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Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets.

Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and... The EMBO Journal vol.6 no.8 pp.2223-2232, 1987 Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets Martin Poenie" 3, Roger Y.Tsien' and Anne-Marie accumulation of vesicular components in the region adjacent to the target cell (Yannelli et al., 1986). The delivery of the lethal Schmitt-Verhulst2 hit by CTLs may involve secretion of prestored granules or of 'Department of Physiology and University of California, Anatomy, Golgi products directed at the target cell (Henkart and Henkart, Berkeley, CA 94720, USA and 2Centre d'lmmunologie INSERM-CNRS de 1982; Zagury, 1982). Evidence is accumulating in support of Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France the view that the lethal hit involves the release of a calcium- 3Present address: Department of Zoology, University of Texas, Austin, TX dependent pore-forming protein called cytolysin (Henkart et al., USA 78712, 1984) or perforin (Podack and Konisberg, 1984; Podack et al., by B.Jordan Communicated 1985; Masson and Tschopp, 1985). Changes in cytosolic free calcium ([Ca2 ]1) have been con- It is important to distinguish the roles of Ca2+ in CTL activa- tinuously imaged during the interaction of the H-2Kb specific tion, delivery of the lethal hit and killing of the target cell. Ad- cytotoxic T cell lymphocyte (CTL) BM 3.3, with either the dition of appropriate targets, antibody to the TcR, or certain H-2K" EL4.BU or the H-2Kk RDM4 cell lines. Activation of lectins can all cause transients in various CTL prepara- [Ca2+]i raises [Ca2+] to several hundred the CTLs by EL4.BU tions or cell lines. In some T cells these stimuli have been shown CTL. Frequently is preferentially nanomolar in the to induce formation of the PIP2 breakdown products, IP3 and [Ca2+]i elevated in the region of the CTL furthest from the site of diacyglycerol, and release of calcium from intracellular stores target contact. These responses require external Ca2+ sug- (Weiss et al., 1986). However, in murine T lymphocytes or clon- gesting that they are generated by the plasma membrane and ed cell lines used by us and others, influx of extracellular calcium not internal stores. Inappropriate targets such as RDM4 evoke component of transients though some is the major [Ca2+]i no changes in Activation of the BM 3.3 CTL is [Ca2W]1. release of intracellular calcium can be detected (Tsien et al., 1982; followed by increases of to several micromolar or our observations). Although classical voltage- [Ca2+]i and unpublished higher in the EL4.BU targets. This massive increase can be calcium channels have not been detected in T lym- dependent by direct application of cytolytic granules isolated mimicked et al., 1984; Matteson and Deutsch, 1984; phocytes (DeCoursey rat natural killer cells. The increase in plasma mem- from al., 1984) a mitogen-regulated calcium channel has Fukushima et is ion-specific since external Mn2+ can brane permeability in human helper T cells (Kuno et al., been recently identified readily enter cells that have been 'hit', as evidenced also target serve to open these channels is not known 1986). How mitogens the selective quenching of fura-2 in those targets. by rapid is probably an intermediate messenger linking recep- but there The flood of Ca2+ into the target cell is followed by a leakage to channel opening (ibid). tor binding fura-2. Since both processes continue after the of the trapped We have employed fura-2 (Grynkiewicz et al., 1985) in con- has they provide a useful assay for the lethal CTL disengaged, with digital image processing (see Poenie et al., 1986) junction this technique can be used to follow com- hit. Furthermore, in intracellular free calcium in both to study changes ([Ca2+]i) of CTL activation and lethal hit delivery, which plete cycles effector and target cells throughout the killing process. We find can be as rapid as 6 min per cycle. under some circumstances and characteristic changes in [Ca2]+ occur in that distinctive T cells/cytosolic free calcium/digital image Key words: cytolytic killer cell and target cells. Killer cells which contact a both gradients/T cell activation processing/calcium show a moderate rise in ]+ close to the susceptible target [Ca2 of contact. In elongated CTLs the initial rise in cor- time [Ca2+]i the change in shape as the CTL rounds up next to relates with Introduction an gradient forms when the target. Frequently, apparent [Ca2+]i makes initial contact with the target such that the CTL T lymphocyte (CTL) mediated cytolysis is a process [Ca2+]i Cytotoxic in the of the CTL most remote from the site of of target cell antigens by the T cell is high region involving specific recognition cell -cell contact. These gradients disappear immediately u on The latter is a heterodimeric protein (Allison and receptor (TcR). removal of extracellular calcium. The period of elevated [Ca et Haskins et al., 1983) express- Lanier, 1985; Allison al., 1982; ]i is variable lasting from a few seconds to several in both its a and chains, associated in the CTL clone specific sequences ing the rise in the effector, following with the CD3 (Meuer et al., 1983; minutes. Typically, on the T cell surface complex [Ca2+]i to levels sufficient to saturate In addition to TcR-antigen interaction, abruptly jumps Samelson et al., 1985). target [Ca2+]i in the killer cell normally returns to fura-2. Whereas cell formation involves other T cell sur- conjugate CTL-target [Ca2+]i remains in the target suggesting that levels, high et This of conjugate for- resting face molecules (Shaw al., 1986). step [Ca2+]i contribute to of the target cell. calcium toxicity may cytolysis is on external magnesium but not mation, which dependent rises in the the fura-2 fluorescence signal After calcium target, and 1977), is followed by calcium- calcium (Golstein Smith, that the cell has become permeable to small declines indicating of the lethal hit and CTL independent lysis delivery dependent is now out of the cell. and that leaking molecules trapped dye cell CTL recognition of target cells of the (Martz, 1977). target et al., 1984) of granules (Henkart Application purified cytolytic an extensive cytoplasmic rearrangement in is accompanied by a similar elevation of calcium causes rapid to fura-2-loaded targets reorientation of the microtubule organiz- CTL which includes the s of contact with the These ex- within 6 granules. in the targets et 1983, 1985) and and Golgi (Kupfer al., ing center (MTOC) IRL Press Limited, Oxford, England M.Poenie, R.Y.Tsien and A.-M.Schmitt-Verhulst 2 0 CY o0 Time (sec) Fig. 1. An of example the use of fluorescence ratio to imaging measure changes in the stimulation of fura-2 during loaded BM 3.3 [Ca2+]i effector cells by EL-4 targets. Here fluorescence ratios are coordinated with pseudocolor hues and calibrated in terms of micromolar Panel Ca2 . shows the for a field of fura-2-loaded resting BM 3.3 cells [Ca2+]i before .0- addition of targets. Panel B shows the BM 3.3 cells after just addition of RDM4 unloaded EL-4 cells. The unloaded EL-4 cells are invisible in the fluor- CU escence image. Panel C shows the BM 3.3 cells as to they begin respond 4q to the added target cells. The of CTLs percentage indicated responding, by their in change hue, continues to increase D (panels and until most E) of the cells have responded. Note that within of many the CTLs [Ca2+]] is elevated unevenly indicating the formation of calcium gradients. Panel shows the of population unloaded the targets overlaying fura-2-loaded CTLs. 0 -0 0 350 periments 700 therefore support the proposal that cytolytic granules and CTL-mediated lysis both cause rapid to the Time damage target (sec) cell plasma membrane. The observation of multiple sequential hits by a single CTL shows that each hit is associated with a The data shown in Fig. is here imaging Figure 1 displayed graphically. separate activation event and transient. Furthermore, the A circle was over each cell in the [Ca2+]i placed 10 in field, typically pixels sequential hit of cells in the vicinity of the CTL diameter and the stresses the short +]i and intensity values average [Ca2 calculated for the area are defined the circle. In range of action the was of the lytic by determined for molecules and is (A) average [Ca2+]i compatible with the each individual cell. Each cell is then a solid block represented by square notion of polarity in the delivery of the lethal hit. to its positioned (y axis) at a according average [Ca2+]i given time (x axis). to three cells with the same can be Up average +j displayed at a [Ca2 given time blocks period If more than three Results by placing side-by-side. cells had the same will not they be Thus in average [Ca2 +1i distinguishable. some cases in the Changes to and not to specific effectors graph all the cells used to obtain [Ca2+]i targets may appear represent the plot when the is especially uniform and When BM population fairly values 3.3 effector cells are average [Ca2 +]i stimulated with EL-4 targets they are clustered close Thus the distribution of the together. [Ca2 +]i population undergo a rise in from nM 130 to an can average of be ascertained for a (right y axis) time vertical [Ca2+]i given by distribution of 500 nM. This can be seen in a individual cells Figure 1, series of fura-2 fluor- squares. Although calcium undergo significant transients the mean for ]j the whole escence [Ca2 population (solid line) shows a small ratio in which only images [Ca2+]- is as various represented When such change. as the RDM-4 cell nonstimulatory targets line are used hues ranging from blue (low calcium) through green, yellow, in a (B) similar to that above no experiment in BM 3.3 change [Ca2+], is orange, red to purple at maximal calcium detectable either in values. This individual cells shown experi- the scatter or by plot in the ment population began with a average (solid monolayer of fura-2 loaded BM line). 3.3 cells, adhered by to a polylysine coverslip. Before addition of target the cells, field of effector cells appears low in marized uniformly the by plotting average of each individual [Ca2+]i cell [Ca2+]i (panel A). When unloaded EL-4 targets in are added (panel B) the field there (Figure or of 2A) the entire population. Both sorts is no immediate effect on in the of data effectors. However, indicate that the effector population undergoes [Ca2+]i a moderate within a few minutes begins to rise in the effector cells increase [Ca2+]i stimulation [Ca2+]i upon with targets. (panel and C) by 10 min D (panels and E) most of This the effector reaction of the BM 3.3 to targets is specific for targets cells have experienced a calcium transient. An with interesting aspect H-2 antigens. Inappropriate targets such as cells appropriate of the rise in the effectors is that it of the H-2 [Ca2+]i often appears as a thymoma, RDM4, are not killed by BM 3.3 effec- gradient across the effector cell (see below). Finally, tor cells panel F Table and (see II) do not evoke an increase in shows the [Ca2+]i transmitted light image of the target cells, in large in the BM 3.3 ex- cells (Figure 2B). cess, layered over the effectors. The data can To be sum- investigate the effect of CTL cytolytic activity on [Ca2+], target 2224 Imaging Ca2+ in cytolytic T cells and targets 1 .10 t UL - - EL41IFura-2 0 900 Time (sec) 2.0 Fig. 3. By loading targets with fura-2 and using unloaded BM 3.3 effector cells the response of the targets can be isolated. This therefore is the reciprocal experiment to that of Figure 1. Panel A shows the resting for a field of EL-4 targets before addition of the BM 3.3 cells. In [Ca2+], panel B the unloaded BM 3.3 cells are added but no response is yet 1 .0o detected. Panel C shows the earliest sign of EL-4 response to the added I Fura-2 RDM4 Cy BM 3.3 cells. Target cells show a rapid and usually permanent rise in to levels sufficient to saturate fura-2. Panel D shows a later time u [Ca2+], point where the respose of the EL-4 cells is more fully developed. The average fluorescence of the targets diminishes rapidly after their has [Ca2+]i risen. This results from dye loss, presumably from leakage through pores formed by cytolysin. Thus in panel D it is evident that some of the targets i,i- that turned purple in panel C have already lost sufficient dye to fade from view. 0 300 600 Time (sec) unloaded BM 3.3 cells were added to a monolayer of [Ca2+]i, fura-2-loaded EL-4 targets (Figure 3), the converse of the ex- 4. representation of the pictorial data from Figure 3 shows Fig. Graphical for individual rises much than that of the CTL in Figure 1. Fluorescence ratio images revealed that targets higher periment that [Ca2+]i after a longer lag period than that observed for CTL activation. In usually are distinctively different from those changes in target [Ca2+]i virtually all of the fura-2-labelled EL-4 targets were this experiment (A) with CTL activation. rises abruptly to very associated for the of observation so that average [Ca2+]i 'hit' within the period [Ca2+], in individual targets after a variable delay period. line) as a whole also rises to high values. When high levels population (solid targets such as RDM-4 are used (B) in the same assay again inappropriate is