No abstract

© 2019 American Association for Cancer Research T cells T cells aPDL1 MHCI and MHCII ARGI iNOS CD40 CD86 proinflammatory cytokines Phagocytosis pathways Proinflammatory Suppressive MacrophageWithout treatment, tumor macrophages maintain a suppressive phenotype.Following anti-PD-L1 treatment, increased IFN signaling remodels the macrophage compartment towards a more proinflammatory phenotype, which can enhance T-cell responses.Remodeling of the macrophage compartment is driven by IFN following anti-PD-L1 treatment. Macrophage Cancer cells ARGI IFNγIFNγ Checkpoint inhibitors like anti-PD1/PD-L1 have demonstrated significant therapeutic efficacy in a subset of patients partly through reinvigoration of CD8 T cells. However, their impact on myeloid cells remains largely unknown. 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 proinflammatory phenotype. This phenotype was characterized by a decrease in Arginase-I (ARG1) expression and an increase in iNOS, MHCII, and CD40 expression. Whole-transcriptome profiling further confirmed extensive polarization of both tumor monocytes and macrophages from a suppressive to a proinflammatory, immunostimulatory phenotype. This polarization was driven mainly through IFNg and was associated with enhanced T-cell activity. Transfer of monocytes into anti-PD-L1treated tumor-bearing mice led to macrophage differentiation into a more proinflammatory phenotype, with an increase in CD8 T cells expressing granzyme-B and an increase in the CD8/Treg ratio compared with control-treated mice. Although in responsive tumor models, anti-PD-L1 treatment remodeled the macrophage compartment with beneficial effects on T cells, both macrophage reprogramming and depletion were needed to maximize anti-PD-L1 responses in a tumor immune contexture with high macrophage burden. Our results demonstrate that anti-PD-L1 treatment can favorably remodel the macrophage compartment in responsive tumor models toward a more proinflammatory phenotype, mainly through increased IFNg levels. They also suggest that directly targeting these cells with reprogramming and depleting agents may further augment the breadth and depth of response to anti-PD-L1 treatment in less responsive or more macrophage-dense tumor microenvironments.

CD96 is a member of the poliovirus receptor (PVR, CD155)‐nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co‐stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK‐ERK pathway. CD96‐mediated signaling led to increased frequencies of NUR77‐ and T‐bet‐expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co‐stimulatory role in CD8+ T cell activation and effector function.

SignificanceCD226 is an important activating receptor involved in mediating natural killer (NK) cell responses against tumors, but how CD226 exerts control over NK cell function is not fully understood. CD226 belongs to the poliovirus receptor (PVR)-nectin family that includes TIGIT and CD96, with TIGIT garnering much attention as a key checkpoint in T cell and NK cell antitumor responses and as an immunotherapy target. Thus, it is imperative to determine how CD226 counteracts the actions of TIGIT and CD96 with which it competes for binding to its ligands such as CD155 (PVR). We demonstrate that CD226 engagement of CD155 is required for phosphorylation of transcription factor FOXO1, resulting in inactivation of its negative regulatory control over NK cell effector function.

Exhausted T cells have been described in cancer patients and murine tumor models largely based on their expression of various inhibitory receptors. Understanding of the functional attributes of these cells is limited. Here, we report that among CD8+ T cells in commonly used syngeneic tumor models, the coexpression of inhibitory receptors PD-1, LAG3, and TIM3 defined a group of highly activated and functional effector cells. Coexpression of these receptors further enriched for antigen-specific cells with increased T-cell receptor clonality. Anti–PD-L1 treatment increased the number and activation of these triple-positive CD8+ T cells without affecting the density of PD-1− cells. The intratumoral density of CD8+ T cells coexpressing inhibitory receptors negatively correlated with tumor burden. The density ratio and pretreatment phenotype of CD8+ T cells coexpressing inhibitory receptors was positively correlated with response across a variety of tumor models. Our results demonstrate that coexpression of inhibitory receptors is not a signifier of exhausted T cells, but rather can define a group of activated and functional effector cells in syngeneic tumor models. In the cancer setting, these cells could represent a heterogeneous population of not only exhausted but also highly activated cells responsive to treatment.