Wiedemann et al. 10.1073/pnas.0600651103. |
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Supporting Text
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Fig. 6.
Quantification of the time of lytic granule polarization in CTL interacting with targets pulsed with 10 mM or 1 nM peptide concentration. The time of polarization was scored by visual inspection on snapshot series. The time occurring between cell-cell contact and stable enrichment of granules at the contact site was calculated. The mean time for granule polarization was 196 s for 10 mM and 194 s for 1 nM. Thirty-four conjugates formed between CTL, and 10 mM targets were scored; 27 conjugates formed between CTL, and 1 nM targets were scored. Data are from 16 independent movie sessions.Fig. 7.
Analysis of CD3 fluorescence intensity at the CTL/target contact site and of calcein fluorescence intensity in target cells. A line was drawn across the CTL and the target, allowing a comparison between the level of TCR expression at the contact site and at the opposite site. The intensity of green (calcein) and blue (CD3) fluorescence was measured on unprocessed images along this line. The time indicated in Left indicates conjugate formation. It should be noted that, for conjugate shown in B, the CTL was slightly out of focus at the time of contact; for this reason the CD3 staining appears low. Data are from three independent experiments out of six.Fig. 8.
Quantification of sustained [Ca2+]i increase and of the time required for target cell death in CTL-target conjugates. For target cell death the time required to detect target blebs was calculated by visual inspection. For [Ca2+]i the sustained increase in Fluo-4 green fluorescence emission was evaluated by visual inspection; furthermore, to have a nonsubjective evaluation of sustained [Ca2+]i increase a region was drawn on individual CTL, and the green fluorescence intensity was measured by using METAMORPH software. The two methods of measurements gave similar results. The mean duration of sustained [Ca2+]i increase was 3,505 s. In four conjugates the targets were already dead before encounter with CTL. It should be noted that the results presented in the figure are actually an underestimate of sustained [Ca2+]i increase. Indeed, after target annihilation the CTL frequently went out of focus or entered in contact with a second target cell; when one of these events occurred the measurement was stopped. Statistical significance of difference between groups was evaluated by an unpaired Student t test using PRISM software (GraphPad). ***, P < 0.0001. Thirty-two conjugates formed between CTL, and 10 mM targets were scored. Data are from nine independent movie sessions.Fig. 9.
CTL polarize lytic granules toward different targets encountered simultaneously. (A) Snapshots depict lytic granule polarization in CTL interacting simultaneously with target cells pulsed with 10 mM (blue) or 1 nM (green) peptide concentration. (B) Snapshots depict lytic granule polarization in CTL interacting with two target cells pulsed with the same antigenic concentration. Data are from one representative experiment of nine.Fig. 10.
CTL kill heterogeneous targets encountered simultaneously. CTL were conjugated with target cells either unpulsed or pulsed with 10 mM or 1 nM peptide concentration. Cytotoxic activity was evaluated by measuring propidium iodide (PI) uptake 4 h after conjugate formation. When two target cell populations were conjugated simultaneously with CTL, one was marked with DDAO Far Red to distinguish the two target populations (see Supporting Materials and Methods). The couple of targets used are specified in the legends of the x axes. The values presented refer to the % of cell death measured in the target population marked in blue. The results are from three independent experiments (mean ± SD). In each experiment the percentage of propidium iodide-positive cells (dead cells) measured in samples where CTL were conjugated with unpulsed JY cells or Daudi cells was subtracted from the percentage of dead cells measured in each other sample. Spontaneous death of unpulsed JY cells was 11.4 ± 4.4% in the three experiments. Spontaneous death of unpulsed Daudi cells was 9.2 ± 2.9%. Statistical significance of difference between groups was evaluated by an unpaired Student t test using PRISM software. *, P < 0.05.Movie 1.
A CTL (loaded with LysoTracker red to visualize lytic granules) is shown during interaction with a target cell pulsed with 10 mM peptide. Lytic granules (red) rapidly polarize toward the target cell (green). Target cell death is detected by the loss of calcein. Quantitative evaluation of granule polarization time is shown in Fig. 6.Movie 2.
A CTL is shown during interaction with one target pulsed with 1 nM peptide. Lytic granules (red) rapidly polarize toward the target cell (green). Target cell death is detected by the loss of calcein. Quantitative evaluation of granule polarization time is shown in Fig. 6.Movie 3.
Two CTL are shown during interaction with unpulsed target cells loaded with calcein. CTL crawl over the target cell surface without a clear evidence of stable granules polarization and target death. Data are from one representative movie of three. Similar results were obtained in two movies in which CTL were interacting with target cells pulsed with an irrelevant peptide..
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Movies 4 and 5.
The two movies show a target cell pulsed with 10 mM peptide bound by two CTL. TCR/CD3 (blue) polarize toward the target cell (green). Target cell death is detected by the loss of calcein. Movie 4 depicts overlapping of differential interference contrast microscopy images with CD3 and calcein staining. Movie 5 Left depicts green and blue fluorescence. In Movie 5 Right TCR staining intensity is shown using a pseudocolor scale. Data are from one representative experiment of six.Movie 6.
