Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior

  1. Fanny Lafouresse  Is a corresponding author
  2. Romain Jugele
  3. Sabina Müller
  4. Marine Doineau
  5. Valérie Duplan-Eche
  6. Eric Espinosa
  7. Marie-Pierre Puisségur
  8. Sébastien Gadat
  9. Salvatore Valitutti
  1. INSERM U1037, Centre de Recherche en Cancérologie de Toulouse (CRCT), Université de Toulouse III-Paul Sabatier, France
  2. Toulouse School of Economics, CNRS UMR 5314, Université Toulouse 1 Capitole, France and Institut Universitaire de France, France
  3. INSERM, UMR1043, Centre de Physiopathologie de Toulouse Purpan, France
  4. Department of Pathology, Institut Universitaire du Cancer-Oncopole de Toulouse, France
8 figures, 3 tables and 1 additional file

Figures

Figure 1 with 4 supplements
Lytic components are asymmetrically distributed in dividing CD8+ T cells.

(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 …

Figure 1—source data 1

CD107a, perforin, and granzyme B distribution between daughter cells.

https://cdn.elifesciences.org/articles/62691/elife-62691-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Gating strategy for imaging flow cytometry (IsX) acquisition.

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 …

Figure 1—figure supplement 2
Analysis and representation of the repartition of markers of interest in dividing cells.

(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 …

Figure 1—figure supplement 3
CD8+ T cells are efficiently stimulated on coated anti-CD3/anti-CD28/ICAM1.

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 …

Figure 1—figure supplement 4
Uneven lytic granule segregation in telophase in CD8+ memory T cells.

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 …

Figure 2 with 1 supplement
CD107a+ vesicle uneven segregation in telophase is confirmed by confocal laser scanning microscopy.

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 …

Figure 2—source data 1

Fold increase of CD107a staining in the brighter nascent daughter cell as compared to the other nascent daughter cell.

https://cdn.elifesciences.org/articles/62691/elife-62691-fig2-data1-v3.xlsx
Figure 2—video 1
3D visualization of CD107a repartition in a telophasic CD8+ T 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.

Fate determining transcription factors do not undergo uneven distribution in telophase.

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 …

Figure 3—source data 1

T-bet and c-Myc distribution between daughter cells.

https://cdn.elifesciences.org/articles/62691/elife-62691-fig3-data1-v3.xlsx
A polarity cue is not necessary for asymmetric repartition of lytic machinery.

(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 …

Figure 4—source data 1

CD107a distribution between daughter cells after polarized and non-polarized stimulation.

https://cdn.elifesciences.org/articles/62691/elife-62691-fig4-data1-v3.xlsx
Asymmetric repartition of CD107a+ vesicles resets at each division event.

(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 …

Figure 5—source data 1

CD107a distribution between daughter cells in undivided cells, at first division and second division.

https://cdn.elifesciences.org/articles/62691/elife-62691-fig5-data1-v3.xlsx
Figure 6 with 6 supplements
Lytic granules randomly distribute on the two sides of the cleavage furrow.

(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 …

Figure 6—figure supplement 1
Lysotracker randomly distribute on the two sides of the cleavage furrow.

(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…

Figure 6—video 1
Symmetric repartition of granzyme B during cell division.

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 …

Figure 6—video 2
Asymmetric repartition of granzyme B during cell division.
Figure 6—video 3
Symmetric repartition of LTRvesicles during cell division.

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 …

Figure 6—video 4
Asymmetric repartition of LTR+ vesicles during cell division.
Figure 6—video 5
LTR+ vesicles repartition during cell divisions.
CTL expressing high level of lytic granules have better killing capability.

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…

Author response image 1
CD107a fold increase measured by comparing the two nascent daughter cells in individual dividing CD8+ T cells (Figure 1A and 2A) and Lysotracker fold increase measured by comparing Lysotrackerhigh and Lysotrackerlow sorted CTL (Figure 7A).

