Extended Data Fig. 3: Myeloid APC differentiation and activation, antigen cross-presentation and T cell proliferation following ISAC stimulation.
a-c, Freshly isolated human myeloid APCs were cultured with rituximab, T785, rituximab and T785 or the rituximab T785-ISAC in the presence of CFSE-labeled CD20+ Toledo tumor cells at a 3:1 ratio. The rituximab concentration is depicted on the X-axis; T785 concentration is equivalent to the amount of T785 conjugated to the rituximab T785-ISAC. Myeloid APCs were analyzed after 18 hours via (a, b) flow cytometry or (c) cytokine bead array. d, Frozen myeloid APCs were thawed, rested for two hours and incubated with trastuzumab or trastuzumab T785-ISAC for 18 hours prior to assessment of cell surface markers by flow cytometry. a-d, Data are shown (mean and SEM) with 3 independent donors and are representative of 3 experiments; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 calculated by two-way ANOVA with Tukey’s multiple comparison correction. e, Mouse splenocytes were isolated and cultured in vitro with ovalbumin antigen complexed with anti-OVA CL264-ISAC or anti-OVA mAb. Antigen cross-presentation was measured using multicolor flow cytometry through detection of MHC-I bound SIINFEKL peptide on total CD11c+ splenic cells. CD8+ T cells with OVA-specific TCR were isolated from OT-1 transgenic mice and co-cultured with anti-OVA mAb or anti-OVA ISAC stimulated APCs. T cell proliferation was measured by final T cell count with the initial number of APCs subtracted.