Analysis of CD3 fluorescence intensity and of calcein fluorescence intensity in target cell using the Linescan function of METAMORPH software. Data are from Movies 4 and 5. Additional TCR/CD3 dynamics data are presented in Fig. 7.Movie 7.
A CTL (stained with anti-TCR/CD3, blue) interacting with a target cell pulsed with 1 nM peptide and marked with calcein (green) is shown. Data are from one representative experiment of two. Similar results were observed in two movies in which target cells were pulsed with 50 nM peptide.Movie 8.
Detection of [Ca2+]i increase in CTL loaded with Fluo-4 AM. A CTL loaded with Fluo-4 AM is shown during its interaction with one target cell pulsed with 10 mM peptide concentration. Green staining depicts [Ca2+]i increase in the T cell. Time of recording: 54 min. Quantitative evaluation of the time required for target cell death detection and of sustained [Ca2+]i increase is shown in Fig. 8.Movie 9.
Detection of [Ca2+]i increase in CTL loaded with Fluo-4 AM. CTL loaded with Fluo-4 AM are shown during their interaction with target cells pulsed with 10 mM peptide concentration. Increase of green staining in the CTL depicts [Ca2+]i increase. Time of recording: 113 min.
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Movies 10 and 11.
CTL address lytic granules (red) toward a target pulsed with 10 mM peptide (blue) and a target pulsed with 1 nM peptide (green) encountered simultaneously. Data are from one representative experiment of eight.
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Movie 12 and 13.
While TCR (blue) polarize toward a target cell pulsed with 10 mM peptide (red) an adjacent target pulsed with 1 nM peptide receives a lethal hit (green). Target cells pulsed with 1 nM are loaded with Fluo-4 AM; target cell death is detected by a transient [Ca2+]i increase. Movie 12 depicts overlapping of differential interference contrast microscopy images with red, green, and blue fluorescence. In Movie 13 only blue (TCR/CD3), red (CMTMR), and green (Fluo-4 AM) fluorescence is shown (Left). TCR staining intensity is shown using a pseudocolor scale (Right). Data are from one representative experiment of three.Supporting Text
Supporting Results
Killing of Bystander Target Cells. Our results showing multiple killing of heterogeneous targets raise the question of whether the encounter of CTL with targets offering a strong antigenic stimulus may lower the threshold for detection and killing of targets offering weak antigenic stimuli. This may result in an augmented response against these latter targets and even in killing of innocent bystander cells (1, 2).
To address this question we measured by FACS analysis the bystander death of nonspecific targets interacting with CTL in the presence of cognate targets. As shown in Fig. 10, unpulsed JY cells mixed with JY cells pulsed with 10 mM or 1 nM peptide concentration were killed in a bystander fashion. Bystander killing was very sensitive because it was similar in the presence of specific targets pulsed with 1 nM or 10 mM peptide concentration. The mechanisms for such efficient bystander killing could be manifold and may include uptake by innocent targets of antigenic peptides released from dying cognate targets and/or peptide transfer through gap junctions during homotypic adhesion among cells (3).
To investigate whether killing of bystander targets could also occur in conditions in which target sensibilization via transfer of antigenic peptides could be excluded, we used as innocent targets Daudi cells that are deficient in b2-microglobulin and therefore defective in MHC class I antigen presentation. Interestingly, by using these innocent targets a moderated bystander killing was also observed, indicating that some bystander cytotoxicity can be triggered in a TCR-independent fashion (Fig. 10 and Supporting Materials and Methods).
Supporting Materials and Methods
Measurement of Cytotoxicity by FACS Analysis.
CTL were conjugated by rapid centrifugation at 2:1 effector/target ratio with target cells (JY cells). Target cells were either left unpulsed or pulsed with high (10 mM) or low (1 nM) antigenic concentration. CTL were conjugated with unpulsed JY target cells either separately or in combination with pulsed cells. The following combinations were used: unpulsed/10 m M, unpulsed/1 nM. Class I-deficient Daudi cells were used as targets cells either alone or in combination with JY; the following combinations were used: Daudi/JY unpulsed, Daudi/JY 10 mM, Daudi/JY 1 nM. CTL and targets were cocultured for 4 h at 37°C. To differentiate the two target cell populations if used simultaneously, one was left unstained and the other was previously loaded with 10 nM DDAO-SE Far Red (Molecular Probes) in PBS for 15 min at 37°C followed by 15 min at 37°C in 10% FCS RPMI medium 1640. Immediately before FACS analysis, 0.125 mg/ml propidium iodide (Molecular Probes) was added to each sample. CTL were excluded from the analysis because they were loaded before conjugate formation with 100 nM CMFDA (Molecular Probes).Cytotoxicity was evaluated by measuring the entry of propidium iodide in the target cells. In each experiment the percentage of propidium iodide-positive cells (dead cells) measured in samples where CTL were conjugated with unpulsed JY cells or Daudi cells was subtracted.
1. Lanzavecchia, A. (1986) Nature 319, 778-780.
2. Duke, R. C. (1989) J. Exp. Med. 170, 59-71.
3. Neijssen, J., Herberts, C., Drijfhout, J. W., Reits, E., Janssen, L. & Neefjes, J. (2005) Nature 434, 83-88.