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SUMMARY Autotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1–6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities., In brief Through LPA production, ATX modulates the tumor microenvironment in autocrine-paracrine manners. Matas-Rico et al. show that ATX/LPA is chemorepulsive for T cells with a dominant inhibitory role for Gα12/13-coupled LPAR6. Upon anticancer vaccination, tumor-intrinsic ATX suppresses the infiltration of CD8+ T cells without affecting their cytotoxic quality., Graphical Abstract

SummaryAutotaxin (ATX) is secreted by diverse cell types to produce lysophosphatidic acid (LPA) that regulates multiple biological functions via G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis mainly via LPAR1; however, its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T-cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T-cell responses but suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from patient samples are consistent with intra-tumor ATX acting as a T-cell repellent. These studies highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T-cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

Blockade of the PD-1 receptor has revolutionized the treatment of metastatic melanoma, with significant increases in overall survival (OS) and a dramatic improvement in patient quality of life. Despite the success of this approach, the number of benefitting patients is limited and there is a need for predictive biomarkers as well as a deeper mechanistic analysis of the cellular populations involved in clinical responses. With the aim to find predictive biomarkers for PD-1 checkpoint blockade, an in-depth immune monitoring study was conducted in 36 advanced melanoma patients receiving pembrolizumab or nivolumab treatment at Karolinska University Hospital. Blood samples were collected before treatment and before administration of the second and fourth doses. Peripheral blood mononuclear cells were isolated and stained for flow cytometric analysis within 2 h of sample collection. Overall survival and progression-free survival (PFS) were inversely correlated with CD69 expression NK cells. In the myeloid compartment, high frequencies of non-classical monocytes and low frequencies of monocytic myeloid derived suppressor cells (MoMDSCs) correlated with response rates and OS. A deeper characterization of monocytic subsets showed that PD-L1 expression in MDSCs, non-classical and intermediate monocytes was significantly increased in patients with shorter PFS in addition to correlating inversely with OS. Our results suggest that cellular populations other than T cells can be critical in the outcome of PD-1 blockade treatment. Specifically, the frequencies of activated NK cells and monocytic subsets are inversely correlated with survival and clinical benefit, suggesting that their role as predictive biomarkers should be further evaluated.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the chromium release assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the flow cytometry-based cytotoxicity assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the flow cytometry-based cytotoxicity assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the chromium release assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Blockade of the PD-1 receptor has revolutionized the treatment of metastatic melanoma, with significant increases in overall survival, and a dramatic improvement in patient quality of life. Despite the success of this therapeutic approach, the number of responding patients is limited and there is a need for predictive biomarkers and a deeper mechanistic analysis of the cellular populations involved in a clinical response. With this objective in mind, an in-depth immune monitoring study was conducted in advanced melanoma patients undergoing treatment with pembrolizumab or nivolumab. 29 patients receiving pembrolizumab (n=5) or nivolumab (n=24) treatment at Karolinska University Hospital were included in this study. Blood samples were collected from patients at the following time points: Before treatment and at the time of the second and fourth doses. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stained for flow cytometric analysis within two hours of sample collection. In the 29 patients included, median OS was 884 days from the start of treatment. Adverse events were observed in 18 patients (62%), including three grade 3-4 (10%). Patients were classified according to their response as PD (progressive disease, 45%), SD (stable disease, 3%) MR (mixed response, 7%) PR (partial response, 31%) and CR (complete response, 14%). For analytic purposes, patients were divided into two groups: 19 (59%) patients with clinical benefit (includes responders and patients with stable disease) and no clinical benefit (12 patients with progressive disease). Patients with clinical benefit had a median overall survival (MOS) of 1013 days, significantly longer than the 303 day MOS in the no clinical benefit group. After analyzing the patients PBMC with a broad flow cytometry panel that included major immune subsets (T cells, NK cells, dendritic cells and myeloid derived suppressor cells (MDSCs)), our results showed that, before treatment, the frequency of two populations is related to clinical benefit: On one hand, patients with clinical benefit had significantly lower frequencies of monocytic MDSCs. On the other hand, NK cell frequencies were higher in these patients. Furthermore, Kaplan Meier analysis showed that patients with high frequencies of NK cells had significantly longer MOS times when compared to patients with low NK cell frequencies. Similarly, patients with low frequencies of monocytic MDSCs, showed significantly better MOS. Our results suggest that cellular populations other than T cells can be critical in the outcome of PD-1 blockade treatment. Specifically, the frequencies of NK cells and monocytic MDSCs are correlated with survival and clinical benefit and their role as predictive biomarkers should be further evaluated. Citation Format: Yago Pico De Coaña, Maria Wolodarski, Irene van der Haar Avila, Giuseppe V. Masucci, Johan Hansson, Rolf Kiessling. Myeloid derived suppressor cells and NK cells are correlated with clinical benefit and survival in advanced melanoma patients treated with PD-1 blocking antibodies nivolumab and pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-116.