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.