Immunomodulatory Effects of a Cell Processing Device: Insights from Single Cell RNA Sequencing and Gene Set Enrichment Analysis

Abstract

The Selective Cytopheretic Device (SCD), marketed as QUELimmuneTM, is an autologous leukocyte processing extracorporeal device which promotes immunomodulation of a systemic hyperinflammatory states. To date, interrogation of the mechanism of action (MoA) of this cell-directed therapy has provided insight into therapeutic impact on neutrophils. However, characterization of monocytes has proven to be more difficult due to their dynamic plasticity, requiring more sophisticated analysis methods. Single cell ribonucleic acid sequencing (scRNAseq) methodology using 10x Flex is reported to further elucidate MO MoA in SCD therapy. Analysis strategies of unsupervised clustering and interrogation of specific genes of interest based on previous mechanistic insights from cell surface marker analysis and bulk RNA sequencing were utilized. SCD-treated monocytes were demonstrated to have lower expression of several proinflammatory gene products including TNF and IL-6 and enrichment in anti-inflammatory gene markers, suggesting that SCD monocyte processing results in the release of monocytes with a less inflammatory phenotype. Calcium-dependent gene pathways are also downregulated, consistent with the low iCa environment of SCD therapy. Furthermore, analysis points to the involvement of key transcription factors such as AP-1 and NFkB. Taken together with preclinical and clinical data, scRNAseq confirms several aspects of the suspected SCD MoA on monocytes to transform them to a lesser pro-inflammatory phenotype.