Supplementary Figure S13. A. 3D in vitro experimental setup. Human PBMCs and SU-DHL-8 tumor cells were co-cultured in a ratio of 5:1 and treated with 0.01 nM CD20-TCB in the presence or absence of 5 μg/mL adalimumab for 24 hrs. Supernatant from the co-cultures was transferred to the bottom channel of the Mimetas OrganoPlate®. PBMCs derived from the same donor as in the co-cultures, are fluorescently labeled and seeded in the upper channel of the Mimetas OrganoPlate®, where endothelial tubules have been previously formed. Mimetas OrganoPlate® are then imaged with the Operetta System to quantify fluorescent cell transmigration at 24 hrs. B. Shown is the cell migration towards cytokine-rich supernatants in the Mimetas OrganoPlate®. TNF-α pretreatment of the endothelial tubule is a positive control. Means of technical replicates from 2 independent experiment +/- SD with ****p<0.0001, **p<0.001 by 2 way ANOVA.
ARTICLE ABSTRACT
Purpose: Target-dependent TCB activity can result in strong and systemic release of cytokines that may develop into Cytokine Release Syndrome (CRS), highlighting the need to understand and prevent this complex clinical syndrome. Experimental design: We explored the cellular and molecular players involved in TCB-mediated cytokine release by single cell RNA sequencing of whole blood treated with CD20-TCB together with bulk RNA sequencing of endothelial cells exposed to TCB-induced cytokine release. We used the in vitro whole blood assay and an in vivo DLBCL model in immunocompetent humanized mice to assess the effects of dexamethasone, anti-TNF-a, anti-IL-6R, anti-IL-1R and inflammasome inhibition, on TCB-mediated cytokine release and anti-tumor activity. Results: Activated T cells release TNF-α, IFN-γ, IL-2, IL-8 and MIP-1β, which rapidly activate monocytes, neutrophils, DCs and NKs along with surrounding T cells to amplify the cascade further, leading to TNF-α, IL-8, IL-6, IL-1β, MCP-1, MIP-1α, MIP-1β and IP-10 release. Endothelial cells contribute to IL-6 and IL-1β release and at the same time release several chemokines (MCP-1, IP-10, MIP-1α and MIP-1β). Dexamethasone and TNF-α blockade efficiently reduced CD20-TCB-mediated cytokine release while IL-6R blockade, inflammasome inhibition and IL1-R blockade induced a less pronounced effect. Dexamethasone, IL-6R blockade, IL-1R blockade and the inflammasome inhibitor did not interfere with CD20-TCB activity, in contrast to TNF-α blockade, which partially inhibited anti-tumor activity. Conclusion: Our work sheds a new light on the cellular and molecular players involved in cytokine release driven by TCBs and provides rationale for the prevention of CRS in patients treated with TCBs.
