The Involvement of STAT/SOCS Signaling in miRNA-Induced T Cell Exhaustion in Cutaneous T-Cell Lymphoma

Background: Cutaneous T-cell lymphoma (CTCL), collectively known as mycosis fungoides (MF) and Sézary syndrome (SS) arises from CD4+ T cells in a background of chronic inflammation. The chronic inflammation fosters the growth of CTCL cells and may facilitate T-cells exhaustion that is characterized by deprived effector function and sustained expression of inhibitory receptors. As a result, malignant CTCL cells escape immune surveillance and are not eliminated. At present, the roles of miRNAs or signaling pathways involved in the expression of immune checkpoints in CTCL has yet to be elucidated. Therefore, this project is aimed to elucidate how immune checkpoints are regulated by miRNAs, and how this regulation contributes to T-cell exhaustion and the development of CTCL.

Methods: We first conducted miRNAseq analysis to assess the miRNA profile of 50 CTCL patient tumor samples. The sequencing data analysis was performed at the City of Hope Integrative Genomics Core. Next, we verified the expression of 3 highly upregulated miRNAs (miR-155, -21 and -130) from the miRNAseq analysis in 5 CTCL cell lines and tumor samples using qRT-PCR and in situ hybridization (ISH). Finally, we transfected the CTCL cell line Myla 2059 and HuT 78 with anti-miR-155, -21, -130 or Scramble (Scr) control using the Lonza nucleofection kit and nucleofector machine. Cell lysates were prepared 72 hours after transfection and then subjected to Western Blot analysis. We probed the blot with antibodies against SOCS, PTEN, pSTAT3 and GAPDH as a loading control.

Results: The data analysis revealed that miR-155 had the highest correlation with CTLA-4 (r = 0.59, P < 0.0001), PD1 (r = 0.42, P = 0.0021), PD-L1 (r = 0.42, P = 0.0025), TIM3 (r = 0.63, P < 0.0001), LAG3 (r = 0.57, P < 0.0001), and ICOS (r = 0.74, P < 0.0001) mRNA; -21 had the highest correlation with PD-L1 (r = 0.44, P = 0.0012), TIM3 (r = 0.52, P < 0.0001), and ICOS (r = 0.37, P < 0.0073) mRNA; and -130 had the highest correlation with CTLA-4 (r = 0.5, P = 0.0002), PD-L1 (r = 0.54, P < 0.0001), TIM3 (r = 0.69, P < 0.0001), LAG3 (r = 0.49, P = 0.0003), and ICOS (r = 0.68, P < 0.0001) mRNA. qRT-PCR and ISH revealed that miRs-155, -21 and -130 were upregulated in all 5 CTCL cell lines and primary tumor samples. There was a dramatic increase in SOCS proteins and significant decrease in pSTAT3 expression in Myla 2059 and HuT 78 cells transfected with anti-miRs-155, -21 or -130, compared to cells transfected with Scr.

Conclusions: Immune checkpoints and ligands like PD-L1 expression in CTCL are regulated by miRs-155, -21 and -130. The SOCS family of proteins, negative regulators of STAT signaling, are involved in miRs-155, -21 and -130-induced immune checkpoints expression in CTCL. Taken together, these results demonstrate the mechanisms of miRNA-induced T cell exhaustion and pave the way for the development of miRNA therapeutics in CTCL.