usually permanent, though targets Elevation of target [Ca2+]i mean is detectable (C individual cell or the population no change in [Ca2+]i oscillate up and down for a time before ultimately occasionally and D). high. Targets reach much higher remaining permanently [Ca2+]i than do CTLs, and this is generally followed by a loss levels not measures a much earlier event an indication that some degree of plasma 51Cr. Imaging only of fluorescence signal, [Ca2+]i from cells, it also allows correlation bet- has taken place. This typical pattern of than 51Cr release target membrane damage in individual CTL and in its associated in the cell population can be readily ween the changes an change target [Ca2+]i fluorescence ratio imaging of cell data is graphed. Figure 4A shows a scatter This is achieved by observed when the target. where both effectors and targets are loaded with EL-4 population exposed to unloaded BM plot of a fura-2-loaded populations conditions it is important to retain the iden- BM 3.3 effector cells were added at the arrow. fura-2. Under these 3.3 cells. The in the field. By adding the effector and which represents the time for ef- of both cell types After an initial delay period, tity it is to and record the positions with the individual targets possible map cells to settle and interact targets, targets sequentially fector before the second cell type. Provid- values. The solid line of the first cell type adding to rise to very high begin [Ca2+]j are not too great, individual conjugate pairs for all the cells in the field. If the ed that cell densities represents the mean [Ca2+]i and the of events in both cell types is using fura-2-loaded RDM4 targets can be followed sequence same experiment repeated the of cell type is determin- BM 3.3 cells, in the RDM4 reconstructed. Although assignment (4B), which do not activate [Ca2W]i the cells' order of appearance, changes in low and unaffected the ofBM 3.3 cells. ed by cells remains by presence [Ca2+]i cell are so different effector and target conjugate pairs EL4.BU cells undergo a high rise in responding Thus most target [Ca2+]j to an alternative means of iden- characteristic as after of target and effector cells and provide between 8 and 16 min mixing tification. which contrasts with the kinetics of release 4, A and B), (Figure an of measurements obtained from 5A shows example 'Cr from the EM4.BU target cells: at an effector to target Figure of of numbers of effector mixed approximately equal of 10:1, 20-30% Cr was released after 40 and 60 a population cell ratio both of which were labeled with fura-2. The 2 h were to release 45 % whereas at a ratio of 2:1, required and target cells, min, 2225 M.Poenie, R.Y.Tsien and A.-M.Schmitt-Verhulst 2-0 1-4 were obtained By contrast, an excess of Figures using whichever cell constituted the unloaded partner. 0 0 0 0 A of in 0 an individual plot within this 0 conjugate pair [Ca2+]i in is shown Here population 5B. the CTL is seen to Figure a moderate elevation of undergo then the first, [Ca2+]i target cell £ to elevate < min 1 after peaks in the begins [Ca2+]2 [Ca2+]i effector cell. 1o-0 EL4 4Fura-2 0 It should be mentioned that different CTL %U effector cells with similar cell in target specificity vary greatly killing efficiency. Various parameters including antigen-receptor and (TcR) avidity of other molecules density, participation accessory on the T cells, Ifi . 8a* oL and intrinsic contribute 'killing machinery' to these dif- may ferences. The BM 3.3 effector chosen for this behaved as study an CTL efficient, various criteria 0.0 yet specific by (Hua et al., When a less active CTL 0 effector of similar 1986). target specific- 200 400 was used Hua et the ity (KB5-C20 cells, al., 1986), same se- Time of CTL activation and quence target permeabilization was (sec) observed but less within a 20-min frequently observation period. Calcium and in CTLs gradients shape change Additional information is obtained from the actual of images -, y l, When responsive conjugate pairs. elongate CTLs contact a target round to the characteristic they typically up mor- conjugate This A phology. occurs in shape change conjunction with a cytoplasmic rearrangement which the places Golgi apparatus and 1 -0 - of the in the of the many organelles CTL to the region adjacent tZ & <e4 - cell. of target Images this of during early period [Ca2+]i l I0- tf! ' 1- *~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ interaction reveal an effector-target increase in CTL [Ca2+] :4:~~~~~~~~~~~ this and in during rounding-up period, cases ] is many [Ca2+ in the CTL highest cytosol most remote from the target. This is illustrated in Figure 6 which focuses on a single CTL as it contacts and to a responds target. Here, is low initially [Ca2+]i in the CTL prior to the contacting target (panel A). Upon con- 1 00 250 to rise tact, [Ca2+]i appears preferentially at the end furthest from the target After the initial (panel B). elevation the BM 3.3 Time (sec) cell begins to round C up (panels -F). Although are spatial [Ca +]i gradients common, other patterns Fig. 5. The entire sequence of changes observed in effectors of CTL and targets can activation are also seen. For example, in Figure 7 be observed when both targets and effectors are loaded with fura-2. By resting rounded BM 3.3 effector makes contact with a target sequentially adding effectors followed by targets, the positions of each cell (panel A). Though the contacting surface area appears to be type small, were determined and individual conjugate pairs followed. In panel A a the result is a large elevation scatter plot is given showing the throughout the effector sequential activation of [Ca2+]i cell BM 3.3 cells followed by the elevation of target The BM 3.3 cells are here (panel B). Subsequently, the CTL forms a [Ca2+]_ gradient [Ca2+]i. and represented by solid squares and the EL-4 cells by open circles. At the undergoes dramatic change in shape (panels C-E). Follow- arrow, fura-2-loaded EL-4 cells were added. As these cells settle and make the formation of ing the typical conjugate configuration, contact with the BM 3.3 cells the initial elevation characteristic of [Ca2+]i [Ca2+1i the BM 3.3 rises in the activation is detected. target cell indicating delivery Subsequently, a second elevation of of the lethal hit (panel calcium is detected this time characteristic of the reaction of the and the EL-4 target F; following section). cells. Solid squares represent the BM 3.3 cells, open circles represent EL-4 The correlation of the calcium gradient with the direction of targets. In panel B for one taken from effector-target pair, the [Ca2+]i attack is displayed spectacularly when panel A data, is shown. in the a single CTL BM 3.3 cell (solid and [labelled line) EL-4 [Ca2+]i cell (broken line) starts at typical resting levels. As killer time progresses a (K)] attacks several cells in vicinity. the In Figure 8 a single transient is detected in the BM 3.3 cell which is followed shortly [Ca2+]i BM 3.3 cell attacks and kills three neighboring EL-4 targets. In thereafter by a much larger calcium increase in the target cell. This panel A the effector cell is touching the first particular EL-4 target conjugate pair was not in cell contact with any other cells and the graph therefore [labelled 1st target represents the interaction of a (Tl)]. Elevation of in single CTL and the killer cell associated [Ca2+]i target. is not instantaneous but begins in panel B. The red pseudocolor in the upper right quadrant of the CTL gives evidence of a calcium solid squares represent CTL while open circles gradient. As seen earlier, this denote gradient is highest in the [Ca2+]i typically targets. The scatter plot shows that CTL region elevation of the CTL remote to the target. [Ca remains elevated +]i [Ca2+]i precedes a much larger rise in the targets, though a for 1 sizeable frac- nearly min before any change is detected in the target (panels tion of each population still remains at rest at the C and In 400-s point. E). panel F, 56 s after the initial rise in CTL [Ca2+]i, The percentage of participating cells was lower here the target abruptly than in jumps to high indicating that the [Ca2+], Figures 1-4 probably because we used lower cell densities lethal hit has occurred. Subsequently, the target fades from view. overall and effector:target ratios near unity to facilitate The killer continues the attack on identifica- a second target (T2) and again tion of each cell type and monitoring of isolated effector-target a [Ca2 ]1 to the gradient appears opposite target. Subsequently cell pairs. At such reduced cell densities, some effectors and in the increases second [Ca2+]j This cycle target. occurs a third targets fail to contact each other and thus do not time show a response. in K and L. panels 2226 Imaging Ca2' in cytolytic T cells and targets Fig. 6. Fluorescence ratio images taken while effectors and targets interact of gradients and morphological changes reveals a striking correlation [Ca2+]i in the effector as it contacts the target cell. Here a particularly elongate BM 3.3 effector is approaching an EL-4 target (panel A). As the front of shown rises dramatically, especially at the the effector contacts the target [Ca2+]i back end (panel B). Subsequently, the back part of the CTL moves up toward the target giving rise to the typical conjugate morphology. the experiment from which this sequence was derived ended Unfortunately a rise in the target was recorded. before multiple hits. 8. A single BM 3.3 effector cell is capable of delivering Fig. activation event as Each hit appears to be associated with a separate rise and directional In this a BM 3.3 a gradient. figure indicated by [Ca2+]i A shows the BM 3.3 effector attacks three target cells in sequence. Panel EL-4 cell (TI). Calcium is cell (labelled K) juxtaposed to the first target activation has not yet taken low in both cell types indicating that CTL increase in the CTL. Panel B shows the first detectable place. [Ca2+]i continues to increase and a then becomes gradient (panel C) [Ca2+]i rises in the cell thereafter, [Ca2+]i target (panel apparent (panel D). Shortly to fade F-H). At the and subsequently the first target begins (panels E) into with the second cell same time the effector is moving contact target Fig. 7. There is sufficient variability in the nature of CTL activation that it rises a second time with the shifted and cor- gradient (T2). [Ca2+]i is impossible to illustrate with one sequence the typical or stereotyped CTL to the position of the second target (panel I). Subsequently, responding response to a target. In this sequence the BM 3.3 effector begins in a rises in the second J) which then also loses its (panel dye [Ca2+]i target rounded-up state with an EL-4 target nearby (panel A). The small region of of formation and hit appears to signal (panel K). A third cycle gradient effector-target contact (panel B) is sufficient to trigger a relatively large, K and L. here it is to know if take in panels However, impossible place long-lasting and initially uniform rise in effector [Ca+],. Subsequently, the of the effector cell or an artifact due to the very the gradient is a function effector undergoes a radical change in shape (panels C and D). Eventually of the adjacent target (T2). at the rear of the effector does form high [Ca2+] a (panel E). gradient of high [Ca2+]i rises that this the EL-4 target enormously indicating cell Finally, [Ca2+]i has been hit. with In some cases lose contact not targets. may actually previous rise in the lethal hit? the after the Is the targets CTL the disengages target [Ca2+]i however, completely not Thus events after the has risen that after calcium has risen in the target the CTL shown). Figure 8 shows target (data [Ca2+]i must be events. under crowded it lytic conditions, rise in target CTL-independent moves to another target though, [Ca2+]i 2227 and M.Poenie, R.Y.Tsien A.