Abstract 970: Single-cell mRNA sequencing analysis of the synergistic impact of double-stranded RNA (dsRNA) and IFNα on human monocyte-derived macrophages
Authors: Giamo Vincent , Grimm Melissa , Pabla Sarabjot , Karasik Ellen , Luce Jesse , Glenn Sean T. , Conroy Jeffrey , Conroy Jeffrey , Foster Barbara , Taniguchi Kiyomi , Kalinski Pawel , Shirai Masataka 
Published: September 2020

Abstract
Purpose of Study: Single-cell mRNAseq (scRNAseq) is a promising technology allowing for unbiased analysis of gene expression at the cellular level. In this study, scRNAseq was used to identify cellular sub-types and differential gene expression functional units in monocyte-derived macrophages in response to double-stranded RNA (dsRNA, viral mimic, TLR3 ligand), IFNα or their combination.
Procedure: Human monocyte-derived macrophages were cultured overnight in the absence (U) or presence of dsRNA (rintatolimod/Ampligen-A), IFNα (I) or their combination (IA), which we previously identified as synergistic in induction of chemokines attracting CTLs, Th1 and NK cells. The cells were harvested after 1 day, captured at the single-cell level and immobilized in a vertical flow array chip (VFAC), which contains 100-microchambers packed with 105 beads immobilizing 1010 oligo(dT) probes with unique cell-ID, UMI, and PCR-tags. Each single cell was captured and lysed on the small hole (3-micro meter in diameter) above the microchamber on the VFAC and cDNA libraries were synthesized and tagged with unique cell-IDs post mRNAs hybridization. ScRNAseq using a custom 94- gene inflammation RNA-seq panel was then performed. Post-clustering (Scanpy), unsupervised cell-type identification (louvain), and downstream analyses were performed on the gene expression data to determine impact of A, I and IA treatment on the cellular composition of the induced macrophages.
Results: PCA and unsupervised clustering revealed sample groups with distinct phenotypic signatures. The analyses revealed the expected synergy between the dsRNA and IFNα in the induction of CXCR3- and CCR5-binding chemokines, but antagonism in the induction of Treg attractants. Unexpectedly, the single cell analyses revealed striking heterogeneity of the responses of the individual myeloid cells within the morphologically uniform macrophage population activated by the individual stimuli and their IA combination.
Conclusion: scRNAseq was able to identify distinct single-cell macrophage phenotypes caused by induction of dsRNA and IFNα. Of the 5 subclusters identified, inducement by IFNα alone or in combination with TLR3 identified subsets of cell populations representing lineage and activation markers. These unexpected observations facilitate the identification of intracellular signaling pathways underlying the heterogenous response of individual myeloid cells to exogenous and endogenous activators, helping to develop improved vaccine adjuvants and cancer immunotherapies.
Citation Format: Vincent Giamo, Melissa Grimm, Sarabjot Pabla, Ellen Karasik, Jesse Luce, Sean T. Glenn, Jeffrey Conroy, Jeffrey Conroy, Barbara Foster, Kiyomi Taniguchi, Pawel Kalinski, Masataka Shirai. Single-cell mRNA sequencing analysis of the synergistic impact of double-stranded RNA (dsRNA) and IFNα on human monocyte-derived macrophages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 970.