134 Expansion of Th17 Cells in CD3/CD28-activated Human PBMC: Monocyte Apoptosis and Skin-Homing T-cell Bias

T-cells undergo polyclonal expansion in cutaneous psoriasis (PsC) and PsA, with persistence of "driver clones" after effective treatment (JCI 127:4031; JI 172:1935). A recent scRNA-seq study found roughly equal compartments of clonal and non-clonal Th17 and Tc17 cells in PsC (Science 371:364). We and others have shown that Th17 expansion from PBMC requires contact between monocytes and memory T-cells in the context of TCR ligation (PNAS 104:17034; SID 139:1245). We stimulated PBMC (n=153) with anti-CD3/CD28 beads for 0 or 24h followed by flow cytometry (CD3+CD45RO+,CD4/CD8,CLA+/CLA-). Activation-related DEGs featured marked up-regulation of Th17 signature mRNAs (IL17A, IL17F, IL22, and CCL22) along with the Th1 cytokine IFNG, with a corresponding induction of IL-17A and IL-22 proteins by flow cytometry. These findings were confirmed by cluster analysis of scRNA-seq libraries of CD3/CD28-activated PBMC (n=4 subjects). Stratified analysis of skin homing in CD3/CD28-activated cells revealed 2.9 to 12.1-fold upregulation of IL17A, IL17F, IL22, and CCL22 in CLA+ vs CLA-, without a corresponding difference in IFNG. IL17A and IL17F were overexpressed in activated T-cells from psoriatics vs. controls (each 1.9-fold, p=4.7x10-4). As revealed by scRNA-seq of lesional psoriatic skin, IL17A was overexpressed (2.2-fold, p= 0.003) in skin-homing (FUT7+) vs non-skin-homing (FUT7-) T-cells, whereas IFNG was not. As reported in a recent CITE-seq study (Front Immunol 12:636720), our bulk- and sc-RNA-seq experiments revealed dramatic disappearance of monocytes within 24h of CD3/CD28 activation, which was confirmed by imaging flow cytometry and morphologically identified as apoptosis by time-lapse microscopy. Taken together with data showing IL-23 expression by inflammatory monocyte-like cells in dermal clusters in PsC (JID 141:1707), our experiments suggest a contact-dependent interplay between activated T-cells and monocyte-derived cells in dermal clusters, which maintains polyclonal activation of skin-homing Th17 cells in psoriatic lesions