Single-Cell RNA Sequencing Identifies a Novel Population of CD4+CD8+ T Cells that are Regulated by COX-2 During Allergic Lung Inflammation

Abstract

Abstract To better understand the relative contributions of different cell types during allergic lung inflammation and the role of cyclooxygenase (COX)-derived eicosanoids in this process, we compared vehicle (control) and ovalbumin (OVA)-exposed lungs from wild type (WT) and COX-2−/− mice by single-cell RNA sequencing (scRNA-Seq). Twenty-two unique cell clusters were identified in allergic lungs based on cell signature markers. In addition to known cell types, we identified 2 clusters of CD4+CD8+. Both populations were mitotically active (high % in S/G2/M phases) and exhibited a unique molecular identifier (UMI) index. FACS analysis confirmed the presence of these two distinct cell populations (CD4+HCD8+H and CD4+LCD8+H) that were increased after OVA-induced allergic lung inflammation. Interestingly, isolated CD4+HCD8+H T cells stimulated with phorbol ester expressed IL-4, IL-5 and IL-13, but not IFNγ. In contrast, isolated and stimulated CD4+LCD8+H T cells primarily expressed IL-5. Mass cytometry (CyTOF) using a 37-antibody panel confirmed the increase in CD4+CD8+ T cells after OVA sensitization/exposure, as well as the induction of IL-4, IL-5 and IL-13. Importantly, CD4+CD8+-1 and CD4+CD8+-2 cell populations identified by scRNA-Seq were increased in lungs from OVA-treated COX-2−/− mice compared to lungs from OVA-treated WT mice. Furthermore, COX-2−/− CD4+CD8+ T cells preferentially produced more IL-4 and IL-5 than WT CD4+CD8+ T cells. Thus, using scRNA-Seq, CyTOF and FACS analyses, we identified two populations of CD4+CD8+ T cells that produce immunoregulatory cytokines and are regulated by COX-2. These cells may play an important, previously unappreciated role in the pathogenesis of allergic lung disease.