GSE193255
Heterogeneity in antigen presenting cells to infection revealed by single cell RNAseq
Antigen presenting cells (APC) play a central role in the host immune response to pathogen invasion by triggering early innate responses and facilitating the subsequent adaptive immune system for effective control and clearance of the infection. Accordingly, APC critically impact the outcome of an immune response. In vitro bone marrow (BM) derived APC facilitate studies aimed at dissecting their biology and function. BM derived cell cultures differentiated with granulocyte-macrophage colony stimulating factor (GM-CSF) consists of a heterogeneous mix of both dendritic cells (DC) and macrophages (MØ). Here we addressed the question of the impact of heterogeneous APC populations in their response to RNA viruses using the well characterised lymphocytic choriomeningitis virus (LCMV) infection model of BMDC populations. We compared LCMV infection and treatment with the dsRNA mimic poly (I:C)) to characterise the response of different subsets of BMDC using single cell transcriptomic analysis. Our results identified differences in the transcriptional response of BM derived APC to LCMV and poly (I:C). BM-DC were more effective at antigen presentation to T cells, whereas BM-MØ were efficient cytokine producers. Overall design: Sorted CD11c+MHC class II+ bone marrow derived cell populations (dendritic cells [BMDC], macrophages [BM-M], double negative cells [DN]) were infected with LCMV clone 13 expressing GFP or stimulated with 100 µg poly (I:C)/ml  , as previously described for 12hr and 48hr for single cell (from the 12 h culture) sorting into customised BD WTA single cell encoding plates for RNA extraction and reverse transcription (primary infection) according to manufacturer's instructions (PN 910000014 Rev. 03). Furthermore, a subset of infected cells (from the 48 h culture) were stimulated with 100 µg poly (I:C)/ml and incubated for another 6hr before single cell sorting into BD WTA single cell encoding plate as previously described (two plates per BM group). In the primary infection, a cell was considered to be infected if there was at least one GFP sequence read count. Differential gene expression was measured between infected and uninfected cells in each subset (Adjusted p-value less than 0.05). In the secondary stimulation with poly (I:C) gene expression was compared between infected cells alone and infected cells plus stimulation among the three BM groups. Selected antiviral ISGs and other genes from a curated database (http://www.informatics.jax.org/vocab/gene_ontology/GO:0002532) were compared between the three BM derived cells (CCL5, CD40, CD86, CXCL10, DDX58, GBP2, IFIT1,IFIT2, IFIT3, IFITM3, IL1b, IL6, IRF7, ISG15, ISG20, MX1, OASL1, OASL2, PML, RSAD2).