Functional remodeling of macrophages with anti-PD-L1 and lenalidomide in cutaneous T cell lymphoma

Background: M2-like tumor-associated macrophages (TAMs) are the most abundant phenotype that promote the growth of cutaneous T cell lymphoma (CTCL) by inducing immunosuppression. Although PD1/PD-L1blockade has demonstrated efficacy in CTCL, little is known about the effects on tumor-associated macrophages. Here, we report that anti–PD-L1 treatment favorably impacts the phenotype and function of tumor macrophages by polarizing the macrophage compartment toward a more pro-inflammatory phenotype. Understanding the mechanisms of macrophage reprogramming will identify novel therapeutic targets to reverse impaired immunity. Methods and results: We used multiplex immunofluorescence staining of lesional skin samples of CTCL patients demonstrating co-localization of PD1 on CD163+ M2 macrophages. Moreover, RNA-seq analysis performed on tissue sections from same CTCL specimens revealed an up-regulation of the TLR, NF-κB and JAK/STAT signaling pathways. To further confirm PD1 expression on M2 TAMs in CTCL, we cultured CD14+ cells from healthy donor-derived peripheral blood in conditioned media (CM) from MyLa cells. We observed macrophage differentiation towards an PD1+ M2 phenotype, with significant CD163, CD206 and PD1 upregulation but not CD80 expression. The TRIF-stimulated NF-κB and JAK/STAT signaling pathways, downstream of both TLR3 and TLR4 were activated in PD1+ M2-like TAMs. Furthermore, our RT-PCR results show significantly elevated IL-10 levels, but decreased IL-1β, CXCL-10 and CXCL-11 compared to control. To determine whether PD1+ M2-like TAMs could be reprogrammed, anti-PD-L1 (durvalumab) and lenalidomide were used for treatment of MyLa-conditioned media induced human peripheral blood monocyte-derived PD1+ M2-like TAMs. The results show that anti-PD-L1 and lenalidomide synergistically increased IL-1β, CXCL-10, and CXCL-11 expression, but significantly decreased IL-10 level compared with the untreated control in vitro through ablation of TLR3, TLR4 and the downstream signaling pathways, which was linked with functional changes in phagocytic activity and cell migration.