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AssigneesInserm (Institut National De La Santé Et La Recherche MéDicale), Universite Paul Sabatier Toulouse III
InventorsSalvatore VALITUTTI, Liza FILALI, Sabina MUELLER, Marie-Pierre PUISSEGUR
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To overcome the difficulty in achieving a quantitative assessment of CTL function in clinical settings, the inventors aimed at developing a new method inspired by their knowledge of the CTL/tumor target biology and based on flow cytometry. In particular, to directly detect the earliest steps of tumor cell resistance to CTL attack at the lytic synapse the inventors developed a method to monitor CTL/tumor cells interaction in the presence of FM1-43, a fluorescent lipophilic dye that rapidly intercalates into lipid bilayer during the membrane turnover that accompanies plasma membrane reparation. This assay allows the inventors to measure reparative membrane turnover of tumor cells under CTL attack by FACS analysis at the whole tumor cell population level. They initially applied this approach to compare the response of melanoma cell (D10 cells) to CTL attack as compared to conventional target cells that are sensitive to CTL mediated cytotoxicity (EB V-transformed B cells, JY cells). The methodology allows to rapidly assessing the synaptic resistance of tumor target cells to CTL attack and the intrinsic capacity of CD8+CTL to efficiently kill their target cells. Thus, the present invention relates to methods and kit for assaying lytic potential of immune effector cells and uses thereof in diagnostic assays.
A method of assaying the lytic potential of a population of immune effector cells comprising the steps of i) contacting the population of immune effector cells with a population of target cells for a sufficient period of time and under conditions suitable for allowing the population of immune effector cells to induce a cytotoxic response, and ii) measuring the reparative membrane turnover level in the population of target cells wherein said level correlates with the lytic potential of the population of immune effector cells.
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A method of screening a test compound for the ability to modulate the lytic potential of a population of immune effector cells comprising the steps of i) contacting the population of immune effector cells with a population of target cells for a sufficient period of time and under conditions suitable for allowing the population of immune effector cells to induce a cytotoxic response in presence of the test compound, ii) measuring the reparative membrane turnover level in the population of target cells, iii) comparing the reparative membrane turnover level determined at step ii) with the reparative membrane turnover level measured in absence of the test compound and iv) selecting the test compound when a difference is detected between the reparative membrane turnover level determined at step ii) and the reparative membrane turnover level measured in the absence of the test compound.
A method of determining whether a patient suffering from cancer will achieve a response with an immune checkpoint inhibitor and treating the patient, comprising the steps of i) isolating a population of immune effector cells from a biological sample obtained from the patient, ii) contacting the population of immune effector cells with a population of target cells for a sufficient period of time and under conditions suitable for allowing the population of immune effector cells to induce a cytotoxic response in the presence of the immune checkpoint inhibitor, iii) measuring the reparative membrane turnover level in the population of target cells, and when the reparative membrane turnover level determined at step iii) is higher than the reparative membrane turnover level measured in the absence of the immune checkpoint inhibitor, then iv) treating the patient with the immune checkpoint inhibitor.
Dependencies are inferred from the claim text.