Supplementary Figure S6. A. Bulk-rna-sequencing of neutrophils purified from whole blood 4 hrs and 20 hrs following treatment with 0.2 μg/mL CD20-TCB or 0.2 μg/mL DP47-TCB for N=4 donors. B. Heat map showing the enriched hallmark pathways in neutrophils, 4 hrs and 20 hrs following treatment with CD20-TCB or DP47-TCB. C. Heat map showing the expression of TNF-α (TNF), IL-1β (IL1B), IL-6 (IL6), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4) and IP-10 (CXCL10) genes in neutrophils, 4 hrs and 20 hrs following treatment with CD20-TCB or DP47-TCB (untargeted TCB). Volcano plots depicting the cytokine genes in neutrophils D. 4 hrs and E. 20 hrs after treatment with CD20-TCB. Each dot represents one gene. The x axis shows the logarithmic fold change and the y axis shows the negative log of the p value for each gene by comparing samples treated with CD20-TCB to samples treated with DP47-TCB (untargeted TCB).
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.See related commentary by Luri-Rey et al., p. 4320