-M.Schmitt-Verhulst in Fig. 9. The existence of a the membranes of unique pore plasma targets 11. The Fig. apparent [Ca2+1] gradient observed during CTL activation after the hit is illustrated the of fura-2 in those could conceivably arise as an artifact of by rapid quenching fura-2 compartmentation. For targets Panel A of by manganese. shows a field fura-2-loaded of example, under conditions of targets many poor dye loading unhydrolyzed ester might which have been hit as indicated their color. Within 5 s of accumulate and remain in hydrophobic by purple compartments. The fluorescence 1 mM addition of most of the have signal from this compartment would manganese, purple-colored targets not change when the cytosol and 25 s disappeared (panel B) are experienced a transient and if localized completely quenched by (panel C). [Ca2+]i at one end of the cell might show that the rest of the give rise to an apparent gradient. In order Subsequent images to determine whether population quenches relatively apparent and 102 s later are still fluorescent calcium gradients be due to slowly brightly might compartmentalized dye, 20 !ZM (panel D). digitonin in calcium-free medium was applied to a CTL exhibiting a gradient during activation. The of this concentration ability of digitonin to selectively permeabilize the membrane of BM plasma 3.3 cells while leaving intracellular membranes intact was determined by comparison of lactate dehydrogenase release to that of acridine orange (see Materials and At 20 methods). tiM digitonin, adhered cells can take several minutes for complete membrane plasma permeabilization to take place. This sequence begins with a CTL undergoing activation (panels A-C). At the peak of the gradient the cell was washed with calcium-free lysis solution (without digitonin) and the gradient immediately disappears (panel D). Subsequently, the cells were washed with 20 MM digitonin-containing lysis solution (panel E). Lysis takes place over 1 min 20 s with the image fading below the detection limits under the conditions of gain (750 V) and background subtraction employed (panels F and The G). rapid loss of fura-2 here indicates that the dye was mainly in the cytosol and free to diffuse away. However, at higher gain (1000 V) unlysed dye-containing granules are still evident and their relative fluorescence excitation efficiencies at 350 nM (panel H) and 385 nM (panel I) indicates that little of this compart- mentalized fura-2 is bound to calcium. These pictures show that only a small fraction of the intact cell fluorescence can be attributed to the compartmentalized dye in these granules. Furthermore, the granules were not in a suitable region of the cell to account for the gradient seen in panel C. ching of fura-2 fluorescence in targets that have been hit, when external Mn2+ (Hallam and is Fig. 10. The cytolysis of EL-4 cells rat NK Rink, 1985) applied (Figure by purified cell derived 9). cytolytic granules shows remarkable to the Manganese ions quench similarity action of BM 3.3 cells. fura-2 fluorescence within seconds in The rat NK cytolytic granules were obtained as a from gift Pierre Henkart targets that have been hit, presumably entering the cell by the at the NIH. Panel A shows a layer of fura-2-loaded EL-4 cells before same pathway as Ca2+ does, whereas quenching occurs slowly addition of granules. Three microliters of suspension of cytolytic granules at over several minutes in intact a concentration targets or CTLs. equaling the content obtained from 106 cells suspended in 1 ml PBS was then = The loss of injected over the EL-4 cells (final fura-2 from EL-4-loaded targets is volume 50 Ml). another indica- addition enters the Upon of cytolytic granules EL4.BU cells rapidly tion that pores have [Ca2+]i been made in the plasma membrane of the (panels B and C). Subsequently, fura-2 leaks out of the EL4.BU cells, an target cell. Not only can a foreign cation such as manganese rapid- effect that mimicks the CTL-mediated permeability changes (panel D). ly enter the cell but - also the fura-2 penta anion of 750 mol. wt rapidly leaves the cell. Most of the targets lose their fluor- CTLs cause an increase in target permeability to external ions escence within -60 s of the calcium rise with a half-time of The rise in target calcium appears to be an influx of external 15-20 s. Clearly, the rise in target precedes the loss [Ca2+]i calcium into the target cells. This is evidenced by the rapid quen- of fura-2 though the two events are closely correlated. 2228 Ca2I in cytolytic T cells and targets Imaging with EGTA medium, in the absence of digitonin (panel D). As Comparison with cytolytic granules can be seen, the apparent gradient immediately disap- [Ca2+]i Purified cytolytic granules from rat NK cells have an effect on pears. The rapid collapse of the elevation suggests that [Ca2+]j target cells similar to that of CTLs. When cytolytic granules are it is sustained by Ca2+ influx across the plasma membrane, not added to fura-2-loaded EL-4 targets, [Ca2 ]i rises rapidly in the by release from internal stores, nor is it an artifact of dye seeing target and dye fluorescence is subsequently lost. This can be seen high Ca2' inside organelles. Next, the cells were washed with in Figure 10. Panel A shows fura-2-loaded EL-4 cells before ad- the same medium containing 20,uM digitonin (panel E). By 1 dition of cytolytic granules. In panel B the granules were added the loss of dye from the target and effector cells becomes min and within a few seconds the entire field shows an enormous obvious (panel F). By 1 min 20 s both cells have disappeared rise in (panels C and D). To obtain a better temporal [Ca2+]i from the field (panel G) at the gain settings used for panels A-F. correlation of the rise and the loss of fluorescence due [Ca2+]i By increasing the camera gain 2-fold to its maximum, three weak- to dye leakage a photometer was used to quantitate the fluor- ly fluorescent granules are detectable which are resistant to escence ratios and average intensity from a single CTL exposed digitonin lysis. The fluorescence of these granules persist for at to cytolytic granules. The results show that a rise in [Ca2+]i least 10 min. Their relative fluorescence at 350 nm (H) and precedes the drop in intensity. The target cell attains 385 nm (I) indicates low fluorescence ratios consistent with those 350nm/385nm excitation ratios of - 25 before the signal becomes observed in intact cells. The position of these granules in what so noisy (due to the loss of signal) as to be unreliable. These would be the middle of the cell as shown in panel C would rule ratios are indicative of fura-2 saturation (data not shown). them out as a cause of the apparent transverse calcium gradient. Are the calcium gradients real? Furthermore, their low fluorescence intensity makes them We were concerned that the apparent gradients of [Ca2W+i across relatively insignificant in the intact cell although such granules CTLs could have been artifacts due to compartmentation of can become conspicuous under conditions of more severe com- fura-2. For example, Almers and Neher (1985) have reported partmentation. Thus it is unlikely that all CTL gradients [Ca2+]i that mast cells loaded with fura-2/AM give smaller apparent can be explained as an artifact of dye compartmentation. transients than when cells are loaded with the fura-2 [Ca2+]i When dye compartmentation is more severe, local bright spots anion directly through a patch pipette. This apparently was due of fluorescence are seen in the cell which tend not to change their to accumulation of incompletely hydrolyzed ester into mast cell ratio when the rest of the cell experiences a change in calcium. secretory granules. Incompletely hydrolyzed ester gives low Thus when cells are intentionally loaded so as to produce com- 350 nm/385 nm excitation ratios and is relatively unresponsive partmentalized dye, cells may contain numerous and obvious to calcium. If CTL secretory granules were likewise to ac- granules (data not shown). cumulate significant fura-2/AM ester and to congregate near the plane of conjugation with the target, a genuinely uniform high in the CTL could be partially masked near the target. Discussion [Ca2+]i if fully hydrolyzed fura-2 were to accumulate in a Conversely, The CTL effector function is generally measured by release of high compartment remote from the target, a [Ca2+]i from target cells. The chroamte being bound to in- [Ca2+]i 51CrO42- would be simulated. Evaluation of fura-2 compartmen- gradient tracellular proteins is released only after extensive plasma mem- tation within cells was carried out as described in Materials and brane damage. Earlier increases in target permeability could be methods. CTLs showing calcium gradients were washed with measured by leakage of smaller molecules such as 86Rb or ATP, calcium-free, EGTA-containing medium followed by the same but the ratio of CTL-induced to spontaneous release was very with digitonin. Digitonin is known to selectively lyse medium small in these assays (Henney, 1973; Russell, 1983). Here, we plasma membrane while leaving intracellular membranes the have used cytosolically trapped fura-2 as both a non-perturbing intact when used at appropriate concentrations. indicator of and a marker for membrane integrity of the [Ca2+]i To determine the right concentration of digitonin, monolayers target cell. ofBM 3.3 cells on polylysine-coated Petri dishes were incubated In the effectors, fura-2 indicates increases in [Ca2+]i resulting 10 jiM acridine orange, a fluorescent dye which accumulates with from CTL activation through the binding of the relevant target intracellular organelles. After three washes in EGTA- in acidic cell H-2 antigens to the TcR/CD3 complex. After one activation medium cells were lysed in the EGTA-medium containing various event, the effector cells slowly return to resting unless [Ca2+]j, digitonin concentrations for 5 min. The amount of released LDH they are reactivated by additional target cells. orange were then measured fluorimetrically. Subse- and acridine Fura-2-loaded targets show within 5 min of effector addition, 1% Triton X-100 lysis solution was added to complete quently, large increases in followed by leakage of dye and corre- [Ca2+]i the lysis and the assays repeated. Using 20 digitonin in a AM sponding loss of signal from the cell. This striking difference lysis solution it is possible to release 100% of the calcium-free in response between the two populations of cells permitted the LDH and only 10-12% of the total acridine orange released total two cell types to be easily distinguished in mixed populations digitonin + Triton X-100. This digitonin concentration can by of cells where both types were loaded with fura-2. >95 % of the intracellular fura-2 in freshly loaded cells. release Is calcium the toxic agent? cells that have been allowed to compartmentalize their However, The high levels of calcium experienced by targets open the way can retain 25-30% of the intracellular fura-2 after digitonin dye for extensive cell damage (Wrogemann and Pena, 1976; Camp- (data not shown). lysis bell and Luzio, 1981). Sustained elevation of [Ca2+] is A visual analysis of CTLs containing calcium gradients such reportedly toxic for many cell types. Mitochondrial damage, ATP as in Figure 11 shows that the low fluorescence intensity and deletion, electrolyte imbalances and activation of esterases all central position of granules in the cell cannot account for the gra- follow from sustained elevation of [Ca2+]i. Thus the large eleva- dient. Here, the CTL develops a normal response to the EL4 caused by CTLs or purified cytolytic granules tion of Ca2+ target showing a typical calcium rise and gradient formation to irreversible cell injury. These aspects of cell damage could lead A-C). Immediately thereafter, the cells were washed (panels 2229 M.Poenie, R.Y.Tsien and A.-M.Schmitt-Verhulst are similar to those which produced by and be complement, sufficient to elevate similarities tins, are not may [Ca2+]i always between CTL and sufficient complement-mediated cell have to full lysis been activation. For sug- Clevers et al. give example, gested (Campbell etal., 1979, 1981; Campbell and showed that the Luzio, 1981). lectin wheat (1986) germ agglutinin nonmitogenic There are certain unique aspects of cell seen in causes elevation damage of CTL- and PI (WGA) [Ca +]i However, hydrolysis. mediated cytolysis not seen in a complement lysed cells such was as obtained when a proliferative non- response only low, the degradation of target cell DNA (Russell et dose of al., PMA was added in 1980). addition to mitogenic WGA. It is However, even this DNAase activity is to note that calcium-dependent the sug- levels obtained due interesting to WGA [Ca2+]i gesting that calcium is an important stimulation component of cell were as target as those lysis. obtained from the high mitogenic Unfortunately, it is difficult to evaluate the lectin relative importance Similar results have phytohemagglutinin been ob- (PHA). of Ca2± influx compared with generalized colloid tained osmotic monoclonal swell- to the T cell using Soluble antibody receptor. ing or other mechanisms of damage since to monoclonal prevent Ca2' antibodies can influx elicit calcium transients in T cells but we presently have to remove external Ca2+, but activation as external Ca measured 2+ interleukin secretion optimal or by pro- or Sr2+ is also required for the initial liferation pore formation so all such as obtained requires greater crosslinking using pathways for cell damage would be blocked (Henkart et or the al., addition of PMA. Sepharose-coupled as cur- antibody If, 1984). However, the fact that a CTL seems can disengage from the the effects from or rently likely, resulting antibody target target cell even before the fura-2 has leaked to the completely TcR/CD3 away are mediated antigen binding complex through suggests that the initial calcium rise of (pore inositol formation) coincides then the difference hydrolysis phospholipids, between with the lethal hit described in the soluble literature and (Martz, 1977). be Sepharose-coupled one of antibody may largely This has Patterns of from [Ca2+]i increase in CTL of effectors: clues to degree. elevation support comparisons acti- [Ca2+]i evoked vation anti-TcR ? with or without the by ad- antibody subsequent dition of A fascinating crosslinking observation from these antibody (in preparation). experiments is the Perhaps then, ap- the cell can evoke a pearance of a by analogy, target increase in transient calcium in rapid global gradient CTLs as many they a soluble that calcium channels while contact their target cells. messenger local- The common feature opens