(A–C) Freshly isolated polyclonal CD8+ T cells or (D) CTL clones were stimulated by immobilized anti-CD8/anti-CD28/ICAM-1 during 72 hr and stained with antibodies directed against the indicated …
CD107a, perforin, and granzyme B distribution between daughter cells.
Based on the brightfield illumination, all events were plotted for their aspect ratio (length/width, equal one for perfectly round cells) and their area. Cells in telophase were defined as those …
(A) Analysis of individual cells in telophase. All IsX generated TIFF files were analyzed using the Fiji software. For each telophase cell, we used three TIFF image corresponding to (1) CTV …
Freshly isolated polyclonal CD8+ T cells previously stained with CTV were stimulated 72 hr using immobilized anti-CD3/anti-CD28/ICAM1. (A) Imaging flow cytometry shows that stimulated cell undergo …
The panels show staining for CD107a and perforin in human CD8+ memory T cells stimulated and analyzed as in Figure 1A,B. CD107a n = 978 from three independent experiments; perforin n = 1127 from …
Freshly isolated polyclonal CD8+ T cells were stimulated by immobilized anti-CD8/anti-CD28/ICAM-1 during 72 hr and stained with antibodies directed against CD107a. Cells in telophase were identified …
Fold increase of CD107a staining in the brighter nascent daughter cell as compared to the other nascent daughter cell.
The video shows 3D reconstruction of a cell in telophase. CD107a (green), α-tubulin (red), and DAPI (cyan). The images presented in Figure 2B has been extracted from this video.
Freshly isolated polyclonal CD8+ T cells were stimulated by immobilized anti-CD8/anti-CD28/ICAM-1 during 72 hr and stained with antibodies directed against T-bet (A) or c-Myc (B). (A) T-bet analysis …
T-bet and c-Myc distribution between daughter cells.
(A) Freshly isolated polyclonal CD8+ T cells were stimulated using immobilized anti-CD8/anti-CD28/ICAM-1 (left) or with PMA/ionomycin (right) during 72 hr and stained with antibodies directed …
CD107a distribution between daughter cells after polarized and non-polarized stimulation.
(A, B) Freshly isolated polyclonal CD8+ T cells were stimulated using immobilized anti-CD8/anti-CD28/ICAM-1 during 72 hr and stained with antibodies directed against CD107a. Cells in telophase were …
CD107a distribution between daughter cells in undivided cells, at first division and second division.
(A,B) Snapshots depict typical cells in division undergoing even (A) or uneven (B) repartition of lytic granules (mCherry-tagged GrzB, red) in telophase as detected by live-cell imaging. Images are …
(A,B) Snapshots depict Imaris software reconstructions of typical cells undergoing uneven (A) and even (B) repartition of LTR+ (red) lytic granules in division as detected by 4D live-cell imaging. Im…
Human CTL were transfected during their expansion phase with mcherryGrzB. 18 hr after transfection, cells were inspected by time-lapse laser scanning confocal microscopy for additional 5–6 hr using …
G2/M sorted CTL were loaded with Hoechst (blue) and LysoTracker Red (LTR, red) and inspected by time-lapse laser scanning confocal microscopy (Figure 6—video 3 and Figure 6—video 4) or spinning-disk …
Clonal CTL were FACS sorted on the basis of their LysoTracker Blue staining. (A) Representative FACS histograms showing LysoTracker Blue staining levels on LysoTrackerhigh and LysoTrackerlow sorted C…
Cytotoxicity assay.
CD107a mean fluorescence intensity (MFI) at the surface of CD8+ T cells.
CD8 mean fluorescence intensity (MFI).
