Colony-stimulating factor 1 (CSF1) is a key regulator of monocyte/macrophage differentiation that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). We show that CSF1 is expressed in human melanoma, and patients with metastatic melanoma have increased CSF1 in blood compared to healthy subjects. In tumors, CSF1 expression correlated with the abundance of CD8 + T cells and CD163 + TAMs. Human melanoma cell lines consistently produced CSF1 after exposure to melanoma-specific CD8 + T cells or T cell-derived cytokines in vitro, reflecting a broadly conserved mechanism of CSF1 induction by activated CD8 + T cells. Mining of publicly available transcriptomic data sets suggested co-enrichment of CD8 + T cells with CSF1 or various TAM-specific markers in human melanoma, which was associated with nonresponsiveness to programmed cell death protein 1 (PD1) checkpoint blockade in a smaller patient cohort. Combination of anti-PD1 and anti-CSF1 receptor (CSF1R) antibodies induced the regression of BRAF V600E-driven, transplant mouse melanomas, a result that was dependent on the effective elimination of TAMs. Collectively, these data implicate CSF1 induction as a CD8 + T cell-dependent adaptive resistance mechanism and show that simultaneous CSF1R targeting may be beneficial in melanomas refractory to immune checkpoint blockade and, possibly, other T cell-based therapies.

Despite the success of immunotherapy using checkpoint blockade, many patients with solid tumors remain refractory to these treatments. In human cancer, the experimental options to investigate the specific effects of antibodies blocking inhibitory receptors are limited and it is still unclear which cell types are involved. We addressed the question whether the direct interaction between T cells and tumor cells can be enforced through blocking a set of inhibitory receptors including PD-1, TIM-3, BTLA and LAG-3, blocked either individually or in dual combinations with the anti-PD-1 antibody, and to determine the condition that induces maximal T cell function preventing tumor cell proliferation. Using short-term Melan-A-specific or autologous re-stimulations, checkpoint blockade did not consistently increase cytokine production by tumor-derived expanded T cells. We next set up a 5-day co-culture assay with autologous melanoma cell lines and expanded tumor infiltrating T cells, originating from tumor specimens obtained from 6 different patients. Amongst all combos tested, we observed that blockade of LAG-3 alone, and more strongly when combined with PD-1 blockade, enforced T cell responses and tumor cell growth control. The combination of anti-LAG-3 plus anti-PD-1 acted through CD8 T cells and led to increased IFNγ production and cytotoxic capacity. Our results show that LAG-3 and PD-1 are regulating the direct interaction between tumor cells and autologous T cells, suggesting that therapy effects may be promoted by enhanced access of the corresponding blocking reagents to the tumor microenvironment. ARTICLE HISTORY

Bladder cancer is one of the most common malignancies and has poor prognosis for patients with locally advanced, muscle-invasive, disease despite the efficacy of immune checkpoint blockade. To develop more effective immunotherapy strategies, we studied a genetic mouse model carrying deletion of Tp53 and Pten in the bladder, which recapitulates bladder cancer tumorigenesis and gene expression patterns found in patients. We discovered that tumor cells became more malignant and the tumor immune microenvironment evolved from an inflammatory to an immunosuppressive state. Accordingly, treatment with anti-PD1 was ineffective, but resistance to anti-PD1 therapy was overcome by combination with a CD40 agonist (anti-CD40), leading to strong antitumor immune responses. Mechanistically, this combination led to CD8+ T-cell recruitment from draining lymph nodes. CD8+ T cells induced an IFNγ-dependent repolarization toward M1-like/IFNβ-producing macrophages. CD8+ T cells, macrophages, IFN I, and IFN II were all necessary for tumor control, as demonstrated in vivo by the administration of blocking antibodies. Our results identify essential cross-talk between innate and adaptive immunity to control tumor development in a model representative of anti-PD1–resistant human bladder cancer and provide scientific rationale to target CD40 in combination with blocking antibodies, such as anti-PD1/PD-L1, for muscle-invasive bladder cancer.

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