of these only gradients kinase C. The local activation is ly stimulating of that kinase appears highest on the side of protein [Ca2+]i the CTL furthest of from the interaction and target cell at might require about the same greater degree time that a major perturbation of the CTL cell membrane or cytoplasmic be slower rearrangement than the takes place. In other simply cell types it has for calcium been messenger responsible channels. hypothesized that opening This would calcium gradients, such as might form be consistent with the results shown in due to 7 chemoattractant where a small gradients or weak Figure electric fields, might contact area leads to a uniform elevation be of involved in cell but as the orientation or directed [Ca2+]i motility (Robinson, contact area 1985). Such becomes more a behavior increases, gradient evident. In this shows a considerable resemblance to the localized model, kinase C would be polarization and reorientation protein activity of CTLs in response to target cells. responsible for a sense of to the CTL. In Perhaps, by analogy, a calcium giving polarity gradient in the many CTL may also neurosecretory cells and cells of be part of, or PMA result from, the hemopoietic treatment directional signal lineage, leading to CTL power- with elevation polarization. fully synergizes to [Ca2+]i promote exocytosis even while to rises The mechanism tending dampen et of [Ca2+]j (Rink elevation appears al., [Ca2+]i to involve open- 1983; et ing of DiVirgilio Albert and calcium al., 1984; channels in the CTL Tashjian, 1985). PMA has since removal of extracellular Ca2+ causes immediate decline in and loss of the gra- [Ca2+]i dients. Current evidence indicates that elevation of in Table Effects of fura-2 [Ca2+]i I. of BM 3.3 CTLs on of loading efficiency of the CTL lysis is caused by perturbation of the TcR or the associated EL4.BU after 180 targets min T3 complex by binding of lectin, anti-TcR antibody or antigen Effector Treatment (Schmitt-Verhulst et % 5'Cr al., 1987). If released at binding of the specific effector: TcR to the target BM 3.3 ratio of cell (fura-2/AM) leads to channel opening in target remote regions of the cell mem- (#M) 1.8:1 0.45:1 brane, 0.11:1 there must be an intermediate event between receptor bind- ing and channel opening. None Similar 93.4 conclusions were 68.3 reached by 28.0 Kuno et al. 11.0 (1986) who 93.5 detected 66.0 opening of calcium 26.0 channels 5.5 when mitogens 90.4 were added 64.3 external to the 26.0 patch pipette. They 1.1 90.2 argued that the 66.5 gigaohm seal of 28.0 the patch pipette would prevent 0.11 89.5 the entry 56.5 of mitogen 23.0 into the patch area and therefore the mitogen had to act through an interme>diate metabolic event to open calcium channels. It is unlikely that a Table diffusible intermediate H. Kinetics of which opens specific 51Cr release from the calcium EL4.BU target by the BM 3.3 CTL clone channels will cause the formation of a gradient in the [Ca2+]i CTL. Other factors must operate to allow preferential calcium Time Target % specific 51Cr released at effector:target elevation in the region of the CTL remote to the target. (min) An ex- cells ratio of planation might be that one global signal causes eleva- [Ca2+]i 10:1 2:1 0.4:1 0.08:1 tion while another locally lowers through calcium [Ca2+], channel inactivation, 20 calcium EL4.BU sequestration or 7.5 1.5 0.0 pumping to the ex- 1.6 terior. 40 Evidence EL4.BU 24.0 already exists for 9.0 3.4 separable but interacting 1.2 60 EL4.BU 27.0 pathways for CTL 8.5 4.0 activation. Expression of IL-2 2.6 receptors and EL4.BU 72.0 interferon 45.0 19.0 secretion are obtained 7.0 using ionophore to raise EL4.BU 83.0 60.0 28.0 and 11.0 [Ca2+]1 PMA to activate kinase C (Nishizuka, 1984) but not 240 EL4.BU 100.0 88.0 45.0 16.0 either alone by et agent (Truneh al., 1985; Albert et al., 1985). RDM4 12.0 7.0 1.7 Furthermore 4.0 of the TcR perturbations or some lec- by antibody 2230 Imaging Ca2' in cytolytic T ceHls and targets Evaluation in cells been shown to increase the pumping activity of Ca2+-ATPases offura-2 compartmentation and of was determined (Lagast et al., 1984) and the activity of inositol 1,4,5-trisphos- The of quality loading degree dye compartmentation by of the release of from intracellular acridine phatases (Connolly et al., 1986). Thus might be locally comparison orange organelles (Arslan [Ca2+]i con- et lactate and fura-2 at threshold al., 1985), dehydrogenase (LDH) digitonin decreased through protein kinase C activity to the for- leading Triton X-100. BM 3.3 cells were in- centrations to the total releasable 1% by mation of a [Ca gradient. cubated with 10 acridine or 1 fura-2/AM and adhered to i]i orange AM AM The possibility exists that yet other signals may be involved Petri dishes. Cells were then washed three in- 5-min polylysine-coated through cubations in a solution of 125 mM 10 mM 1 mM and 1.1 in CTL activation and polarization. Activation via the T cell KCI, NaCl, MgCl2 mM 7.2 Calcium was low to the in- EGTA pH (EGTA medium). kept preserve receptor may involve a number of surface proteins, associated such as the mitochondria which suffer of intracellular tegrity organelles might or not with the TcR which may contribute to binding of targets in the of Ca2+. cells were a ex- 5-min presence high Subsequently, lysed by or transmembrane signalling. Thus there exists the possibility to fresh EGTA medium 20 LDH was measured posure containing 1M digitonin. an of the to a mixture of multiple signals, some global and others local, rise to giving by adding aliquot (0.25 ml) digitonin lysate containing 1 mM 10 mM sodium lactate in solution and NAD and (2.5 the CTL response. ml) lysis measuring increase at 340-nm excitation and 440-nm emis- the rate of NADH fluorescence The approach presented here permits the observation of spatial- was measured in a second the sion. Acridine orange aliquot by scanning ly distributed activation events as well as the direct temporal and at 480-nm excitation. The material 500-600-nm emission intensity remaining spatial relationship between an activation event in the effector Triton in EGTA medium and on the dish was then extracted with 1% X-100 the and the lethal hit delivery in the target cell. These results are assays repeated. in individual cells complementary to biochemical of Measurements analysis signalling pathways of [Ca2i] used to adhere cells were a 1 which show that TcR/CD3 activation leads to the phosphoryla- Polylysine-coated coverslips prepared using mg/mil mol. wt -500 in 1 M KCI. Clean solution poly-L-lysine (from Sigma, 000) tion of CD3 proteins (Samelson et and that activation al., 1986) were coated with a thin film of solution and then 18-mm coverslips polylysine for killing or of interferon occur under induction gamma synthesis two beakers of distilled water and dried. Prior to rinsed immediately through conditions where different CD3 proteins are phosphorylated cell the were washed with HBSS/3% FCS. adding suspensions coverslips were attached a thin film of silicone to a (Boyer et al., 1987). An extension of the studies presented here slotted, Coverslips by grease rectangular insert which fitted into a holder on the of a stainless-steel to various systems of induction of CTL water-jacketed stage killing through lectins, IM-35 was at Zeiss constant, microscope. Temperature kept 37°C, by circulating anti-TcR/CD3 antibodies or phorbol esters will test the hypothesis water the insert holder and coils the Nikon 40x /1.3NA through tubing surrounding brought the of various CTL forward here. Additionally, analysis 50 of cell was added to the chamber lens. objective Approximately Al suspension clones different efficiencies for the same the After the cells had settled and adhered exhibiting killing target containing polylysine-coated coverslip. unattached cells were washed off with HBSS-3% FCS. After the cell will provide information as to which activation step (CTL collecting measurements a second cell was then added. baseline type hit is the of or lethal delivery) limiting process killing. Fluorescence ratio 32 hues to the range images using pseudocolor represent ratios were obtained as described et of fluorescence al., 1986). (Poenie previously have been normalized to that of a thin film of solu- All fluorescence ratio images 120 mM 10 mM 20 mM 7.5 and tion KCI, Hepes pH containing K2H2EDTA, Materials and methods The 10 mM fura-2 salt Probes, OR). (Molecular City, pentapotassium Oregon to account for the effect of and cells: and culture conditions ratios and have been Effector target origin viscosity Rmin Rmax adjusted and in (M.Poenie R.Y.Tsien, BM 3.3 is an alloreactive H-2Kb T cell clone derived from CBA/J mice preparation). specific and values The were (Hua et al., This line was maintained as described Hua et al. (1986) intensity 1986). by images analyzed by taking average [Ca2+]i each cell in the field. The mean within the area of a over spot placed intensity stimulation with 2500 rad irradiated cells from H-2b mice and by weekly spleen in the various formats in were then used and graph display presented from the EL4.C16 cell. The tumor cell [Ca2+]i IL-2 containing supematant thymoma the text. as were the H-2b EL4.BU and the H-2k lines used targets lymphoma thymoma line RDM-4. Cells were cultured in RPMI 1640 8% fetal calf serum containing and as described et Acknowledgements (FCS), supplemented (Hua al., 1986). for his her sabbatical 5ICr release thanks Dr Weissman Measure of cytotoxic activity by A.-M.S.-V. hospitality during Irving Henkart for his of in his We also thank Pierre leave classical 51Cr release cells cells/ml RPMI-5% FCS) gift cytolytic In the assay, target (107 laboratory. Henkart for their discussions. and Pierre Henkart and were labelled with 0.1 mCi Na2 1CrO4 for 1 h at 370C followed 3 washes helpful by granules Maryanna from NIH the Searle Scholars This work was cells cells (GM31004), FCS. of 51Cr-loaded well) in RPMI-5% Triplicate samples (104 per supported by grants of Califor- and the Cancer Research numbers of effector cells in 200 of RPMI-5% Committee, were then incubated with various University yd Program, Coordinating was undertaken the tenure of an nia. The work in this FCS at 37°C in microtitre after a 2-min cen- reported during V-shaped plates (Greiner, FRG) paper -Eleanore Roosevelt International award- American Cancer 200 At the time in Fellowship of the at Society trifugation microplate g. appropriate points (given Union Cancer to ed the International were then harvested and counted on a A.-M.S.-V. by Against the tables) supernatants (0.1 ml) gamma 51Cr release was determined as follows: The % counter. specific [experimental - release (1 N - medium References medium HCI) c.p.m.] c.p.m. c.p.m./maximum c.p.m. of the BM 3.3 effector cells also Hua et al., 1986) x 100. The specificity (see and Schmitt-Verhulst,A.-M. (1985) Hua,C., Truneh,A., Pierres,M. Albert,F., of 5'Cr release from the H-2k RDM4 tumor the absence target is indicated by 3649-3655. J. Immunol., 134, of BM 3.3 cells was not altered the cells. The effectiveness by target lysis by J. 8746-8759. Biol. and Chem., 260, (1985) Albert,P.R. Tashjian,A.H.,Jr here Table The kinetics of as fura-2 used lysis levels of loading (see I). target 107-109. and Nature, 314, Lanier,L.L. (1985) Allison,J.P. release is also for (Table II). 51Cr demonstrated by given comparison and J. 2293-2300. 129, Bloch,D. (1982) Immunl., Allison,J.P., Mclntyre,B.W. Fura-2 loading of cells FEBS 13-18. and 192, Lett., Almers,W. Neher,E. (1985) and J. Biol. Pozzan,T. (1985) Cells and were loaded with fura-2 by incubation with 0.5-1 Beltrame,M., Tsien,R.Y. (effectors targets) Arslan,P., DiVirgilio,F., 2719 - 2727. ester for 30 min at in RPMI-1640 fura-2 Chem., 260, acetoxymethyl (fura-2/AM) 37°C ,OM and Schmitt- at a of 106 cells/ml. cells were Buferne,M., Hua,C. 2% serum Subsequently, Guimezanes,A., containing density Boyer,C., Langlet,C., Ann. 65-82. x 2% serum and at room Before Immunol., 137, diluted 5 in RPMI temperature. Verhulst,A.M. (1987) containing kept FEBS 55-60. and 107, and at - 3 X I07 cells/ml Lett, use loaded cells were Daw,R.A. Luzio,J.P. (1979) centrifuged pellets resuspended Campbell,A.K., 1110-1112. and 37, Balanced Salt Solution 1 mM Ca2+ and 3% FCS. in Hank's (HBSS) containing Luzio,J.P. (1981) Experienta, Campbell,A.K. and Biochem. J., 194, Luzio,J.P. which with time and is more rapid (1981) To avoid dye compartmentation, progresses Daw,R.A., Hallet,M.B. Campbell,A.K., 551-560. small cells) were loaded and used quickly. at higher temperatures aliquots (106 951-958. and Cell, 46, (1986) showed were discarded and a new batch If cells dye compartmentation they Majerus,P.W. Connolly,T.M., Lawing,W.J.,Jr and Nature, 307, Cahalan,M.D. (1984) CTL clones incubated with 1 fura-2/AM attained intracellular con- zM Chandy,S., Gupta,S. prepared. Decoursey,T.E.., of 300-350 fura-2. with to 10 iM extemal fura-2/AM 465-468. centrations Loading up zM 691-693. and Nature, 310, Pozzan,T. effect on the with which BM 3.3 cells killed EL4 targets Lew,D.P. 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(1986) Im- J. 377-382. munol., 136, Zagury,D. (1982) Adv. Exp. Med. Biol., 146, 149-165. Received on February 20, 1987; revised on May 19, 1987 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets.

