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Supplementary Figure S11 from Dissecting the mechanisms underlying the Cytokine Release Syndrome (CRS) mediated by T Cell Bispecific Antibodies

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posted on 2024-09-16, 11:27 authored by Gabrielle Leclercq-Cohen, Nathalie Steinhoff, Llucia Albertí-Servera, Sina Nassiri, Sabrina Danilin, Emily Piccione, Emilio Yángüez, Tamara Hüsser, Sylvia Herter, Stephan Schmeing, Petra Gerber, Petra Schwalie, Johannes Sam, Stefanie Briner, Sylvia Jenni, Roberta Bianchi, Marlene Biehl, Floriana Cremasco, Katerina Apostolopoulou, Hélène Haegel, Christian Klein, Pablo Umana, Marina Bacac

Supplementary Figure S11. Single cell RNA-sequencing of whole blood treated with 0.2 μg/mL CD20-TCB was performed using the BD Rhapsody platform. A. UMAP plots of neutrophils colored by treatment. B. UMAP plots showing IL-1β (IL1B), IL-1Ra (IL1RN), IL-6 (IL6), IP-10 (CXCL10), IL-8 (CXCL8), TNF-α (TNF), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4) gene expression within neutrophil clusters identified above. C. Violin plots showing the kinetics of gene expression distribution in neutrophils. The cytokine and cytokine receptor genes are listed on the y axis. The color code on the right side of the violin plots represents the median gene expression. The untreated condition corresponds to the pooled baseline and untreated samples. N= 4 donors (baseline, and 20 hrs) and n=2 donors (2 hrs, 4 hrs and 6 hrs).

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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.

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