Results are from the indicated figures of the revised manuscript. The red line indicates the 1.5 threshold for asymmetric repartition of CD107a (corresponding to the 40-60% range) between daughter …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (Homo sapiens) | HLA-A2 restricted CD8+ T cell clone (VLAELVKQI) | Khazen et al., 2016 | ||
Cell line (Homo sapiens) | HLA-A2 restricted CD8+ T cell clone (NLVPMVATV) | Khazen et al., 2016 | ||
Cell line (Homo sapiens) | HLA-A2 restricted CD8+ T cell clone (VLAELVKQI) | Khazen et al., 2016 | ||
Cell line (Homo sapiens) | JY (EBV-transformed B cells) | Khazen et al., 2016; Vasconcelos et al., 2015 | ||
Biological sample (Homo sapiens) | Buffy coats of Healthy donors | EFS, Toulouse, France | With consent and approval AC-2014–2384 | |
Antibody | Anti-human CD3 (Human monoclonal, TR66) | Enzo | Cat# ALX-804–822 RRID:AB_2051037 | (1 µg/ml) |
Antibody | Anti-human CD28 (Mouse monoclonal, CD28.2) | eBioscience | Cat# 16-0289-81 RRID:AB_468926 | (1 µg/ml) |
Recombinant protein | Recombinant human ICAM-1-Fc fusion protein | R and D Systems | Cat# 720-IC | (0.5 µg/ml) |
Antibody | Anti-human CD107a (Mouse monoclonal, H4A3) | BD Pharmingen | Cat# 555798 RRID:AB_396132 | (10 µg/ml) |
Antibody | Anti-human CD107a AlexaFluor 647 (Mouse monoclonal, H4A3) | BD Pharmingen | Cat# 562622 RRID:AB_2737684 | (Diluted at 1/100) |
Antibody | Anti-human Granzyme B (Mouse monoclonal, GB11) | Thermo Scientific | Cat# MA1-80734 RRID:AB_931084 | (10 µg/ml) |
Antibody | Anti-human Granzyme B AlexaFluor 647 (Mouse monoclonal, GB11) | BD Pharmingen | Cat# 561999 RRID:AB_10897997 | (10 µg/ml) |
Antibody | Anti-human T-bet (Rabbit polyclonal, Tbx21) | Abcam | Cat# ab181400 | (10 µg/ml) |
Antibody | Anti-human C-myc (Mouse monoclonal, 9E10) | Thermo Scientific | Cat# MA1-980 RRID:AB_558470 | (10 µg/ml) |
Antibody | Anti-human α-tubulin (Rabbit polyclonal) | Abcam | Cat# ab15246 RRID:AB_301787 | (Diluted at 1/100) |
Antibody | Anti-mouse IgG1 Alexa Fluor 647 (Goat polyclonal) | Invitrogen | Cat# A21240 RRID:AB_2535809 | (10 µg/ml) |
Antibody | Anti-mouse IgG1 Alexa Fluor 488 (Goat polyclonal) | Invitrogen | Cat# A-21121 RRID:AB_2535764 | (10 µg/ml) |
Antibody | Anti-rabbit (H+L) AlexaFluor488 (Goat polyclonal) | Invitrogen | Cat# A11034 RRID:AB_2576217 | (10 µg/ml) |
Antibody | Anti-rabbit (H+L) AlexaFluor647 (Donkey polyclonal) | Invitrogen | Cat# A31573 RRID:AB_2536183 | (10 µg/ml) |
Antibody | Anti-rabbit AlexaFluor555 (Goat polyclonal) | Invitrogen | Cat# A21428 RRID:AB_2535849 | (10 µg/ml) |
Antibody | Anti-mouse IgG Abberior Star 580 (Goat polyclonal) | Abberior Instruments | Cat# 52403 | (10 µg/ml) |
Antibody | Anti-human CD107a-PEcy7 (mouse monoclonal, H4A3) | BD Pharmingen | Cat# 561348 RRID:AB_10644018 | (Diluted at 1/50) |
Antibody | Anti-human CD8-FITC (mouse monoclonal, HIT8A) | BD Pharmingen | Cat# 555634 RRID:AB_395996 | (Diluted at 1/50) |
Recombinant DNA reagent | MGC Human GZMB Sequence verified cDNA (Clone Id: 5223876) | GE Healthcare BIO Sciences | Cat# MHS6278-202801737 | |
Recombinant DNA reagent | mCherry-SEpHluorin | Koivusalo et al., 2010 | Addgene cat# 32001 | |
Recombinant DNA reagent | pT7-GZMB-mCherry-SEpHluorin | This paper | ||
Sequence-based reagent | Primer: XhoI-T7-GzB Forward caaCTCGAGTAATACGACTC ACTATAGGGAGACCCGGTA CCatgcaaccaatcctgcttctgcc | This paper | ||
Sequence-based reagent | Primer: EcoRI-GzB-noSTOP-R caaGAATTCcggcgtggcgtttcatggttttctttatccag | This paper | ||
Peptide, recombinant protein | CMV peptide p65 (NV-9) | GeneCust | Cat# 181329 | |
