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
Target-dependent TCB activity can result in the 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.
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-TNFα, anti-IL6R, anti-IL1R, and inflammasome inhibition, on TCB-mediated cytokine release and antitumor activity.
Activated T cells release TNFα, IFNγ, IL2, IL8, and MIP-1β, which rapidly activate monocytes, neutrophils, DCs, and NKs along with surrounding T cells to amplify the cascade further, leading to TNFα, IL8, IL6, IL1β, MCP-1, MIP-1α, MIP-1β, and IP-10 release. Endothelial cells contribute to IL6 and IL1β release and at the same time release several chemokines (MCP-1, IP-10, MIP-1α, and MIP-1β). Dexamethasone and TNFα blockade efficiently reduced CD20-TCB–mediated cytokine release whereas IL6R blockade, inflammasome inhibition, and IL1R blockade induced a less pronounced effect. Dexamethasone, IL6R blockade, IL1R blockade, and the inflammasome inhibitor did not interfere with CD20-TCB activity, in contrast to TNFα blockade, which partially inhibited antitumor activity.
Our work sheds new light on the cellular and molecular players involved in cytokine release driven by TCBs and provides a rationale for the prevention of CRS in patients treated with TCBs.