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Copyright © European Molecular Biology Organization 1987
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10.1002/j.1460-2075.1987.tb02494.x
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Abstract

The EMBO Journal vol.6 no.8 pp.2223-2232, 1987 Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets Martin Poenie" 3, Roger Y.Tsien' and Anne-Marie accumulation of vesicular components in the region adjacent to the target cell (Yannelli et al., 1986). The delivery of the lethal Schmitt-Verhulst2 hit by CTLs may involve secretion of prestored granules or of 'Department of Physiology and University of California, Anatomy, Golgi products directed at the target cell (Henkart and Henkart, Berkeley, CA 94720, USA and 2Centre d'lmmunologie INSERM-CNRS de 1982; Zagury, 1982). Evidence is accumulating in support of Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France the view that the lethal hit involves the release of a calcium- 3Present address: Department of Zoology, University of Texas, Austin, TX dependent pore-forming protein called cytolysin (Henkart et al., USA 78712, 1984) or perforin (Podack and Konisberg, 1984; Podack et al., by B.Jordan Communicated 1985; Masson and Tschopp, 1985). Changes in cytosolic free calcium ([Ca2 ]1) have been con- It is important to distinguish the roles of Ca2+ in CTL activa- tinuously imaged during the interaction of the H-2Kb specific tion, delivery of the lethal hit and killing of the target cell. Ad- cytotoxic T cell lymphocyte (CTL) BM 3.3, with either the dition of appropriate targets, antibody to the TcR, or certain H-2K" EL4.BU or the H-2Kk RDM4 cell lines. Activation of lectins can all cause transients in various CTL prepara- [Ca2+]i raises [Ca2+] to several hundred the CTLs by EL4.BU tions or cell lines. In some T cells these stimuli have been shown CTL. Frequently is preferentially nanomolar in the to induce formation of the PIP2 breakdown products, IP3 and [Ca2+]i elevated in the region of the CTL furthest from the site of diacyglycerol, and release of calcium from intracellular stores target contact. These responses require external Ca2+ sug- (Weiss et al., 1986). However, in murine T lymphocytes or clon- gesting that they are generated by the plasma membrane and ed cell lines used by us and others, influx of extracellular calcium not internal stores. Inappropriate targets such as RDM4 evoke component of transients though some is the major [Ca2+]i no changes in Activation of the BM 3.3 CTL is [Ca2W]1. release of intracellular calcium can be detected (Tsien et al., 1982; followed by increases of to several micromolar or our observations). Although classical voltage- [Ca2+]i and unpublished higher in the EL4.BU targets. This massive increase can be calcium channels have not been detected in T lym- dependent by direct application of cytolytic granules isolated mimicked et al., 1984; Matteson and Deutsch, 1984; phocytes (DeCoursey rat natural killer cells. The increase in plasma mem- from al., 1984) a mitogen-regulated calcium channel has Fukushima et is ion-specific since external Mn2+ can brane permeability in human helper T cells (Kuno et al., been recently identified readily enter cells that have been 'hit', as evidenced also target serve to open these channels is not known 1986). How mitogens the selective quenching of fura-2 in those targets. by rapid is probably an intermediate messenger linking recep- but there The flood of Ca2+ into the target cell is followed by a leakage to channel opening (ibid). tor binding fura-2. Since both processes continue after the of the trapped We have employed fura-2 (Grynkiewicz et al., 1985) in con- has they provide a useful assay for the lethal CTL disengaged, with digital image processing (see Poenie et al., 1986) junction this technique can be used to follow com- hit. Furthermore, in intracellular free calcium in both to study changes ([Ca2+]i) of CTL activation and lethal hit delivery, which plete cycles effector and target cells throughout the killing process. We find can be as rapid as 6 min per cycle. under some circumstances and characteristic changes in [Ca2]+ occur in that distinctive T cells/cytosolic free calcium/digital image Key words: cytolytic killer cell and target cells. Killer cells which contact a both gradients/T cell activation processing/calcium show a moderate rise in ]+ close to the susceptible target [Ca2 of contact. In elongated CTLs the initial rise in cor- time [Ca2+]i the change in shape as the CTL rounds up next to relates with Introduction an gradient forms when the target. Frequently, apparent [Ca2+]i makes initial contact with the target such that the CTL T lymphocyte (CTL) mediated cytolysis is a process [Ca2+]i Cytotoxic in the of the CTL most remote from the site of of target cell antigens by the T cell is high region involving specific recognition cell -cell contact. These gradients disappear immediately u on The latter is a heterodimeric protein (Allison and receptor (TcR). removal of extracellular calcium. The period of elevated [Ca et Haskins et al., 1983) express- Lanier, 1985; Allison al., 1982; ]i is variable lasting from a few seconds to several in both its a and chains, associated in the CTL clone specific sequences ing the rise in the effector, following with the CD3 (Meuer et al., 1983; minutes. Typically, on the T cell surface complex [Ca2+]i to levels sufficient to saturate In addition to TcR-antigen interaction, abruptly jumps Samelson et al., 1985). target [Ca2+]i in the killer cell normally returns to fura-2. Whereas cell formation involves other T cell sur- conjugate CTL-target [Ca2+]i remains in the target suggesting that levels, high et This of conjugate for- resting face molecules (Shaw al., 1986). step [Ca2+]i contribute to of the target cell. calcium toxicity may cytolysis is on external magnesium but not mation, which dependent rises in the the fura-2 fluorescence signal After calcium target, and 1977), is followed by calcium- calcium (Golstein Smith, that the cell has become permeable to small declines indicating of the lethal hit and CTL independent lysis delivery dependent is now out of the cell. and that leaking molecules trapped dye cell CTL recognition of target cells of the (Martz, 1977). target et al., 1984) of granules (Henkart Application purified cytolytic an extensive cytoplasmic rearrangement in is accompanied by a similar elevation of calcium causes rapid to fura-2-loaded targets reorientation of the microtubule organiz- CTL which includes the s of contact with the These ex- within 6 granules. in the targets et 1983, 1985) and and Golgi (Kupfer al., ing center (MTOC) IRL Press Limited, Oxford, England M.Poenie, R.Y.Tsien and A.-M.Schmitt-Verhulst 2 0 CY o0 Time (sec) Fig. 1. An of example the use of fluorescence ratio to imaging measure changes in the stimulation of fura-2 during loaded BM 3.3 [Ca2+]i effector cells by EL-4 targets. Here fluorescence ratios are coordinated with pseudocolor hues and calibrated in terms of micromolar Panel Ca2 . shows the for a field of fura-2-loaded resting BM 3.3 cells [Ca2+]i before .0- addition of targets. Panel B shows the BM 3.3 cells after just addition of RDM4 unloaded EL-4 cells. The unloaded EL-4 cells are invisible in the fluor- CU escence image. Panel C shows the BM 3.3 cells as to they begin respond 4q to the added target cells. The of CTLs percentage indicated responding, by their in change hue, continues to increase D (panels and until most E) of the cells have responded. Note that within of many the CTLs [Ca2+]] is elevated unevenly indicating the formation of calcium gradients. Panel shows the of population unloaded the targets overlaying fura-2-loaded CTLs. 0 -0 0 350 periments 700 therefore support the proposal that cytolytic granules and CTL-mediated lysis both cause rapid to the Time damage target (sec) cell plasma membrane. The observation of multiple sequential hits by a single CTL shows that each hit is associated with a The data shown in Fig. is here imaging Figure 1 displayed graphically. separate activation event and transient. Furthermore, the A circle was over each cell in the [Ca2+]i placed 10 in field, typically pixels sequential hit of cells in the vicinity of the CTL diameter and the stresses the short +]i and intensity values average [Ca2 calculated for the area are defined the circle. In range of action the was of the lytic by determined for molecules and is (A) average [Ca2+]i compatible with the each individual cell. Each cell is then a solid block represented by square notion of polarity in the delivery of the lethal hit. to its positioned (y axis) at a according average [Ca2+]i given time (x axis). to three cells with the same can be Up average +j displayed at a [Ca2 given time blocks period If more than three Results by placing side-by-side. cells had the same will not they be Thus in average [Ca2 +1i distinguishable. some cases in the Changes to and not to specific effectors graph all the cells used to obtain [Ca2+]i targets may appear represent the plot when the is especially uniform and When BM population fairly values 3.3 effector cells are average [Ca2 +]i stimulated with EL-4 targets they are clustered close Thus the distribution of the together. [Ca2 +]i population undergo a rise in from nM 130 to an can average of be ascertained for a (right y axis) time vertical [Ca2+]i given by distribution of 500 nM. This can be seen in a individual cells Figure 1, series of fura-2 fluor- squares. Although calcium undergo significant transients the mean for ]j the whole escence [Ca2 population (solid line) shows a small ratio in which only images [Ca2+]- is as various represented When such change. as the RDM-4 cell nonstimulatory targets line are used hues ranging from blue (low calcium) through green, yellow, in a (B) similar to that above no experiment in BM 3.3 change [Ca2+], is orange, red to purple at maximal calcium detectable either in values. This individual cells shown experi- the scatter or by plot in the ment population began with a average (solid monolayer of fura-2 loaded BM line). 