, CA, USA) or neutralizing anti-human IL-2 monoclonal antibody (10 µg/ml, clone MQ1-17H12, BD Bioscience, NJ, USA ) was added at the onset of PBMC culture. In some experiments, PBMC were cultured for 1 h alone or with strain M and then cultured with or without anti-CD3 (10 µg/ml) and/or anti-CD28 (5 µg/ml) (eBioscience, San Diego, CA, USA) for further 18 h. In parallel, PBMC were simultaneously cultured for 18 h with anti-CD3 and/or CD28 and Mtb strains.

Spleens were isolated from WT and TLR5^−/− chimeras to assess ex vivo proliferation or C57Bl/6 WT mice to assess T-cell polarization. Spleens were gently squeezed through a 70 μm mesh cell strainer (Becton Dickinson, San Diego, CA, USA) to obtain a single cell suspension. Cells were washed and resuspended in RPMI1640 (supplemented with 10% FCS, 20 mM L-glutamine, 100 U/ml penicillin and 100 μg/mL streptomycin) and seeded at a density of 3 × 10⁵ cells/well in a 96 well u-bottom cell culture plate (Greiner Bio One, Alphen aan den Rijn, the Netherlands). Cells were stimulated for 72 hours with medium alone, 1 ng/mL Flagellin Ultrapure, 1 ng/mL Flagellin Ultrapure in combination with 2 μg/mL anti-CD3 (eBioscience) or 2 μg/mL anti-CD3 in combination with 2 μg/mL anti-CD28 as a positive control. To assess proliferation, cells were incubated with 0.5 μCi [3 H]Thymidine during the last 16 hours of 3 days in culture. To quantify thymidine incorporation the cells were washed with PBS and lysed with 0.1 M NaOH and cell-associated radioactivity was determined by liquid scintillation counting. To assess T-cell polarisation, after 72 hours of stimulation, 10 μg/mL Brefeldin-A was added overnight to retain the proteins inside the cell. The next morning cells were harvested for flow cytometric analysis.