Visualizing CTL/melanoma cell interactions: multiple hits must be delivered for tumour cell annihilation
Corresponding Author
Íris Caramalho
INSERM, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactions lymphocytaires, Toulouse, France
Instituto Gulbenkian de Ciência, Apartado, Oeiras, Portugal
Correspondence to: Iris CARAMALHO, INSERM, U563, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactionslymphocytaires, Toulouse, F-31300 France.Tel.: (33) 562 74 83 66Fax: (33) 562 74 45 58E-mail: [email protected]Search for more papers by this authorMustapha Faroudi
INSERM, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactions lymphocytaires, Toulouse, France
Present address: Division of Immune Cell Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Search for more papers by this authorElisabetta Padovan
Instituto Gulbenkian de Ciência, Apartado, Oeiras, Portugal
Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal
Search for more papers by this authorSabina Müller
INSERM, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactions lymphocytaires, Toulouse, France
Search for more papers by this authorSalvatore Valitutti
INSERM, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactions lymphocytaires, Toulouse, France
Université Toulouse III Paul-Sabatier, Toulouse, France
Instituto Gulbenkian de Ciência, Apartado, Oeiras, Portugal
Search for more papers by this authorCorresponding Author
Íris Caramalho
INSERM, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactions lymphocytaires, Toulouse, France
Instituto Gulbenkian de Ciência, Apartado, Oeiras, Portugal
Correspondence to: Iris CARAMALHO, INSERM, U563, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactionslymphocytaires, Toulouse, F-31300 France.Tel.: (33) 562 74 83 66Fax: (33) 562 74 45 58E-mail: [email protected]Search for more papers by this authorMustapha Faroudi
INSERM, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactions lymphocytaires, Toulouse, France
Present address: Division of Immune Cell Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Search for more papers by this authorElisabetta Padovan
Instituto Gulbenkian de Ciência, Apartado, Oeiras, Portugal
Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal
Search for more papers by this authorSabina Müller
INSERM, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactions lymphocytaires, Toulouse, France
Search for more papers by this authorSalvatore Valitutti
INSERM, Centre de Physiopathologie de Toulouse Purpan, Section Dynamique moléculaire des interactions lymphocytaires, Toulouse, France
Université Toulouse III Paul-Sabatier, Toulouse, France
Instituto Gulbenkian de Ciência, Apartado, Oeiras, Portugal
Search for more papers by this authorAbstract
It is well established that cytotoxic T lymphocytes (CTL) can kill target cells offering a very small number of specific peptide/MHC complexes (pMHC). It is also known that lethal hit delivery is a very rapid response that occurs within a few minutes after cell–cell contact. Whether cytotoxicity is efficient and rapid in the context of CTL interaction with target cells derived from solid tumours is still elusive. We addressed this question by visualizing the dynamics of human CTL interaction with melanoma cells and their efficiency in eliciting cytotoxicity. Our results show that in spite of CTL activation to lethal hit delivery, killing of melanoma cells is not efficient. Time-lapse microscopy experiments demonstrate that individual CTL rapidly polarize their lytic machinery towards target cells, yet the apoptotic process in melanoma cells is defective or ‘delayed’ as compared to conventional targets. These results indicate that although CTL activation to lethal hit delivery can be viewed as a ‘digital’ phenomenon rapidly triggered by a few ligands, melanoma cell annihilation is an ‘analogue’ response requiring multiple hits and prolonged contact time.
Supporting Information
Figure S1: Melanoma cells express HLA-A2. Surface expression of HLA-A2 in JY, HBL and D10 cells. Thin lines correspond to the staining obtained with the isotype control Ab. Numbers indicate the median fluorescence intensity of HLA-A2 staining (bold lines). A representative result of two independent experiments is shown.
Figure S2: JY and melanoma cells induce similar levels of TCR down-regulation and IFN-γ production. (A) Surface TCR expression levels on CTL after 4 hrs of conjugation with JY, HBL and D10 cells either unpulsed (U) or pulsed with 1 nM or 10 μM pp65 peptide. Results are from three independent experiments performed in duplicates. TCR expression levels on CTL conjugated with unpulsed targets were considered to be 100%. (B) IFN-γ staining in CTL after 4 hrs of conjugation, with JY, HBL and D10 cells either unpulsed (U) or pulsed with 1 nM or 10 μM peptide. Results are from two independent experiments performed in duplicates. In A and B, results are expressed as mean ± standard deviation.
Figure S3: Melanoma cells resist CTL-mediated killing. (A) Efficiency of CTL-mediated killing of HBL and D10 melanomas, as compared to JY cells (considered as 100% lysis). Each dot corresponds to an independent experiment performed in duplicates. To determine killing efficiency, the formula [(P+CTL)/P]/[(U+CTL)/U] was used, where: P and P+CTL are the frequencies of pulsed targets that incorporated PI, when alone or conjugated with CTL, respectively; U and U+CTL are the frequencies of unpulsed targets that incorporated PI when cultured alone or in conjugation with CTL, respectively. ***P < 0.0001. (B) Efficiency of CTL-mediated killing of 3 additional target cells (LG2, T2 and K562-A2) compared to JY (taken as 100% lysis). Each dot corresponds to an independent experiment done in duplicates. Killing efficiency was determined as described in A. **P < 0.01. (C) Frequency of JY, HBL and D10 cells that incorporated PI after 21 hrs of treatment with 90 μM H2O2, or in control cultures. Results of a representative experiment out of 4 performed in triplicates are shown. Numbers indicate the frequency of PI-positive cells ± standard deviation.