Peptide, recombinant protein | Human rIL-2 | Miltenyi Biotec | Cat# 130-097-748 | (150 IU/ml) |
Peptide, recombinant protein | Human rIL-15 | Miltenyi Biotec | Cat# 130-095-766 | (50 ng/mL) |
Commercial assay or kit | EasySep Negative human CD8+ T cell isolation kit | StemCell Technologies | Cat# 17953 | |
Commercial assay or kit | EasySep human Memory CD8+ T cell enrichment kit | StemCell Technologies | Cat# 19159 | |
Software, algorithm | IDEAS SpotCount Threshold (M03,nucleus,60) | Amnis, Luminex | ||
Software, algorithm | IDEAS Area Range Threshold (M02,tubulin,75),50–5000, 0–0.5 | Amnis, Luminex | ||
Software, algorithm | Fiji | Schindelin et al., 2012 | ||
Software, algorithm | Imaris Software | Oxford Instruments | ||
Software, algorithm | ZEN ZEISS Efficient Navigation | |||
Software, algorithm | Huygens Professional version 18.10 using CMLE algorithm with SNR:7 | Scientific Volume Imaging, USA | STED images were deconvolved | |
Software, algorithm | Python software version 3.5 | χ2 of independence test, χ2 of homogeneity test and Kolmogorov-Smirnov goodness-of-fit test | ||
Software, algorithm | GraphPad Prism software version five for windows | Paired Student’s t-test | ||
Software, algorithm | FlowJo software | TreeStar | ||
Other | SYTOXOrange Dead Cell Stain | ThermoFisher Scientific | Cat# S11368 | Manufacturer recommended dilution |
Other | DAPI | Molecular Probes, Invitrogen | Cat# D1306 RRID:AB_2629482 | |
Other | Hoechst 33342 | ThermoFisher Scientific | Cat# 1399 | (200 ng/ml) |
Other | CellTrace Violet Cell Proliferation kit | ThermoFisher Scientific | Cat# C34557 | (5 µM) |
Other | LysoTraker Blue (DND22) Dye | Molecular probes | Cat# L7525 | (200 nM) |
Other | LysoTraker Red (DND99) Dye | Molecular probes | Cat#L7528 | (200 nM) |
Other | 7-Aminoactinomycin D (7-AAD) | BD Pharmingen | Cat# 559925 | (0.25 µg) |
Other | Ibidi μ-slide chambered coverslips Angiogenesis | Ibidi, Biovalley | Cat# 81506 | |
Other | Ibidi μ-slide chambered coverslips eight well | Ibidi, Biovalley | Cat# 80821 | |
Other | Nunc Lab-Tek chamber slides eight wells | Nunc, ThermoFisher Scientific | Cat#1 54526 | |
Other | Micromesh array (100 μm) | Microsurface, Tebu- Bio | Cat# MMA-0500-100-08-01 |
Independence chi-square test between heterogeneous cells and all cells | Test statistic () | p-value (p) | Degree of freedom (dl) | |
---|---|---|---|---|
CD107a, Experiment 1, 0 division CD107a, Experiment 1, 1 division CD107a, Experiment 1, 2 divisions | 4.060439 3.565087 1.614763 | 11.07 11.07 7.815 | 0.540748 0.613563 0.656047 | 5 5 3 |
CD107a, Experiment 2, 0 division CD107a, Experiment 2, 1 division CD107a, Experiment 2, 2 divisions | 0.278928 0.413804 | 7.815 7.815 | 0.963942 0.937376 | 3 3 |
CD107a, Experiment 3, 0 division CD107a, Experiment 3, 1 division CD107a, Experiment 3, 2 divisions | 2.36867 2.092976 0.655225 | 15.51 9.488 9.488 | 0.967574 0.718663 0.956734 | 8 4 4 |
Experiment 1 | Kolmogorov–Smirnov test | Zero division | One division | Two divisions | |||
---|---|---|---|---|---|---|---|
p-value | p-value | p-value | |||||
1 | 0 division | ||||||
1 division | 0.13148 | 0 | |||||
2 divisions | 0.220034 | 0 | 0.116283 | 0 | |||
2 | 0 division | ||||||
1 division | 0.087873 | 0 | |||||
2 divisions | 0.0891924 | 0 | 0.04634 | 0.03582 | |||
3 divisions | 0.054621 | 0.0159 | 0.067702 | 0.001185 | 0.047275 | 0.116534 | |
3 | 0 division | ||||||
1 division | 0.14714 | 0.002607 | |||||
2 divisions | 0.209553 | 0 | 0.143594 | 0 | |||
3 divisions | 0.190642 | 0 | 0.121757 | 0 | 0.038549 | 0.3545 |