PD-L1 Is Regulated By Interferon Gamma and Interleukin 6 through STAT1 and STAT3 Signaling in Cutaneous T-Cell Lymphoma

Abstract

Background: Signaling interactions within the cutaneous T cell lymphoma (CTCL) microenvironment often create a permissive environment for CTCL growth. In response to oncogenic signals from proliferating tumor cells, the tumor microenvironment changes throughout disease progression. Studies have shown that dendritic cells and macrophages are critical tumorigenic regulators of the microenvironment in hematologic malignancies. Tumor cells escape immune surveillance by manipulating immune checkpoint receptors like PD1, and activation of the PD1 receptor by PD-L1 transduces a signal that leads to the inhibition of T-cell functions. Tumor cells, resting dendritic cells, and macrophages in the tumor microenvironment express PD-L1; and proinflammatory cytokines like IFN-γ and IL 6 upregulate PD-L1 on tumor cells. However, the mechanisms of PD-L1 upregulation in CTCL remain unknown.Methods: Immunoblots: Primary patient peripheral blood mononuclear cells (PBMCs), CTCL cell lines (Mac1 and Mac2a) and mature monocyte-derived dendritic cells (MoDCs) from healthy patients were treated with IFNγ, IL-6, a a pan JAK/STAT inhibitor (tofacitinib), and a PD-L1 inhibitor for 72 hrs. Cell lysates were collected and subjected to immunoblot analysis with antibodies against PD-L1, pSTAT1, and pSTAT3. Immunohistochemistry: Paraffin sections of patients' skin biopsies were incubated with mAbs against CD163 PD-L1, and pSTAT3 at 4°C overnight. Sections were incubated with appropriate secondary Abs for 2 hrs at room temperature. The images were taken with a Zeiss 700 confocal microscope. Flow Cytometry: Migrated skin cells from skin explant cultures taken from CTCL patients were incubated with fluorochrome-conjugated mAbs and analyzed on an LSR Fortessa (BD) flow cytometer. The gating parameters were as follows: epidermal LCs (CD1a+, CD14neg, HLA-DRbright), dermal CD1a+ DCs (CD1a+, CD14neg, HLA-DRbright, CD11b+, CD11c+), and dermal CD14+ DCs (CD1aneg, CD14+, HLA-DRbright, CD11b+, CD11c+). Gates were set for collection and analysis of at least 10,000 live events, and the data were analyzed with FlowJo 9.7.6 software (TreeStar).Results: Our idata indicate that T cells in CTCL have an exhausted phenotype, presumably due to chronic Ag stimulation by cutaneous DCs. Macrophages and dendritic cell subsets in lesional skin from mycosis fungoides patients as well as PBMCs from Sézary syndrome (SS) patients express PD-L1. To elucidate the mechanism of PD-L1 regulation, we treated PBMCs from SS patients and CTCL cell lines with IFN-γ, IL-6, a pan JAK/STAT inhibitor (tofacitinib), and a PD-L1 inhibitor (durvalumab). We found upregulation of PD-L1 protein expression by PBMCs when treated with IFN-γ and IL-6, through upregulated STAT1 and STAT3 signaling detected by Western blots. A pan JAK/STAT inhibitor abrogated the increased expression of PD-L1. Monocyte-derived dendritic cells from healthy donors exhibit upregulated PD-L1 expression by IFN-γ, but not with IL-6. Durvalumab treatment reduced pSTAT3 and PD-L1 expression. We have therefore initiated a clinical trial with a PD-L1 inhibitor (durvalumab) in patients with mycosis fungoides (MF)/Sézary syndrome (SS) and observed promising initial responses.Conclusions: PBMCs from Sezary syndrome patients and moDCs from healthy donor upregulate PD-L1 through STAT signaling initiated by proinflammatory cytokines IFN-γ and IL-6. Durvalumab also reduced PD-L1 and pSTAT3 expression in patient PBMCs. PD-L1 expression may result from the proinflammatory background of the tumor microenvironment and activation of the PD1 receptor by PD-L1 transduces a signal that leads to the inhibition of T-cell functions in CTCL. DisclosuresKadin:Allergan, Inc: Honoraria. Querfeld:Soligenix: Research Funding; Actelion: Honoraria, Research Funding; Medivir: Honoraria; Mallinckrodt: Honoraria; Kyowa: Research Funding; Elorac: Research Funding; Celgene: Honoraria, Research Funding; MiRagen: Honoraria, Research Funding; Trillium Therapeutics: Research Funding; City of Hope: Employment; Mindera: Consultancy.