Tnfα Promotes an Immunosuppressive Microenvironment in Cutaneous T Cell Lymphoma and Regulates PD-L1 Expression

Background: Tumor‐associated macrophages (TAMs) play a key role in cutaneous T cell lymphoma (CTCL) growth and neoplastic T cells escape immune surveillance via PD1-PD-L1 axis (Querfeld, C., et al., Blood 2019; Khodadoust, M.S., et al., J Clin Oncol, 2020). There remains a lack of knowledge about how cytokines regulate the mechanisms controlling tumor-growth and polarize the tumor microenvironment (TME).

Methods and Results: To investigate PD-L1 and PD1 expression on TAMs and T cells in mycosis fungoides (MF) and the leukemic variant Sézary syndrome (SS) patients, we performed multiplex immunofluorescence (IF) staining of lesional skin samples of MF patients that demonstrated co-localization of PD-L1 on CD163⁺ M2 macrophages and PD1 expression on CD4⁺ and CD8⁺ T cells. In addition, significant enrichment of CD14⁺ and CD16⁺/CD14^dim CD163⁺ M2-like monocytes/macrophages with upregulated PD-L1 expression in SS patients compared to healthy donors (HDs) was found via FACS analysis. We also performed 30-plex Luminex cytokine assay on plasma samples, which showed significantly increased IL-6, IL-10, IFNγ and TNFα levels in plasma of MF/SS compared to HDs. To investigate whether polarization towards an M2-like macrophage phenotype with increased PD-L1 expression correlated with the cytokine expression from CTCL-TME, we cultured total PBMCs from HDs with conditioned media (CM) from well established CTCL cell lines MyLa and HuT78 and analyzed PD-L1 mRNA, total PD-L1 protein and PD-L1 surface expression on M2-like macrophages. Significantly increased expression of PD-L1 protein in total PBMCs, especially on CD14⁺ and CD16⁺/CD14^dim M2-like macrophages was seen. To understand whether distinct cytokines are associated with PD-L1 upregulation on CD163⁺ M2-like populations, total PBMCs from HDs were stimulated with human recombinant IL-6, IL-10, IFNγ or TNFα. Antibody blocking studies were conducted by adding anti human IL-6, IL-10, IFNγ or TNFα to the cultures with CM. TNFα stimulation significantly increased the CD14⁺ M2-like subset, but did not affect CD16⁺/CD14^dim M2-like subset. We observed increased PD-L1 expression on both M2-like populations with TNFα compared to other cytokines. In contrast, blockade of TNFα significantly decreased the CD14⁺ M2-like subset with reduced PD-L1 expression and increased CD16⁺/CD14^dim M2-like cells with upregulated PD-L1 expression. To explore whether the STAT pathway regulates PD-L1 expression through cytokines from CTCL TME, we incubated total PBMCs from HDs in CM of MyLa and HuT78 cells with/without a pan-STAT inhibitor, and in media alone. Inhibition of STAT signaling decreased CD14⁺ M2-like macrophage population, but did not alter the CD16⁺/CD14^dim M2-like population. In addition, pan-STAT inhibition significantly reduced surface expression of PD-L1 on both CD14⁺ and CD16⁺/CD14^dim M2-like macrophages. The effects of cytokines on STAT signaling components in regulating PD-L1 expression were also investigated by FACS and immunoblots. TNFα blockade significantly downregulated PD-L1, but also pSTAT1, pSTAT3 and pNF-κB levels, illustrating the role of TNFα on STAT1, STAT3 and NF-κB pathways in conjunction with PD-L1 expression. Stimulation with TNFα increased pSTAT3 level in CD14⁺ M2-like macrophages, while it did not significantly change pSTAT3 in CD16⁺/CD14^dim M2-like macrophages. Anti-TNFα reduced pSTAT3 levels in CD14⁺ M2-like macrophages, but profoundly increased PD-L1 in CD16⁺/CD14^dim M2-like macrophages, which aligns with our data of increased PD-L1 expression on CD16⁺/CD14^dim M2-like macrophages following TNFα blockade.

Conclusion: We profiled immune alterations of monocyte/macrophages populations and PD-L1 expression in CTCL regulated by selected cytokines. Our results support the dominant role of TNFα in the CTCL microenvironment. Here we show that TNFα potentiates the immunosuppressive TME through macrophage polarization and STAT-mediated PD-L1 regulation. Our results identify potential targets for combination immunotherapy.