Fix and perm buffer set

Monocytes were stimulated with RPMI, IL4 or different concentrations of IL4-aNPs (indicated in Extended Data Fig. 6a) for 20 min at 37 °C. The cells were transferred to a V-bottom 96-well plate and kept on ice for the duration of the staining procedure. After staining for viability and CD14 (in the manner described above), the cells were fixed and permeabilized using the fix and perm buffer set (eBioscience) for 45 min at 4 °C in the dark. The cells were washed twice with perm buffer and incubated overnight in freezer-chilled absolute methanol at −20 °C. Following two more washes in perm buffer the cells were stained for phospho-STAT6 using the antibody described in Supplementary Table 3, for 45 min at 4 °C in the dark. The cells were washed two more times in perm buffer and finally re-suspended in PBA for acquisition on the Cytoflex cytometer. The gating strategy was largely similar to the one for macrophage surface marker with the addition of a selection for CD14-positive events.

For mixed lymphocyte reaction experiments, collected macrophages were used for subsequent T cell polarization assays. Allogeneic naive T cells were seeded with macrophages in a ratio of 10 T cells for every macrophage. The cells were cultured in flat-bottom 96-well plates for 7 days in standard cell culture medium. In this model, human leukocyte antigen mismatch causes non-specific activation of the T cell receptor. On the final day, the cells were stimulated with phorbol 12-myristate 13-acetate (25 ng ml⁻¹) + ionomycin (0.5 µg ml⁻¹) for 4 h in the presence of 100 ng ml⁻¹ Brefeldin A, a ‘golgi plug’. The cells were collected and split over two flow-cytometry antibody panels (one for CD4 T cells and one for CD8; see also Supplementary Table 3). The cells were stained in a similar manner as described above, with an extra step for permeabilization of the T cells to allow for intracellular cytokine staining. This was performed using the fix and perm buffer set (eBioscience), according to the manufacturer’s instructions. The gating strategy was similar to what is described above, with the addition of a selection for CD3-positive events. The percentage of cells positive for hallmark cytokines of T cell polarization were calculated to estimate T cell subset proportions.