Rhtgf β1

To analyze CD103-induced T cells, specified cell populations were first resuspended in full RPMI, mixed with anti-CD2/3/28 activation beads (Miltenyi biotec) in a 1:2 bead to cell ratio and cultured for 3 days in the presence or absence of 10 ng/ml rhTGFβ1 (Miltenyi Biotec) to induce CD103 expression.
To quantify cytokine expression capabilities, stimulation beads were removed from the cultures as per manufacturer’s instructions and cells were rested for 24 h in full RPMI 1640 medium (+10% FCS, +1% P/S, Gibco). Afterwards, rested cells were restimulated for 4 h with 1 mM inomycin (Cayman Chemical) and 50 ng/ml PMA (Sigma) in the presence of a combination of Brefeldin A (Applichem) and Monensin (Biolegend) at a concentration of 0.5 µg/ml each, to inhibit cytokine release.
To elucidate CD103-dependent interactions with E-Cadherin, induced cells were rested in the same way. Subsequently, cells were restimulated in the presence of 10 µg/ml (or 100 µg/ml where specifically indicated) anti-β7 (FIB504, CellXGene), anti-CD103 (Ber-ACT8, Biolegend) or corresponding isotype controls (Sigma Aldrich, Biolegend) by transferring them to wells previously coated with anti-CD3 (UCHT2, Biolegend) at concentrations of 1 or 5 µg/ml with or without E-Cadherin Fc Chimeras (Biolegend) at a concentration of 5 µg/ml for 1 h. Plates were then cultured for 3 days at 37 °C.

CD4⁺ T cells were purified from spleen and lymph nodes of C57BL/6 mice using anti-CD4 microbeads (Miltenyi Biotech) and then stained in PBS with 2% FCS for 15 min at room temperature with anti-CD4-FITC and anti-CD62L-PercP (both Biolegend, San Diego, CA, USA). The naive CD4⁺CD62L^high T cells were sorted using the BD FACSAria II cell sorter. After sorting, cells were activated for Th17 differentiation with anti-CD3 (2 μg/ml, plate-bound), anti-CD28 (2 μg/ml, soluble), rhTGF-β1 (2.5 ng/ml, Miltenyi Biotec), and rmIL-6 (20 ng/ml, Miltenyi Biotec). Cells were cultured for 72 h and analyzed by FACS.

Human microglial isolations were performed as described previously.⁵⁰ Use of human brain tumor and peri-tumoral tissue resected at surgery for research was approved by National Health Service Lothian under protocol number LREC 15/ES/0094 issued to P.M.B. with full informed consent. For isolation of human microglia, the same protocol was used as the mice (CD11b beads) with a few adjustments detailed as follows. For all steps until the Percoll gradient, RPMI (ThermoFisher, 11875093) with 3% FBS +2 mM EDTA was used instead of HBSS. Additionally, as the tissue was not perfused, an additional treatment with red blood cell lysis buffer (Biolegend, 420301) was carried out following the Percoll gradient step. Microglia were counted using a haemocytometer and plated out at 40,000 cells/well onto a black-walled, optical bottom 96-well plate (ThermoFisher Scientific) coated with poly-L-lysine. Cells were cultured in DMEM/F-12 (ThermoFisher Scientific) supplemented with 1% PenStrep, 10% FBS, 50 ng/mL rhTGFβ-1 (Miltenyi), 10 ng/μL mCSF1 (R&D Systems) for 7 days with a half media change on day 3. Phagocytosis assay was performed and analyzed as described above with 9 fields of view per well. Exclusion criteria for a case/donor was poor viability of the cells at the end of the experiment.