A multi-level study of the effects of glucocorticoids on human T cells

Abstract

Abstract Glucocorticoids (GCs) have been the cornerstone of immunosuppressive therapies for the past 70 years, yet the mechanisms by which they regulate human immunity remain poorly understood. Specifically, there remains a need for direct insight into how GCs regulate the functions of human T cells. We have previously demonstrated that response to GCs in humans is highly cell type-dependent. In this project, we combine high-throughput techniques to study the effects of GCs on human CD4+ and CD8+ T cells, in vivo and in vitro. As part of a clinical study in 20 healthy volunteers, T cells were purified before and 2 and 4 hours after a dose of the GC methylprednisolone. RNA-seq was performed to identify GC-responsive genes. We identified a strong transcriptional response in T cells, where increased transcript abundance predominates. While there is a high degree of overlap of GC-responsive genes and in response directionality, CD4+ T cells show a stronger and more unique response than CD8+ T cells. To understand the functional genomic consequences of GC treatment in primary T cells, we developed a protocol to simultaneously assay gene expression (RNA-seq), chromatin accessibility (ATAC-seq), and glucocorticoid receptor binding (CUT&RUN) on cells treated in vitro. We are also performing experiments using tandem mass tagging (TMT) and mass spectrometry to identify which of the differentially expressed transcripts correspond to protein-level changes. Integrating data obtained at the transcript, chromatin, and protein levels will provide us with a means to identify biologically relevant effects of glucocorticoids and will guide the selection of assays to determine the functional consequences of those effects on human T cells.