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 …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional 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 donorsEFS, Toulouse, FranceWith consent and approval AC-2014–2384
AntibodyAnti-human CD3 (Human monoclonal, TR66)EnzoCat# ALX-804–822
RRID:AB_2051037
(1 µg/ml)
AntibodyAnti-human CD28 (Mouse monoclonal, CD28.2)eBioscienceCat# 16-0289-81
RRID:AB_468926
(1 µg/ml)
Recombinant proteinRecombinant human ICAM-1-Fc fusion proteinR and D SystemsCat# 720-IC(0.5 µg/ml)
AntibodyAnti-human CD107a (Mouse monoclonal, H4A3)BD PharmingenCat# 555798
RRID:AB_396132
(10 µg/ml)
AntibodyAnti-human CD107a AlexaFluor 647 (Mouse monoclonal, H4A3)BD PharmingenCat# 562622
RRID:AB_2737684
(Diluted at 1/100)
AntibodyAnti-human Granzyme B (Mouse monoclonal, GB11)Thermo ScientificCat# MA1-80734
RRID:AB_931084
(10 µg/ml)
AntibodyAnti-human Granzyme B AlexaFluor 647 (Mouse monoclonal, GB11)BD PharmingenCat# 561999
RRID:AB_10897997
(10 µg/ml)
AntibodyAnti-human T-bet (Rabbit polyclonal, Tbx21)AbcamCat# ab181400(10 µg/ml)
AntibodyAnti-human C-myc (Mouse monoclonal, 9E10)Thermo ScientificCat# MA1-980
RRID:AB_558470
(10 µg/ml)
AntibodyAnti-human α-tubulin (Rabbit polyclonal)AbcamCat# ab15246
RRID:AB_301787
(Diluted at 1/100)
AntibodyAnti-mouse IgG1 Alexa Fluor 647 (Goat polyclonal)InvitrogenCat# A21240
RRID:AB_2535809
(10 µg/ml)
AntibodyAnti-mouse IgG1 Alexa Fluor 488 (Goat polyclonal)InvitrogenCat# A-21121
RRID:AB_2535764
(10 µg/ml)
AntibodyAnti-rabbit (H+L) AlexaFluor488 (Goat polyclonal)InvitrogenCat# A11034
RRID:AB_2576217
(10 µg/ml)
AntibodyAnti-rabbit (H+L) AlexaFluor647 (Donkey polyclonal)InvitrogenCat# A31573
RRID:AB_2536183
(10 µg/ml)
AntibodyAnti-rabbit AlexaFluor555 (Goat polyclonal)InvitrogenCat# A21428
RRID:AB_2535849
(10 µg/ml)
AntibodyAnti-mouse IgG Abberior Star 580 (Goat polyclonal)Abberior InstrumentsCat# 52403(10 µg/ml)
AntibodyAnti-human CD107a-PEcy7 (mouse monoclonal, H4A3)BD PharmingenCat# 561348
RRID:AB_10644018
(Diluted at 1/50)
AntibodyAnti-human CD8-FITC (mouse monoclonal, HIT8A)BD PharmingenCat# 555634
RRID:AB_395996
(Diluted at 1/50)
Recombinant DNA reagentMGC Human GZMB Sequence verified cDNA (Clone Id: 5223876)GE Healthcare BIO SciencesCat# MHS6278-202801737
Recombinant DNA reagentmCherry-SEpHluorinKoivusalo et al., 2010Addgene cat# 32001
Recombinant DNA reagentpT7-GZMB-mCherry-SEpHluorinThis paper
Sequence-based reagentPrimer: XhoI-T7-GzB Forward caaCTCGAGTAATACGACTC
ACTATAGGGAGACCCGGTA
CCatgcaaccaatcctgcttctgcc
This paper
Sequence-based reagentPrimer: EcoRI-GzB-noSTOP-R caaGAATTCcggcgtggcgtttcatggttttctttatccagThis paper
Peptide, recombinant proteinCMV peptide p65 (NV-9)GeneCustCat# 181329
Peptide, recombinant proteinHuman rIL-2Miltenyi BiotecCat# 130-097-748(150 IU/ml)
Peptide, recombinant proteinHuman rIL-15Miltenyi BiotecCat# 130-095-766(50 ng/mL)
Commercial assay or kitEasySep Negative human CD8+ T cell isolation kitStemCell TechnologiesCat# 17953
Commercial assay or kitEasySep human Memory CD8+ T cell enrichment kitStemCell TechnologiesCat# 19159
Software, algorithmIDEAS SpotCount Threshold (M03,nucleus,60)Amnis, Luminex
Software, algorithmIDEAS Area Range Threshold (M02,tubulin,75),50–5000, 0–0.5Amnis, Luminex
Software, algorithmFijiSchindelin et al., 2012
Software, algorithmImaris SoftwareOxford Instruments
Software, algorithmZEN ZEISS Efficient Navigation
Software, algorithmHuygens Professional version 18.10 using CMLE algorithm with SNR:7Scientific Volume Imaging, USASTED images were deconvolved
Software, algorithmPython software version 3.5χ2 of independence test, χ2 of homogeneity test and Kolmogorov-Smirnov goodness-of-fit test
Software, algorithmGraphPad Prism software version five for windowsPaired Student’s t-test
Software, algorithmFlowJo softwareTreeStar
OtherSYTOXOrange Dead Cell StainThermoFisher ScientificCat# S11368Manufacturer recommended dilution
OtherDAPIMolecular Probes, InvitrogenCat# D1306
RRID:AB_2629482
OtherHoechst 33342ThermoFisher ScientificCat# 1399(200 ng/ml)
OtherCellTrace Violet Cell Proliferation kitThermoFisher ScientificCat# C34557(5 µM)
OtherLysoTraker Blue (DND22) DyeMolecular probesCat# L7525(200 nM)
OtherLysoTraker Red (DND99) DyeMolecular probesCat#L7528(200 nM)
Other7-Aminoactinomycin D (7-AAD)BD PharmingenCat# 559925(0.25 µg)
OtherIbidi μ-slide chambered coverslips AngiogenesisIbidi, BiovalleyCat# 81506
OtherIbidi μ-slide chambered coverslips eight wellIbidi, BiovalleyCat# 80821
OtherNunc Lab-Tek chamber slides eight wellsNunc, ThermoFisher ScientificCat#1 54526
OtherMicromesh array (100 μm)Microsurface, Tebu- BioCat# MMA-0500-100-08-01
Table 1
Results of independence chi-square test in telophase.
Independence chi-square test between heterogeneous cells and all cellsTest statistic
(χ2)
χ1-α,dl2p-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
Table 2
Results of Kolmogorov–Smirnov test on G1.
Experiment 1Kolmogorov–Smirnov testZero divisionOne divisionTwo divisions
Dn,mp-valueDn,mp-valueDn,mp-value
10 division
1 division0.131480
2 divisions0.22003400.1162830
20 division
1 division0.0878730
2 divisions0.089192400.046340.03582
3 divisions0.0546210.01590.0677020.0011850.0472750.116534
30 division
1 division0.147140.002607
2 divisions0.20955300.1435940
3 divisions0.19064200.12175700.0385490.3545

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