Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes

Abstract

Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca2+] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting.

Fig 1. Capacitance steps in stimulated CTLs.

(A) Examples of step capacitance responses in CTLs. The arrows indicate steps associated with granzyme B positive exocytotic events in TIRF images. (B) The series conductance and the membrane conductance are shown (C) Examples of a cluster of positive steps indicating exocytotic events (part 1) and of negative steps (part 2, downward) which indicate endocytosis (arrows).

Fig 2. Fusion of a granzyme B-mCherry containing lytic granule with the plasma membrane.

(A) Consecutive frames from the TIRF video imaging are shown. The video frame numbers are shown below the images. The arrowhead indicates a lytic granule which decreased in fluorescence and generated a cloud of fluorescence while fusing. (B) The fluorescent intensity (arbitrary units) of the fusing granule (filled circles) indicated by the arrow in A was plotted versus frame number. The intensity of the juxtagranular region (halo, open circles) is included. As the granule fused its intensity increased briefly (frame 534) and it then disappeared. At frame 535 the juxtagranular area brightened. In subsequent frames the fluorescence dissipated (frame 537). (C) The whole-cell membrane capacitance trace (C_m, upper trace) and the camera trigger voltage are shown (lower trace, positive steps indicate shutter opening) versus time. The capacitance step in the C_m trace at time 48.2 s occurs at video frame 535 (arrow).

Fig 3. Capacitance steps associated with lytic granule fusion events indicate a population with a diameter near 325 nm.

The frequency histograms of capacitance steps observed in CTLs. The open circles show the frequency histogram of 508 fusion events obtained from 38 CTLs which were not related to LG fusion. The histogram is fit with a triple Log-normal equation (solid line). The filled circles show the histogram of 98 LG fusion events, which is fit with a single Log-normal (solid line).

Fig 4. Lytic granules which undergo fusion can exhibit long dwell times.

(A) The dwell times of 101 LGs which fused are shown. Each LG is represented as a horizontal bar beginning at the start of its sojourn at the membrane and ending at its fusion. Contiguous bars with the same color indicate granules from the same CTL. Most fusing granules appear at the membrane early and most granules which fused exhibited a relatively short dwell time. (B) The frequency histogram of dwell times could be fit with a single exponential distribution (solid line, 95% confidence limits are indicated by the dashed lines) with a mean near 28.5 seconds.

Fig 5. The behavior of lytic granules at the CTL-coverslip interface.

(A) Representative tracks of granules in a single CTL are shown. Seven tracks (F) indicate LGs which underwent exocytosis (blue is the track end). Three tracks (NF) are LGs which did not fuse. The tracks are to scale but have been rearranged to decrease the size of the image and improve visibility. Two representative tracks from fixed beads (B) which were recorded prior to the CTL experiment are included to show intrinsic jitter of the workstation. (B) A plot of the frame-to-frame movement of a representative track. The red trace is a representative track from a fixed bead. The mean movement of 11 fixed fluorescent beads (0.2 μm in diameter, black trace) is superimposed on the plot.

Fig 6. Fusing and non-fusing LGs cannot be distinguished based on their mobility.

(A) The averaged frequency histograms of LG displacements are shown.The frame to frame displacement of the tracks of 58 granules which fused and 816 granules which did not fuse were calculated. There was no difference between the distributions of displacements of fusing LGs and those which did not fuse. (B) The caging diameters (see text) were calculated and the averaged frequency histograms of the largest distances covered in ten frames (1 second) are shown for fusing and non-fusing LGs. There was also no difference between the caging diameters of fusing and non-fusing LG.

Fig 7. Fusion of LGs is biphasic.

Frequency histograms show the latency from calcium perfusion to fusion in CTLs. The filled circles represent the frequency histogram for all capacitance steps. These were fit with a double exponential (solid line) with a fast time constant of 28.2 s and a slower time constant of 148.4 s. The open circles represent the frequency histogram of the capacitance steps associated with LG fusion. This histogram was well fit with a dual exponential (solid line) with a fast time constant of 15.9 s and a slow time constant of 116.3 s. The 95% confidence intervals are indicated by dashed lines in both curves.