An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

Abstract

It is presently assumed that lethal hit delivery by cytotoxic T lymphocytes (CTLs) is mechanistically linked to centrosome polarization toward target cells, leading to dedicated release of lytic granules within a confined secretory domain. Here we provide three lines of evidence showing that this mechanism might not apply as a general paradigm for lethal hit delivery. First, in CTLs stimulated with immobilized peptide-MHC complexes, lytic granules and microtubule organizing center localization into synaptic areas are spatio-temporally dissociated, as detected by total internal reflection fluorescence microscopy. Second, in many CTL/target cell conjugates, lytic granule secretion precedes microtubule polarization and can be detected during the first minute after cell-cell contact. Third, inhibition of microtubule organizing center and centrosome polarization impairs neither lytic granule release at the CTL synapse nor killing efficiency. Our results broaden current views of CTL biology by revealing an extremely rapid step of lytic granule secretion and by showing that microtubule organizing center polarization is dispensable for efficient lethal hit delivery.

Fig. 1.

TIRFM analysis shows dissociation between MTOC and lytic granule dynamics. (A–D) Sequence of snapshots of TIRFM depicting the interaction between pp65-specific CTL and pMHC-coated surfaces. CTLs were loaded with LysoTracker-red and TubulinTracker-green. Results are representative of 59 cells analyzed over four independent experiments. (A) Snapshots are from Movie S1. (B) Snapshots are from Movie S2. (C) Snapshots are from Movie S3. (D) Snapshots are from Movie S4.

Fig. 2.

Spatiotemporal dissociation of lytic granule secretion and MTOC polarization during CTL/target cell interaction. (A) Sequence of snapshots depicting the interaction between pp65-specific CTL and target cells pulsed with antigenic peptide. CTLs were labeled with LysoTracker-red and TubulinTracker-green. Target cells were loaded with Fluo-4 AM to detect [Ca²⁺]_i increase as a marker of lethal hit delivery. Image acquisition (one image per 7 s) was performed using a LSM 510 confocal microscope. Snapshots are from Movie S9. Results are from one representative conjugate of 11 (57% of a total 19 cells analyzed). (B) Sequence of snapshots depicting the interaction between TubulinTracker-green loaded pp65-specific CTLs and target cells pulsed with antigenic peptide and loaded with Fluo-4 AM. Image acquisition (one image per 0.5 s) was performed using a LSM 710 confocal microscope. Snapshots are from Movie S11. Results are from one representative conjugate of 40 (55% of a total 72 cells analyzed). (C) Comparison of the time required for lethal hit reception by target cells and CTL MTOC polarization in individual CTL/target cells conjugates. Statistical significance of difference between groups was evaluated by a paired Student's t test using GraphPad Prism software. ***P < 0.001.

Fig. 3.

Inhibition of PKCζ function affects CTL polarization toward target cells. (A) Target cells unpulsed or TSST-1–pulsed were conjugated with Vβ2⁺ CTLs pretreated or not with PKCζ-PS. After 5 min at 37 °C, cells were stained for α-tubulin (green) and perforin (blue). (B) Measurement of distances between MTOC and the center of the CTL/target cell contact site; 82 unpulsed, 85 pulsed, and 71 pulsed conjugates with PKCζ-PS–pretreated CTLs from from three independent experiments (performed with cells form three different donors) were scored. Bars indicate mean values. Statistical significance of difference between groups was evaluated by an unpaired Student's t test using GraphPad Prism software. ***P < 0.001.

Fig. 4.

Synaptic exposure of CD107a can occur in the absence of MTOC polarization. (A) Vβ2⁺ CTLs pretreated or not with PKCζ-PS were conjugated with unpulsed or TSST-1–pulsed target cells. Surface expression of CD107a on CTLs was measured by FACS analysis at the indicated times. Data are from one representative experiment of three independent experiments performed in duplicate. (B) TSST-1–pulsed target cells were conjugated with Vβ2⁺ CTLs pretreated or not with PKCζ-PS. After 3 min, cells were fixed and stained for CD107a. Cells were then permeabilized and stained for α-tubulin. (C) Quantification of the intensity of CD107a staining per micrometer at the IS and at the distal-IS; 90 untreated conjugates and 83 conjugates in which CTLs were pretreated with PKCζ-PS were scored from three different donors. Statistical significance of difference between groups was evaluated by an unpaired Student's t test using GraphPad Prism software. ***P < 0.001; ^nsP > 0.05.

Fig. 5.

CTL polarization responses are dispensable for cytotoxicity. (A and B) Target cells either unpulsed or TSST-1–pulsed (A) or unpulsed and peptide-pulsed (B) were cocultured for 4 h with Vβ2⁺ CTLs (A) or pp65-specific CTLs (B) pretreated or not with PKCζ-PS at the indicated effector/target ratios. Cytotoxicity was evaluated by flow cytometry using 7-AAD uptake by target cells. Data are from three independent experiments performed in duplicate and are represented as mean ± SEM. Statistical significance of difference between groups was evaluated by an unpaired Student's t test using GraphPad Prism software. ^nsP > 0.05.