3.3 cells, adhered by to a polylysine coverslip. Before addition of target the cells, field of effector cells appears low in marized uniformly the by plotting average of each individual [Ca2+]i cell [Ca2+]i (panel A). When unloaded EL-4 targets in are added (panel B) the field there (Figure or of 2A) the entire population. Both sorts is no immediate effect on in the of data effectors. However, indicate that the effector population undergoes [Ca2+]i a moderate within a few minutes begins to rise in the effector cells increase [Ca2+]i stimulation [Ca2+]i upon with targets. (panel and C) by 10 min D (panels and E) most of This the effector reaction of the BM 3.3 to targets is specific for targets cells have experienced a calcium transient. An with interesting aspect H-2 antigens. Inappropriate targets such as cells appropriate of the rise in the effectors is that it of the H-2 [Ca2+]i often appears as a thymoma, RDM4, are not killed by BM 3.3 effec- gradient across the effector cell (see below). Finally, tor cells panel F Table and (see II) do not evoke an increase in shows the [Ca2+]i transmitted light image of the target cells, in large in the BM 3.3 ex- cells (Figure 2B). cess, layered over the effectors. The data can To be sum- investigate the effect of CTL cytolytic activity on [Ca2+], target 2224 Imaging Ca2+ in cytolytic T cells and targets 1 .10 t UL - - EL41IFura-2 0 900 Time (sec) 2.0 Fig. 3. By loading targets with fura-2 and using unloaded BM 3.3 effector cells the response of the targets can be isolated. This therefore is the reciprocal experiment to that of Figure 1. Panel A shows the resting for a field of EL-4 targets before addition of the BM 3.3 cells. In [Ca2+], panel B the unloaded BM 3.3 cells are added but no response is yet 1 .0o detected. Panel C shows the earliest sign of EL-4 response to the added I Fura-2 RDM4 Cy BM 3.3 cells. Target cells show a rapid and usually permanent rise in to levels sufficient to saturate fura-2. Panel D shows a later time u [Ca2+], point where the respose of the EL-4 cells is more fully developed. The average fluorescence of the targets diminishes rapidly after their has [Ca2+]i risen. This results from dye loss, presumably from leakage through pores formed by cytolysin. Thus in panel D it is evident that some of the targets i,i- that turned purple in panel C have already lost sufficient dye to fade from view. 0 300 600 Time (sec) unloaded BM 3.3 cells were added to a monolayer of [Ca2+]i, fura-2-loaded EL-4 targets (Figure 3), the converse of the ex- 4. representation of the pictorial data from Figure 3 shows Fig. Graphical for individual rises much than that of the CTL in Figure 1. Fluorescence ratio images revealed that targets higher periment that [Ca2+]i after a longer lag period than that observed for CTL activation. In usually are distinctively different from those changes in target [Ca2+]i virtually all of the fura-2-labelled EL-4 targets were this experiment (A) with CTL activation. rises abruptly to very associated for the of observation so that average [Ca2+]i 'hit' within the period [Ca2+], in individual targets after a variable delay period. line) as a whole also rises to high values. When high levels population (solid targets such as RDM-4 are used (B) in the same assay again inappropriate is usually permanent, though targets Elevation of target [Ca2+]i mean is detectable (C individual cell or the population no change in [Ca2+]i oscillate up and down for a time before ultimately occasionally and D). high. Targets reach much higher remaining permanently [Ca2+]i than do CTLs, and this is generally followed by a loss levels not measures a much earlier event an indication that some degree of plasma 51Cr. Imaging only of fluorescence signal, [Ca2+]i from cells, it also allows correlation bet- has taken place. This typical pattern of than 51Cr release target membrane damage in individual CTL and in its associated in the cell population can be readily ween the changes an change target [Ca2+]i fluorescence ratio imaging of cell data is graphed. Figure 4A shows a scatter This is achieved by observed when the target. where both effectors and targets are loaded with EL-4 population exposed to unloaded BM plot of a fura-2-loaded populations conditions it is important to retain the iden- BM 3.3 effector cells were added at the arrow. fura-2. Under these 3.3 cells. The in the field. By adding the effector and which represents the time for ef- of both cell types After an initial delay period, tity it is to and record the positions with the individual targets possible map cells to settle and interact targets, targets sequentially fector before the second cell type. Provid- values. The solid line of the first cell type adding to rise to very high begin [Ca2+]j are not too great, individual conjugate pairs for all the cells in the field. If the ed that cell densities represents the mean [Ca2+]i and the of events in both cell types is using fura-2-loaded RDM4 targets can be followed sequence same experiment repeated the of cell type is determin- BM 3.3 cells, in the RDM4 reconstructed. Although assignment (4B), which do not activate [Ca2W]i the cells' order of appearance, changes in low and unaffected the ofBM 3.3 cells. ed by cells remains by presence [Ca2+]i cell are so different effector and target conjugate pairs EL4.BU cells undergo a high rise in responding Thus most target [Ca2+]j to an alternative means of iden- characteristic as after of target and effector cells and provide between 8 and 16 min mixing tification. which contrasts with the kinetics of release 4, A and B), (Figure an of measurements obtained from 5A shows example 'Cr from the EM4.BU target cells: at an effector to target Figure of of numbers of effector mixed approximately equal of 10:1, 20-30% Cr was released after 40 and 60 a population cell ratio both of which were labeled with fura-2. The 2 h were to release 45 % whereas at a ratio of 2:1, required and target cells, min, 2225 M.Poenie, R.Y.Tsien and A.-M.Schmitt-Verhulst 2-0 1-4 were obtained By contrast, an excess of Figures using whichever cell constituted the unloaded partner. 0 0 0 0 A of in 0 an individual plot within this 0 conjugate pair [Ca2+]i in is shown Here population 5B. the CTL is seen to Figure a moderate elevation of undergo then the first, [Ca2+]i target cell £ to elevate < min 1 after peaks in the begins [Ca2+]2 [Ca2+]i effector cell. 1o-0 EL4 4Fura-2 0 It should be mentioned that different CTL %U effector cells with similar cell in target specificity vary greatly killing efficiency. Various parameters including antigen-receptor and (TcR) avidity of other molecules density, participation accessory on the T cells, Ifi . 8a* oL and intrinsic contribute 'killing machinery' to these dif- may ferences. The BM 3.3 effector chosen for this behaved as study an CTL efficient, various criteria 0.0 yet specific by (Hua et al., When a less active CTL 0 effector of similar 1986). target specific- 200 400 was used Hua et the ity (KB5-C20 cells, al., 1986), same se- Time of CTL activation and quence target permeabilization was (sec) observed but less within a 20-min frequently observation period. Calcium and in CTLs gradients shape change Additional information is obtained from the actual of images -, y l, When responsive conjugate pairs. elongate CTLs contact a target round to the characteristic they typically up mor- conjugate This A phology. occurs in shape change conjunction with a cytoplasmic rearrangement which the places Golgi apparatus and 1 -0 - of the in the of the many organelles CTL to the region adjacent tZ & <e4 - cell. of target Images this of during early period [Ca2+]i l I0- tf! ' 1- *~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ interaction reveal an effector-target increase in CTL [Ca2+] :4:~~~~~~~~~~~ this and in during rounding-up period, cases ] is many [Ca2+ in the CTL highest cytosol most remote from the target. This is illustrated in Figure 6 which focuses on a single CTL as it contacts and to a responds target. Here, is low initially [Ca2+]i in the CTL prior to the contacting target (panel A). Upon con- 1 00 250 to rise tact, [Ca2+]i appears preferentially at the end furthest from the target After the initial (panel B). elevation the BM 3.3 Time (sec) cell begins to round C up (panels -F). Although are spatial [Ca +]i gradients common, other patterns Fig. 5. The entire sequence of changes observed in effectors of CTL and targets can activation are also seen. For example, in Figure 7 be observed when both targets and effectors are loaded with fura-2. By resting rounded BM 3.3 effector makes contact with a target sequentially adding effectors followed by targets, the positions of each cell (panel A). Though the contacting surface area appears to be type small, were determined and individual conjugate pairs followed. In panel A a the result is a large elevation scatter plot is given showing the throughout the effector sequential activation of [Ca2+]i cell BM 3.3 cells followed by the elevation of target The BM 3.3 cells are here (panel B). Subsequently, the CTL forms a [Ca2+]_ gradient [Ca2+]i. and represented by solid squares and the EL-4 cells by open circles. At the undergoes dramatic change in shape (panels C-E). Follow- arrow, fura-2-loaded EL-4 cells were added. As these cells settle and make the formation of ing the typical conjugate configuration, contact with the BM 3.3 cells the initial elevation characteristic of [Ca2+]i [Ca2+1i the BM 3.3 rises in the activation is detected. target cell indicating delivery Subsequently, a second elevation of of the lethal hit (panel calcium is detected this time characteristic of the reaction of the and the EL-4 target F; following section). cells. Solid squares represent the BM 3.3 cells, open circles represent EL-4 The correlation of the calcium gradient with the direction of targets. In panel B for one taken from effector-target pair, the [Ca2+]i attack is displayed spectacularly when panel A data, is shown. in the a single CTL BM 3.3 cell (solid and [labelled line) EL-4 [Ca2+]i cell (broken line) starts at typical resting levels. As killer time progresses a (K)] attacks several cells in vicinity. the In Figure 8 a single transient is detected in the BM 3.3 cell which is followed shortly [Ca2+]i BM 3.3 cell attacks and kills three neighboring EL-4 targets. In thereafter by a much larger calcium increase in the target cell. This panel A the effector cell is touching the first particular EL-4 target conjugate pair was not in cell contact with any other cells and the graph therefore [labelled 1st target represents the interaction of a (Tl)]. Elevation of in single CTL and the killer cell associated [Ca2+]i target. is not instantaneous but begins in panel B. The red pseudocolor in the upper right quadrant of the CTL gives evidence of a calcium solid squares represent CTL while open circles gradient. As seen earlier, this denote gradient is highest in the [Ca2+]i typically targets. The scatter plot shows that CTL region elevation of the CTL remote to the target. [Ca remains elevated +]i [Ca2+]i precedes a much larger rise in the targets, though a for 1 sizeable frac- nearly min before any change is detected in the target (panels tion of each population still remains at rest at the C and In 400-s point. E). panel F, 56 s after the initial rise in CTL [Ca2+]i, The percentage of participating cells was lower here the target abruptly than in jumps to high indicating that the [Ca2+], Figures 1-4 probably because we used lower cell densities lethal hit has occurred. Subsequently, the target fades from view. overall and effector:target ratios near unity to facilitate The killer continues the attack on identifica- a second target (T2) and again tion of each cell type and monitoring of isolated effector-target a [Ca2 ]1 to the gradient appears opposite target. Subsequently cell pairs. At such reduced cell densities, some effectors and in the increases second [Ca2+]j This cycle target. occurs a third targets fail to contact each other and thus do not time show a response. in K and L. panels 2226 Imaging Ca2' in cytolytic T cells and targets Fig. 6. Fluorescence ratio images taken while effectors and targets interact of gradients and morphological changes reveals a striking correlation [Ca2+]i in the effector as it contacts the target cell. Here a particularly elongate BM 3.3 effector is approaching an EL-4 target (panel A). As the front of shown rises dramatically, especially at the the effector contacts the target [Ca2+]i back end (panel B). Subsequently, the back part of the CTL moves up toward the target giving rise to the typical conjugate morphology. the experiment from which this sequence was derived ended Unfortunately a rise in the target was recorded. before multiple hits. 8. A single BM 3.3 effector cell is capable of delivering Fig. activation event as Each hit appears to be associated with a separate rise and directional In this a BM 3.3 a gradient. figure indicated by [Ca2+]i A shows the BM 3.3 effector attacks three target cells in sequence. Panel EL-4 cell (TI). Calcium is cell (labelled K) juxtaposed to the first target activation has not yet taken low in both cell types indicating that CTL increase in the CTL. Panel B shows the first detectable place. [Ca2+]i continues to increase and a then becomes gradient (panel C) [Ca2+]i rises in the cell thereafter, [Ca2+]i target (panel apparent (panel D). Shortly to fade F-H). At the and subsequently the first target begins (panels E) into with the second cell same time the effector is moving contact target Fig. 7. There is sufficient variability in the nature of CTL activation that it rises a second time with the shifted and cor- gradient (T2). [Ca2+]i is impossible to illustrate with one sequence the typical or stereotyped CTL to the position of the second target (panel I). Subsequently, responding response to a target. In this sequence the BM 3.3 effector begins in a rises in the second J) which then also loses its (panel dye [Ca2+]i target rounded-up state with an EL-4 target nearby (panel A). The small region of of formation and hit appears to signal (panel K). A third cycle gradient effector-target contact (panel B) is sufficient to trigger a relatively large, K and L. here it is to know if take in panels However, impossible place long-lasting and initially uniform rise in effector [Ca+],. Subsequently, the of the effector cell or an artifact due to the very the gradient is a function effector undergoes a radical change in shape (panels C and D). Eventually of the adjacent target (T2). at the rear of the effector does form high [Ca2+] a (panel E). gradient of high [Ca2+]i rises that this the EL-4 target enormously indicating cell Finally, [Ca2+]i has been hit. with In some cases lose contact not targets. may actually previous rise in the lethal hit? the after the Is the targets CTL the disengages target [Ca2+]i however, completely not Thus events after the has risen that after calcium has risen in the target the CTL shown). Figure 8 shows target (data [Ca2+]i must be events. under crowded it lytic conditions, rise in target CTL-independent moves to another target though, [Ca2+]i 2227 and M.Poenie, R.Y.Tsien A.