Acta Dermato-Venereologica 99-12CompleteContent | Page 34

SHORT COMMUNICATION Effect of Indoleamine 2,3 Dioxygenase Inhibitor on the Cytotoxic Activity of Tumour-infiltrating Lymphocytes Sarah LE NAOUR 1 , Anne-Chantal KNOL 1 , Marie-Christine PANDOLFINO 2 , Amir KHAMMARI 1 and Brigitte DRÉNO 1,2 1 Dermato-Cancerology Department, CHU Nantes, CIC1413, CRCINA, University Nantes, Place Alexis Ricordeau, FR-44093 Nantes, and 2 Cell and Gene Therapy Unit, CHU Nantes, Nantes, France. E-mail: [email protected] Accepted Aug 12, 2019; E-published Aug 13, 2019 There have been major therapeutic advances in the treat- ment of melanoma in recent years (1, 2). Adoptive T-cell therapy, based on tumour-infiltrating lymphocytes (TILs) obtained from tumour tissues, has been developed in the field of immunotherapy. An objective clinical response of between 40% and 72% in the metastatic stage has been reported (3, 4) but the clinical response remains limited in time. In the metastatic stage of melanoma, the clinical response remains limited and short-term. A major current challenge is to increase the efficacy of TILs by countering the immune tolerance of the tumour microenvironment through identification of the molecules implicated in this phenomenon. Many factors have been suggested, inclu- ding indoleamine 2,3 dioxygenase (IDO), an intracellular enzyme that metabolizes tryptophan to kynurenine. Tryp- tophan deficiency inhibits the activity and proliferation of T cells (5). A recent paper (6) has shown that IDO1 expression in metastatic melanoma lymph nodes is as- sociated with an increased risk of relapse and death. Thus, we hypothesize that the presence of IDO in the tumour microenvironment could inhibit the activity of TILs, and that adding an IDO inhibitor to the TILs could increase their cytotoxic activity against melanoma cells. In a first step, the 7 melanoma cell-lines were incubated either with an IDO inhibitor, 1-methyl-L-tryptophan (1-MT) (in 2 dif- ferent concentrations), alone or after incubation with interferon gamma (INF-γ) for mimicking the chronic inflammatory micro- environment state of a metastasis (associated with an increase in immune tolerance). In a second step, the 7 melanoma cells were distributed on a 96-well plate (300,000 cells per well) with 100,000 autologous TILs. Co-cultures were incubated for 6 h at 37°C, 5% CO 2 . Finally, melanoma cells were analysed by flow cytometry for expression of IDO1, MHC-I, MHC-II and PD-L1 antigens and reactivity of TILs by a double-labelling: membrane CD3 and intracellular interferon (IFN)-γ. The data were analysed using CellQuestPro ® software. Tryptophan and kynurenine levels were determined by enzyme-linked immunoassay (ELISA) (Im- muSmol (BD Biosciences, San Jose, USA)) in order to calculate kynurenine/tryptophan ratios. RESULTS In the basal state (without INF-γ), IDO1 expression was shown for 5 melanoma cell-lines (Fig. 1a). MHC-I was expressed by all the cell-lines. MHC-II was expressed by only 4 cell-lines. No expression of PD-L1 was noted in any of the 7 cell-lines. Intracytoplasmic expression of IFN-γ by TILs was very low in the basal state. Adding the IDO inhibitor decreased the expression of IDO1 in a dose-dependent manner (Fig. 1a). For both MHC-I and MHC-II, IDO inhibitor increased their expression in a dose-dependent manner (Fig. 1b, c). No effect on the cytotoxic activity of TILs was found by adding IDO inhibitor (Fig. 2). The absence of expression of IDO1 by 2 melanoma cell-lines is in agreement with a recent paper showing that not all melanoma metastases express IDO (11). In the inflammatory state (induced by INF-γ), IDO1 was expressed in all melanoma cell-lines: 2 melanoma cell- lines not expressing IDO1 in the basal state, and increased in the other 5 cell-lines in a dose-dependent manner (Fig. 0,00 0,05 0 20 0 0,15 0,10 40 20 0,20 40 d 0,25 80 60 Median 0,30 100 60 b 80 Median 80 70 c 60 50 40 30 20 10 0 Median 120 a Median 100 To investigate this hypothesis we used the model of co-culture of a melanoma cell-line and autologous TILs obtained from the same patient (6), with the addition of IDO inhibitor. The endpoints were the frequency of reactive autologous T cells among the TILs and the expression of molecules involved in the recognition of tumour antigens by T cells with and without IDO inhibitor. Melanoma cells were derived from patients with metastatic melanoma, who were previously included in several distinct clinical trials of TILs (7–10). Seven tumour cell-lines with associated autologous TILs were selected (4 wild-type cell-lines and 3 cell-lines with positive BRAF mutational status, but no difference according this muta- tional status was noted). MATERIAL AND METHODS 1186 Fig. 1. (a) Kynurenine/tryptophan ratios. (b) Mean fluorescence intensity (MFI) for MHC-I expression. (c) MFI for MHC-II expression. (d) Percentage of positive cells for PD-L1: results expressed as median, green  = basal state, red  = inflammatory state. doi: 10.2340/00015555-3285 Acta Derm Venereol 2019; 99: 1186–1187 This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta Journal Compilation © 2019 Acta Dermato-Venereologica.