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Comparative Study
. 2017 Feb;150(2):199-212.
doi: 10.1111/imm.12679. Epub 2016 Nov 2.

Distinct patterns of cytolytic T-cell activation by different tumour cells revealed by Ca2+ signalling and granule mobilization

Melissa Frick  1 , Pierre Mouchacca  1 , Grégory Verdeil  1 , Yannick Hamon  1 , Cyrille Billaudeau  1 , Michel Buferne  1 , Mathieu Fallet  1 , Nathalie Auphan-Anezin  1 , Anne-Marie Schmitt-Verhulst  1 , Claude Boyer  1
Affiliations
Comparative Study

Distinct patterns of cytolytic T-cell activation by different tumour cells revealed by Ca2+ signalling and granule mobilization

Melissa Frick et al. Immunology. 2017 Feb.

Abstract

Cancer-germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer-germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T-cell receptor specific for the P1A35-43 peptide associated with H-2Ld , although able to induce regression of P1A-expressing P815 mastocytoma cells, were much less effective against P1A-expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A-specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H-2Ld . The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video-microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+ ]i) were induced by both types of P1A-expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB-Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB-Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour-CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule-1, the ligand for LFA-1, which was not detected on the melanoma cells.

Keywords: cytolytic T lymphocyte; fluorescent granzyme B; melanoma; tumour.

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Figures

Figure 1
Figure 1
Characteristics of different P1A‐expressing tumour cells. FACS analysis for expression of H‐2Ld, B7.1 (CD80) and ICAM‐1 (CD54) was performed as described in the Material and methods. Numbers correspond to % positive cells and mean fluorescence intensity (MFI) of the positive cells.
Figure 2
Figure 2
In vitro differentiation of TCRP1A‐GZMB‐Tom CD8 T cells. CD8 T cells from P1A‐GZMB‐Tom mice labelled with Cell Tracer Violet (CTV) were cultured with 10−7 m P1Ap‐preloaded splenocytes from congenic rag−/− mice (see Materials and methods) for 1 (blue), 2 (green) or 3 (bordeaux) days. Non‐activated CD8 T cells (red) were cultured for 1 day in the absence of P1Ap. CTV and GZMB‐Tom fluorescence as well as staining for CD25 and CD44 expression were measured by FACS (see Materials and methods) on the gated CD8 T cells. Interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) production were measured by FACS after reactivation of the CD8 T cells for 4 hr with ionomycin and PMA in the presence of monensin followed by fixation and permeabilization. Numbers indicate % positive cells /(in italic) MFI of the positive cells. Results are representative of three and two experiments, respectively, for surface markers and for cytokines.
Figure 3
Figure 3
Differential extent of degranulation of TCRP1A‐GZMB‐Tom CTL induced by different P1A‐expressing tumour cells. CTL were incubated alone (red) or mixed with the tumour cells (untreated: blue, or pre‐loaded with peptide P1Ap: green) at a 1 : 1 ratio in 100 μl culture medium containing the anti‐Lamp1‐APC monoclonal antibody (mAb), before mild centrifugation and 30 min incubation at 37°. FACS analysis of anti‐Lamp1 mAb and GZMB‐Tom fluorescence was performed. (a) Density plots representing the binding of Lamp1 mAb and the expression of GZMB‐Tom on the CTL. (b) CTL degranulation measured by increase in Lamp1 staining on the CTL, expressed as % Lamp1‐positive CTL (upper left graph) and MFI of Lamp1 on all CTL (lower left graph) or by decrease in GZMB‐Tom fluorescence, expressed as % of MFI of GZMB‐Tom on Lamp‐1‐positive CTL (upper right graph, where MFI for all CTL alone was set at 100%) and MFI of GZMB‐Tom on all CTL (lower right graph). Results are the mean of three (without peptide) or two (with peptide) experiments. Statistical analysis was performed using t‐test: *P < 0·05; **P < 0.01.
Figure 4
Figure 4
Visualization of the kinetics of CTL activation and granule re‐localization upon CTL/tumour cell contact by video microscopy. Images from video microscopy show Ca2+ fluxes and fluorescence of GZMB‐Tom containing granules in Fluo‐4‐labelled TCRP1A‐GZMB‐Tom CTL added to various tumour targets. The first image corresponds to that before the first increase in Ca2+ (except for the P1.204 P1A‐negative tumour cells). (a) P511 and P1.204 non‐adherent tumour cells were labelled with Violet Calcein and deposited onto polylysine‐activated Labtek wells, before addition of the CTL. Upper images show Fluo‐4 and GZMB‐Tom fluorescence in the CTL and Calcein Violet fluorescence in the tumour cells. Lower images are devoid of Fluo‐4 fluorescence to better visualize GZMB‐Tom. (b) Adherent tumour cells T‐1236 and T‐RFP‐69 were grown on Labtek wells 2 days before the experiment. T‐1236 cells were labelled with CTV whereas T‐RFP‐69 cells endogenously express DsRed. When indicated (two last lanes), T‐RFP69 cells were loaded with peptide P1Ap. For T‐1236 tumour cells, upper images show Fluo‐4, GZMB‐Tom and Calcein Violet fluorescence and lower images are devoid of Fluo‐4 fluorescence. For T‐RFP‐69 cells, upper images show Fluo‐4, GZMB‐Tom and DsRed fluorescence, lower images are devoid of Fluo‐4 fluorescence. Video microscopy recordings (90 min at 37°) were performed and analysed as described in Materials and methods.
Figure 5
Figure 5
Barcoding the TCRP1A‐GZMB‐Tom CTL Ca2+ fluxes in response to the different tumour cells. The videos corresponding to the CTL responses to tumour cells shown in Fig. 4 were analysed for Fluo‐4‐positive cell tracking with the MAAACS software described by Salles et al.26. The raw fluorescence recordings were normalized to the median of Fluo‐4 fluorescence values for each individual CTL. (a) Normalized Fluo‐4 intensity is displayed for individual cells as a barcoded response over time (colour‐code is function of the fluorescence amplitude (FA) threshold set to FA > 2·5) of the CTL in response to the indicated tumour cells. (b) The pattern of the Ca2+ response of the CTL towards each of the indicated tumour cells is represented in pie charts (non‐activated: black, oscillating: blue, sustained: purple, unique: grey) (see Materials and methods for classification of criteria). (c, d) Statistical representation of the fluorescence signal amplitude (c) and of the proportion of Ca2+ signal above the threshold for each cell per unit time (response fraction) (d). Mean ± SEM is shown. Statistical analysis was carried out with the Mann–Whitney non‐parametric test.
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