-M.Schmitt-Verhulst in Fig. 9. The existence of a the membranes of unique pore plasma targets 11. The Fig. apparent [Ca2+1] gradient observed during CTL activation after the hit is illustrated the of fura-2 in those could conceivably arise as an artifact of by rapid quenching fura-2 compartmentation. For targets Panel A of by manganese. shows a field fura-2-loaded of example, under conditions of targets many poor dye loading unhydrolyzed ester might which have been hit as indicated their color. Within 5 s of accumulate and remain in hydrophobic by purple compartments. The fluorescence 1 mM addition of most of the have signal from this compartment would manganese, purple-colored targets not change when the cytosol and 25 s disappeared (panel B) are experienced a transient and if localized completely quenched by (panel C). [Ca2+]i at one end of the cell might show that the rest of the give rise to an apparent gradient. In order Subsequent images to determine whether population quenches relatively apparent and 102 s later are still fluorescent calcium gradients be due to slowly brightly might compartmentalized dye, 20 !ZM (panel D). digitonin in calcium-free medium was applied to a CTL exhibiting a gradient during activation. The of this concentration ability of digitonin to selectively permeabilize the membrane of BM plasma 3.3 cells while leaving intracellular membranes intact was determined by comparison of lactate dehydrogenase release to that of acridine orange (see Materials and At 20 methods). tiM digitonin, adhered cells can take several minutes for complete membrane plasma permeabilization to take place. This sequence begins with a CTL undergoing activation (panels A-C). At the peak of the gradient the cell was washed with calcium-free lysis solution (without digitonin) and the gradient immediately disappears (panel D). Subsequently, the cells were washed with 20 MM digitonin-containing lysis solution (panel E). Lysis takes place over 1 min 20 s with the image fading below the detection limits under the conditions of gain (750 V) and background subtraction employed (panels F and The G). rapid loss of fura-2 here indicates that the dye was mainly in the cytosol and free to diffuse away. However, at higher gain (1000 V) unlysed dye-containing granules are still evident and their relative fluorescence excitation efficiencies at 350 nM (panel H) and 385 nM (panel I) indicates that little of this compart- mentalized fura-2 is bound to calcium. These pictures show that only a small fraction of the intact cell fluorescence can be attributed to the compartmentalized dye in these granules. Furthermore, the granules were not in a suitable region of the cell to account for the gradient seen in panel C. ching of fura-2 fluorescence in targets that have been hit, when external Mn2+ (Hallam and is Fig. 10. The cytolysis of EL-4 cells rat NK Rink, 1985) applied (Figure by purified cell derived 9). cytolytic granules shows remarkable to the Manganese ions quench similarity action of BM 3.3 cells. fura-2 fluorescence within seconds in The rat NK cytolytic granules were obtained as a from gift Pierre Henkart targets that have been hit, presumably entering the cell by the at the NIH. Panel A shows a layer of fura-2-loaded EL-4 cells before same pathway as Ca2+ does, whereas quenching occurs slowly addition of granules. Three microliters of suspension of cytolytic granules at over several minutes in intact a concentration targets or CTLs. equaling the content obtained from 106 cells suspended in 1 ml PBS was then = The loss of injected over the EL-4 cells (final fura-2 from EL-4-loaded targets is volume 50 Ml). another indica- addition enters the Upon of cytolytic granules EL4.BU cells rapidly tion that pores have [Ca2+]i been made in the plasma membrane of the (panels B and C). Subsequently, fura-2 leaks out of the EL4.BU cells, an target cell. Not only can a foreign cation such as manganese rapid- effect that mimicks the CTL-mediated permeability changes (panel D). ly enter the cell but - also the fura-2 penta anion of 750 mol. wt rapidly leaves the cell. Most of the targets lose their fluor- CTLs cause an increase in target permeability to external ions escence within -60 s of the calcium rise with a half-time of The rise in target calcium appears to be an influx of external 15-20 s. Clearly, the rise in target precedes the loss [Ca2+]i calcium into the target cells. This is evidenced by the rapid quen- of fura-2 though the two events are closely correlated. 2228 Ca2I in cytolytic T cells and targets Imaging with EGTA medium, in the absence of digitonin (panel D). As Comparison with cytolytic granules can be seen, the apparent gradient immediately disap- [Ca2+]i Purified cytolytic granules from rat NK cells have an effect on pears. The rapid collapse of the elevation suggests that [Ca2+]j target cells similar to that of CTLs. When cytolytic granules are it is sustained by Ca2+ influx across the plasma membrane, not added to fura-2-loaded EL-4 targets, [Ca2 ]i rises rapidly in the by release from internal stores, nor is it an artifact of dye seeing target and dye fluorescence is subsequently lost. This can be seen high Ca2' inside organelles. Next, the cells were washed with in Figure 10. Panel A shows fura-2-loaded EL-4 cells before ad- the same medium containing 20,uM digitonin (panel E). By 1 dition of cytolytic granules. In panel B the granules were added the loss of dye from the target and effector cells becomes min and within a few seconds the entire field shows an enormous obvious (panel F). By 1 min 20 s both cells have disappeared rise in (panels C and D). To obtain a better temporal [Ca2+]i from the field (panel G) at the gain settings used for panels A-F. correlation of the rise and the loss of fluorescence due [Ca2+]i By increasing the camera gain 2-fold to its maximum, three weak- to dye leakage a photometer was used to quantitate the fluor- ly fluorescent granules are detectable which are resistant to escence ratios and average intensity from a single CTL exposed digitonin lysis. The fluorescence of these granules persist for at to cytolytic granules. The results show that a rise in [Ca2+]i least 10 min. Their relative fluorescence at 350 nm (H) and precedes the drop in intensity. The target cell attains 385 nm (I) indicates low fluorescence ratios consistent with those 350nm/385nm excitation ratios of - 25 before the signal becomes observed in intact cells. The position of these granules in what so noisy (due to the loss of signal) as to be unreliable. These would be the middle of the cell as shown in panel C would rule ratios are indicative of fura-2 saturation (data not shown). them out as a cause of the apparent transverse calcium gradient. Are the calcium gradients real? Furthermore, their low fluorescence intensity makes them We were concerned that the apparent gradients of [Ca2W+i across relatively insignificant in the intact cell although such granules CTLs could have been artifacts due to compartmentation of can become conspicuous under conditions of more severe com- fura-2. For example, Almers and Neher (1985) have reported partmentation. Thus it is unlikely that all CTL gradients [Ca2+]i that mast cells loaded with fura-2/AM give smaller apparent can be explained as an artifact of dye compartmentation. transients than when cells are loaded with the fura-2 [Ca2+]i When dye compartmentation is more severe, local bright spots anion directly through a patch pipette. This apparently was due of fluorescence are seen in the cell which tend not to change their to accumulation of incompletely hydrolyzed ester into mast cell ratio when the rest of the cell experiences a change in calcium. secretory granules. Incompletely hydrolyzed ester gives low Thus when cells are intentionally loaded so as to produce com- 350 nm/385 nm excitation ratios and is relatively unresponsive partmentalized dye, cells may contain numerous and obvious to calcium. If CTL secretory granules were likewise to ac- granules (data not shown). cumulate significant fura-2/AM ester and to congregate near the plane of conjugation with the target, a genuinely uniform high in the CTL could be partially masked near the target. Discussion [Ca2+]i if fully hydrolyzed fura-2 were to accumulate in a Conversely, The CTL effector function is generally measured by release of high compartment remote from the target, a [Ca2+]i from target cells. The chroamte being bound to in- [Ca2+]i 51CrO42- would be simulated. Evaluation of fura-2 compartmen- gradient tracellular proteins is released only after extensive plasma mem- tation within cells was carried out as described in Materials and brane damage. Earlier increases in target permeability could be methods. CTLs showing calcium gradients were washed with measured by leakage of smaller molecules such as 86Rb or ATP, calcium-free, EGTA-containing medium followed by the same but the ratio of CTL-induced to spontaneous release was very with digitonin. Digitonin is known to selectively lyse medium small in these assays (Henney, 1973; Russell, 1983). Here, we plasma membrane while leaving intracellular membranes the have used cytosolically trapped fura-2 as both a non-perturbing intact when used at appropriate concentrations. indicator of and a marker for membrane integrity of the [Ca2+]i To determine the right concentration of digitonin, monolayers target cell. ofBM 3.3 cells on polylysine-coated Petri dishes were incubated In the effectors, fura-2 indicates increases in [Ca2+]i resulting 10 jiM acridine orange, a fluorescent dye which accumulates with from CTL activation through the binding of the relevant target intracellular organelles. After three washes in EGTA- in acidic cell H-2 antigens to the TcR/CD3 complex. After one activation medium cells were lysed in the EGTA-medium containing various event, the effector cells slowly return to resting unless [Ca2+]j, digitonin concentrations for 5 min. The amount of released LDH they are reactivated by additional target cells. orange were then measured fluorimetrically. Subse- and acridine Fura-2-loaded targets show within 5 min of effector addition, 1% Triton X-100 lysis solution was added to complete quently, large increases in followed by leakage of dye and corre- [Ca2+]i the lysis and the assays repeated. Using 20 digitonin in a AM sponding loss of signal from the cell. This striking difference lysis solution it is possible to release 100% of the calcium-free in response between the two populations of cells permitted the LDH and only 10-12% of the total acridine orange released total two cell types to be easily distinguished in mixed populations digitonin + Triton X-100. This digitonin concentration can by of cells where both types were loaded with fura-2. >95 % of the intracellular fura-2 in freshly loaded cells. release Is calcium the toxic agent? cells that have been allowed to compartmentalize their However, The high levels of calcium experienced by targets open the way can retain 25-30% of the intracellular fura-2 after digitonin dye for extensive cell damage (Wrogemann and Pena, 1976; Camp- (data not shown). lysis bell and Luzio, 1981). Sustained elevation of [Ca2+] is A visual analysis of CTLs containing calcium gradients such reportedly toxic for many cell types. Mitochondrial damage, ATP as in Figure 11 shows that the low fluorescence intensity and deletion, electrolyte imbalances and activation of esterases all central position of granules in the cell cannot account for the gra- follow from sustained elevation of [Ca2+]i. Thus the large eleva- dient. Here, the CTL develops a normal response to the EL4 caused by CTLs or purified cytolytic granules tion of Ca2+ target showing a typical calcium rise and gradient formation to irreversible cell injury. These aspects of cell damage could lead A-C). Immediately thereafter, the cells were washed (panels 2229 M.Poenie, R.Y.Tsien and A.