Regulation of IL-2- and IL-6-dependent signaling in human Tregs by CISH and SOCS3

Abstract

Abstract SOCS proteins function as negative regulators of cytokine signaling. However, the role in of these proteins in human Tregs remains poorly understood. To address this issue, CRISPR-Cas9 genome editing was used to probe the role of SOCS3 and CISH in ex vivo expanded human Tregs. High efficiency editing of SOCS3 and CISH was confirmed at the DNA and RNA levels and the protein level for CISH. Deletion of SOCS3 resulted in sustained activation of STAT3 upon treatment with IL-6 whereas IL-2-induced STAT5 activation and IL-10- induced STAT3 activation were not affected. These results suggest that in Tregs SOCS3 specifically targets signaling initiated through IL-6R. Proliferative capacity and expression of Treg-specific phenotypic markers were not affected by SOCS3 ablation. RNA-seq revealed a common transcriptional signature between IL-6-treated wild-type and SOCS3-edited cells, showing upregulation of pro- and anti-inflammatory response genes and other genes that may affect Treg stability. Other differentially expressed genes in SOCS3-edited cells belong to pathways that may play a role in how Tregs integrate cytokine signals. In contrast, CISH editing resulted in increased IL-2-dependent activation of STAT5 and STAT3 and led to increased proliferation of Tregs. These effects were accompanied by only minor change in the expression of Treg-specific phenotypic markers. Our data indicate a restricted role of SOCS3 and CISH in regulating the responses of Tregs to IL-6 and IL-2, respectively. These activities likely contribute to maintaining Treg homeostasis under normal and inflammatory conditions. Supported by: NIH R01AI131648