50% of patients with cutaneous lupus erythematosus (CLE) respond to antimalarials. Because of a two-month delay in onset of action of antimalarials, however, the other half of these patients continue with disease activity and progressive scarring, only to find they are not responding. The molecular and cellular basis for this heterogeneity of responses to treatment remains uncharacterized, leading to treatment delays and then the use of toxic therapies and multiple therapeutic regimens. Our goal is to understand the biologic determinants in newly diagnosed CLE patients who then do not respond to antimalarials. This information will pave the way for personalized, targeted therapies for this difficult subgroup of patients, to improve their health and quality of life (QoL). We hypothesize that, before treatment, antimalarial non-responders will have differentially expressed T cell subsets and unique pathway markers relative to antimalarial responders. Specific Aims: Aim 1. Immunologic analyses of the cellular infiltrate in lesional CLE skin from well-characterized patients at baseline who subsequently were determined to be antimalarial responders or non-responders. Aim 2. Analysis of cells and corresponding signaling pathways together in lesional CLE skin at baseline who subsequently were determined to be antimalarial nonresponders or antimalarial responders. Research Strategy: Using our prospective database of 470 CLE patients characterized in terms of their subsequent responses to therapy, we will examine baseline lesional skin biopsies obtained before treatment from fifteen hydroxychloroquine-responsive, fifteen quinacrine-responsive, and twenty antimalarial refractory patients. The lesional skin biopsies will be characterized by immunophenotyping of cells in tissue using high-dimensional single-cell CyTOF. This approach allows characterization of 35 cell-surface markers directly in lesional skin, using a panel of metal halide-labeled antibodies that are commercially available from Fluidigm. We have chosen markers that allow quantification of specific subclasses of T cells, B cells, mDCs, and pDCs, as well as other inflammatory cells and transcription factors present in CLE skin. This approach detects differential expression of immune cells and activated pathways in lesional skin without needing to elute or artificially remove cells for analysis. We will use bioinformatics software for in-depth characterization of immune cell subsets that differentiate antimalarial non-responders relative to responders. The proposed studies will provide a systematic approach to better understand critical cells and pathways in antimalarial-refractory CLE. Examining multiple cell types simultaneously, as well as their signaling pathways using CyTOF mass cytometry provides a novel approach to a difficult and unexplored problem of refractory skin disease in lupus, providing insight into future targeted therapy in refractory disease.