Extended Data Fig. 4: ISACs elicit distinct intracellular signaling in monocytes and cDCs.
a-c,e-f, Freshly isolated human PBMC were stimulated with 1 μM of rituximab-ISAC or an equimolar mixture of rituximab and T785 in the presence of CD20+ Toledo tumor cells at a 1:1 ratio for 15 minutes. a, Signaling induction of p-MAPKAPK2, p-p38, p-CREB and p-S6 in monocytes and cDCs was quantified by the arcsinh of the ISAC or mixture as compared to the unstimulated PBMCs (n=6 independent donors). b, cDCs were subsetted as CD141+ cDC1s and CD1c+ cDC2s (n=6 independent donors). c, Signaling through pERK1/2 and p-S6 measured following 5, 15, or 30-minute stimulation with rituximab T785-ISAC or the mixture of rituximab and T785 (n=2 independent donors). d, Monocytes were treated with 1 μM of R406 prior to stimulation with 2 μM T785 overnight. Activation was assessed by flow cytometry, and no statistical significance was seen between the conditions with and without Syk inhibition. The dashed line represents the expression level of the unstimulated control (n=2 technical replicates of pooled myeloid APCs with 6 independent donors per pool). e, Signaling induction of p-MAPKAPK2, pERK1/2 and pIRF7 was quantified by the arcsinh of the ISAC with and without Syk blockade (R406) as compared to the unstimulated control (n=6 independent donors). f, Signaling induction of pIRF7 was quantified by the arcsinh following stimulation with rituximab T785-ISAC or rituximab-TLRnull-ISAC (TLRnull-ISAC) (n=6 independent donors). a, b, d-f, Data shown (mean and SEM) are representative of two independent experiments. P values were calculated by two-tailed paired T tests.
