Anti cd28 antibody

In vitro suppression was performed as previously described^{59 (link)} with some modifications. Naïve CD4+ T cells were sorted as CD3⁺CD4⁺CD25^-CD62L⁺ cells on a BD FACSAria starting from pre-enriched CD3⁺ PBMCs of healthy donors [CD3 human microbeads, Miltenyi Biotec] [Supplementary Figure 3]. Cells were labelled with 1 nM Far Red CellTrace [ThermoFisher], re-suspended in medium containing hIL2 [Proleukin] and anti-CD28 antibody [2 µg/ml, Tonbo] and plated in 96-well plates [NUNC Maxisorp] pre-coated with anti-CD3 antibody [2 µg/ml, Tonbo] at a concentration of 2.5 x 10⁴ cells/well; 2 x 10⁵ cells/well were plated as unstimulated negative control. iNKT cell lines were stimulated with anti-CD3 and anti-CD28 antibodies [2 µg/ml each, Tonbo] on NUNC Maxisorp plates for 5 days. Their supernatant was collected, applied to proliferating naïve CD4+ T cells in the presence or absence of anti-IL10 neutralising antibody [10 µg/ml], and in part used to evaluate cytokine concentration, as described in Supplementary Methods. After 5 days, proliferating naïve CD4⁺ T cells were labelled with Zombie vital dye [Biolegend] and analysed with [BD] FACS Celesta. Proliferation analyses were performed with a FlowJo v10 [BD] proliferation tool. The suppression index was calculated by dividing the proliferation index of maximum proliferation controls by that of the wells under investigation.