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Abstract

CCCA is a poorly understood common cause of alopecia in Black women. Limited reports indicate benefit from JAK/STAT inhibition, which influences Th1 and Th2 cell differentiation. The distinct role of cells mediating this disorder through previously identified genetic mechanisms is largely unclear, limiting focused therapy development. We obtained biopsies from 7 CCCA patients and 8 controls. After bulk RNA sequencing, we conducted spatial proteomics on two cases and one control using 29 fluorescent immune cell markers and JAK/STAT pathway components. We segmented cells using Sopa, then implemented machine-learning-based quality control focusing on markers with at least 50 segmented cells per specimen. We compared per-cell mean JAK/STAT expression of cases against controls. We evaluated 37,972 total cells. There were more cells positive for CD3 (T-cells), CD20 (B cells), CD38 (plasma cells), CD68 and CD163 (histiocytes) in cases. Phosphorylated STAT6 (pSTAT6) expression was significantly less (FDR < 0.05) in cases, with little to no expression in CD3, CD4, CD20, CD38, CD45, CD68, and CD163-positive cells, with general positivity in these cells in controls. These results clarify the cells that mediate CCCA, supporting the role of Th1 and Th2 pathways. As pSTAT6 expression is associated with Th2 immunity, these data suggest Th1 immune mediation of CCCA, supported by the infiltration of inflammatory cells, particularly histiocytes. These results complement our bulk RNA sequencing work demonstrating significant changes in gene expression involved in these pathways, suggesting mediation predominantly by infiltrating immune cells. While analysis is ongoing, these results afford valuable insights into this disease.