Figure S4: Additional melanoma and epithelial tumour cell lines are resistant to CTL-mediated cytotoxicity. (A) Percentage of CTL that expose Lamp-1 at the surface, after 1 hr of conjugation with JY, M17, M44, M113, MCF-7 and HepG-2 cells, either unpulsed (U) or pulsed (P) with 10 μM peptide. Data are from a representative experiment out of 3 performed in duplicates. Numbers indicate the frequency of Lamp-1-positive cells. (B) Frequency of PI-positive JY, M17, M44, M113, MCF-7 and HepG-2 cells either unpulsed (U) or pulsed (P) with 10 μM peptide after 4 hrs of conjugation with CTL. Data are from a representative experiment out of 3 performed in duplicates. Numbers indicate the frequency of PI-positive cells.
Figure S5: Defective CTL-mediated induction of apoptosis in melanoma cells. (A) Frequency of JY, HBL and D10 cells either unpulsed (U) or pulsed (P) with 10 μM peptide expressing active caspase-3, after 1 hr of conjugation with CTL, as determined by flow cytometry. An E/T ratio of 2:1 was used. Data are from a representative experiment out of 3 performed in duplicates. Numbers indicate the frequency of active caspase-3-positive cells. Pooled data obtained in three independent experiments are shown in the right-hand panel. *P < 0.05 and ***P < 0.0001. (B) Frequency of JY, HBL and D10 cells either unpulsed (U) or pulsed with 10 μM pp65 (P) that exhibit DNA breaks, after 4 hrs of conjugation with CTL. One representative result and pooled data from at least four independent experiments performed in duplicates are shown in the left-hand and right-hand panels, respectively. Numbers indicate the frequency of tunel-positive cells. **P < 0.01 and ***P < 0.0001.
Figure S6: Melanoma cells express intracellular caspase-3. The expression of caspase-3 in JY and melanoma cells was determined by intracellular FACS staining (bold lines). Thin lines correspond to the isotype control staining. Numbers indicate the median fluorescence intensity of caspase-3 staining. Plotted is a representative result of 2.
Figure S7: Statistical analysis of morphological data. Distances between CTL MTOC and the CTL/target contact site were measured using the Zeiss software. An example of measurement of a CTL/ unpulsed target cell conjugate is shown in the upper panel. In the lower panel, each dot corresponds to a CTL/target conjugate. ***P < 0.001.
Figure S8: CTL rapidly polarize their lytic machinery towards HBL cells. Sequences of snapshots of Lysotracker red-labelled CTL interacting with either calcein-labelled JY or calcein-labelled HBL target cells pulsed with 10 μM pp65. A representative experiment out of 6 is shown.
Figure S9: 'Delayed' apoptosis in melanoma cells. Plotted is the time required for JY and HBL cells to initiate membrane blebing that precedes cell annihilation after initial contact with CTL, as determined by time-lapse video microscopy. Numbers correspond to the mean time in seconds necessary for the initiation of target membrane blebing (***P = 0.0003).
Figure S10: Expression of serpin PI-9 and XIAP do not account for resistance of melanoma cells to CTL-mediated killing. (A) Histograms show the intracellular expression of PI-9 in JY, HBL and D10 cells (bold lines). CTL are used as a positive control of PI-9 expression. Thin lines indicate the staining using the isotype control Ab. Numbers indicate PI-9 median fluorescence intensity. Data are representative of three independent experiments. (B) Intracellular expression of XIAP in JY, HBL and D10 cells (bold lines). Hela cells are used as a positive control of XIAP expression. Thin lines correspond to the staining obtained with the isotype control Ab. Numbers indicate XIAP median fluorescence intensity. Data are representative of three independent experiments.
Movie S1: Two CTL, loaded with LysoTracker red to visualize lytic granules, are shown during interaction with a JY cell pulsed with 10 μM of pp65 peptide. Lytic granules (red) rapidly polarize towards the target cell labelled with calcein (green). Recording time in minutes is indicated in upper left corner.
Movie S2: CTL labelled as in Movie S1 are shown during interaction with HBL targets pulsed with 10 μM peptide. Lytic granules (red) rapidly polarize towards the target cells labelled with calcein (green).
Movies S3 and S5: Detection of [Ca2+]i increase in a JY cell loaded with Fluo-4 AM. A CTL labelled with Lysotracker red (red) is shown during its interaction with one JY target pulsed with 10 μM of peptide. Green staining depicts [Ca2+]i increase in the target cell. In Movie S5, only Fluo-4 staining intensity is shown using a pseudocolour scale.
Movies S4 and S6: Detection of [Ca2+]i increase in HBL target cells loaded with Fluo-4 AM. CTL labelled with Lysotracker red (red) are shown during its interaction with HBL targets pulsed with 10μM of pp65 peptide. Green staining depicts [Ca2+]i increase in the target cell. In Movie S6, only Fluo-4 staining intensity is shown using a pseudocolour scale.
Movie S7: CTL labelled with Lysotracker red (red) are shown during interaction with 10 μM peptide pulsed-HBL cells labelled with calcein (green).
Movie S8: Two CTL labelled with Lysotracker red (red) are shown during interaction with JY cells labelled with calcein (green), previously pulsed with 10μM peptide.
Movie S9: Three CTL labelled with Lysotracker red (red) are shown during interaction with a 10 μM peptide pulsed-HBL cell labelled with calcein (green).
Movie S10: CTL labelled with Lysotracker red (red) are shown during interaction with both JY and HBL target cells pulsed with 10 μM peptide. JY cells are loaded with calcein (green) and Bodipy 630 (blue) to distinguish them from calcein-labelled HBL cells. Target cell death is indicated by the loss of the calcein staining.
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