-M.Schmitt-Verhulst are similar to those which produced by and be complement, sufficient to elevate similarities tins, are not may [Ca2+]i always between CTL and sufficient complement-mediated cell have to full lysis been activation. For sug- Clevers et al. give example, gested (Campbell etal., 1979, 1981; Campbell and showed that the Luzio, 1981). lectin wheat (1986) germ agglutinin nonmitogenic There are certain unique aspects of cell seen in causes elevation damage of CTL- and PI (WGA) [Ca +]i However, hydrolysis. mediated cytolysis not seen in a complement lysed cells such was as obtained when a proliferative non- response only low, the degradation of target cell DNA (Russell et dose of al., PMA was added in 1980). addition to mitogenic WGA. It is However, even this DNAase activity is to note that calcium-dependent the sug- levels obtained due interesting to WGA [Ca2+]i gesting that calcium is an important stimulation component of cell were as target as those lysis. obtained from the high mitogenic Unfortunately, it is difficult to evaluate the lectin relative importance Similar results have phytohemagglutinin been ob- (PHA). of Ca2± influx compared with generalized colloid tained osmotic monoclonal swell- to the T cell using Soluble antibody receptor. ing or other mechanisms of damage since to monoclonal prevent Ca2' antibodies can influx elicit calcium transients in T cells but we presently have to remove external Ca2+, but activation as external Ca measured 2+ interleukin secretion optimal or by pro- or Sr2+ is also required for the initial liferation pore formation so all such as obtained requires greater crosslinking using pathways for cell damage would be blocked (Henkart et or the al., addition of PMA. Sepharose-coupled as cur- antibody If, 1984). However, the fact that a CTL seems can disengage from the the effects from or rently likely, resulting antibody target target cell even before the fura-2 has leaked to the completely TcR/CD3 away are mediated antigen binding complex through suggests that the initial calcium rise of (pore inositol formation) coincides then the difference hydrolysis phospholipids, between with the lethal hit described in the soluble literature and (Martz, 1977). be Sepharose-coupled one of antibody may largely This has Patterns of from [Ca2+]i increase in CTL of effectors: clues to degree. elevation support comparisons acti- [Ca2+]i evoked vation anti-TcR ? with or without the by ad- antibody subsequent dition of A fascinating crosslinking observation from these antibody (in preparation). experiments is the Perhaps then, ap- the cell can evoke a pearance of a by analogy, target increase in transient calcium in rapid global gradient CTLs as many they a soluble that calcium channels while contact their target cells. messenger local- The common feature opens of these only gradients kinase C. The local activation is ly stimulating of that kinase appears highest on the side of protein [Ca2+]i the CTL furthest of from the interaction and target cell at might require about the same greater degree time that a major perturbation of the CTL cell membrane or cytoplasmic be slower rearrangement than the takes place. In other simply cell types it has for calcium been messenger responsible channels. hypothesized that opening This would calcium gradients, such as might form be consistent with the results shown in due to 7 chemoattractant where a small gradients or weak Figure electric fields, might contact area leads to a uniform elevation be of involved in cell but as the orientation or directed [Ca2+]i motility (Robinson, contact area 1985). Such becomes more a behavior increases, gradient evident. In this shows a considerable resemblance to the localized model, kinase C would be polarization and reorientation protein activity of CTLs in response to target cells. responsible for a sense of to the CTL. In Perhaps, by analogy, a calcium giving polarity gradient in the many CTL may also neurosecretory cells and cells of be part of, or PMA result from, the hemopoietic treatment directional signal lineage, leading to CTL power- with elevation polarization. fully synergizes to [Ca2+]i promote exocytosis even while to rises The mechanism tending dampen et of [Ca2+]j (Rink elevation appears al., [Ca2+]i to involve open- 1983; et ing of DiVirgilio Albert and calcium al., 1984; channels in the CTL Tashjian, 1985). PMA has since removal of extracellular Ca2+ causes immediate decline in and loss of the gra- [Ca2+]i dients. Current evidence indicates that elevation of in Table Effects of fura-2 [Ca2+]i I. of BM 3.3 CTLs on of loading efficiency of the CTL lysis is caused by perturbation of the TcR or the associated EL4.BU after 180 targets min T3 complex by binding of lectin, anti-TcR antibody or antigen Effector Treatment (Schmitt-Verhulst et % 5'Cr al., 1987). If released at binding of the specific effector: TcR to the target BM 3.3 ratio of cell (fura-2/AM) leads to channel opening in target remote regions of the cell mem- (#M) 1.8:1 0.45:1 brane, 0.11:1 there must be an intermediate event between receptor bind- ing and channel opening. None Similar 93.4 conclusions were 68.3 reached by 28.0 Kuno et al. 11.0 (1986) who 93.5 detected 66.0 opening of calcium 26.0 channels 5.5 when mitogens 90.4 were added 64.3 external to the 26.0 patch pipette. They 1.1 90.2 argued that the 66.5 gigaohm seal of 28.0 the patch pipette would prevent 0.11 89.5 the entry 56.5 of mitogen 23.0 into the patch area and therefore the mitogen had to act through an interme>diate metabolic event to open calcium channels. It is unlikely that a Table diffusible intermediate H. Kinetics of which opens specific 51Cr release from the calcium EL4.BU target by the BM 3.3 CTL clone channels will cause the formation of a gradient in the [Ca2+]i CTL. Other factors must operate to allow preferential calcium Time Target % specific 51Cr released at effector:target elevation in the region of the CTL remote to the target. (min) An ex- cells ratio of planation might be that one global signal causes eleva- [Ca2+]i 10:1 2:1 0.4:1 0.08:1 tion while another locally lowers through calcium [Ca2+], channel inactivation, 20 calcium EL4.BU sequestration or 7.5 1.5 0.0 pumping to the ex- 1.6 terior. 40 Evidence EL4.BU 24.0 already exists for 9.0 3.4 separable but interacting 1.2 60 EL4.BU 27.0 pathways for CTL 8.5 4.0 activation. Expression of IL-2 2.6 receptors and EL4.BU 72.0 interferon 45.0 19.0 secretion are obtained 7.0 using ionophore to raise EL4.BU 83.0 60.0 28.0 and 11.0 [Ca2+]1 PMA to activate kinase C (Nishizuka, 1984) but not 240 EL4.BU 100.0 88.0 45.0 16.0 either alone by et agent (Truneh al., 1985; Albert et al., 1985). RDM4 12.0 7.0 1.7 Furthermore 4.0 of the TcR perturbations or some lec- by antibody 2230 Imaging Ca2' in cytolytic T ceHls and targets Evaluation in cells been shown to increase the pumping activity of Ca2+-ATPases offura-2 compartmentation and of was determined (Lagast et al., 1984) and the activity of inositol 1,4,5-trisphos- The of quality loading degree dye compartmentation by of the release of from intracellular acridine phatases (Connolly et al., 1986). Thus might be locally comparison orange organelles (Arslan [Ca2+]i con- et lactate and fura-2 at threshold al., 1985), dehydrogenase (LDH) digitonin decreased through protein kinase C activity to the for- leading Triton X-100. BM 3.3 cells were in- centrations to the total releasable 1% by mation of a [Ca gradient. cubated with 10 acridine or 1 fura-2/AM and adhered to i]i orange AM AM The possibility exists that yet other signals may be involved Petri dishes. Cells were then washed three in- 5-min polylysine-coated through cubations in a solution of 125 mM 10 mM 1 mM and 1.1 in CTL activation and polarization. Activation via the T cell KCI, NaCl, MgCl2 mM 7.2 Calcium was low to the in- EGTA pH (EGTA medium). kept preserve receptor may involve a number of surface proteins, associated such as the mitochondria which suffer of intracellular tegrity organelles might or not with the TcR which may contribute to binding of targets in the of Ca2+. cells were a ex- 5-min presence high Subsequently, lysed by or transmembrane signalling. Thus there exists the possibility to fresh EGTA medium 20 LDH was measured posure containing 1M digitonin. an of the to a mixture of multiple signals, some global and others local, rise to giving by adding aliquot (0.25 ml) digitonin lysate containing 1 mM 10 mM sodium lactate in solution and NAD and (2.5 the CTL response. ml) lysis measuring increase at 340-nm excitation and 440-nm emis- the rate of NADH fluorescence The approach presented here permits the observation of spatial- was measured in a second the sion. Acridine orange aliquot by scanning ly distributed activation events as well as the direct temporal and at 480-nm excitation. The material 500-600-nm emission intensity remaining spatial relationship between an activation event in the effector Triton in EGTA medium and on the dish was then extracted with 1% X-100 the and the lethal hit delivery in the target cell. These results are assays repeated. in individual cells complementary to biochemical of Measurements analysis signalling pathways of [Ca2i] used to adhere cells were a 1 which show that TcR/CD3 activation leads to the phosphoryla- Polylysine-coated coverslips prepared using mg/mil mol. wt -500 in 1 M KCI. Clean solution poly-L-lysine (from Sigma, 000) tion of CD3 proteins (Samelson et and that activation al., 1986) were coated with a thin film of solution and then 18-mm coverslips polylysine for killing or of interferon occur under induction gamma synthesis two beakers of distilled water and dried. Prior to rinsed immediately through conditions where different CD3 proteins are phosphorylated cell the were washed with HBSS/3% FCS. adding suspensions coverslips were attached a thin film of silicone to a (Boyer et al., 1987). An extension of the studies presented here slotted, Coverslips by grease rectangular insert which fitted into a holder on the of a stainless-steel to various systems of induction of CTL water-jacketed stage killing through lectins, IM-35 was at Zeiss constant, microscope. Temperature kept 37°C, by circulating anti-TcR/CD3 antibodies or phorbol esters will test the hypothesis water the insert holder and coils the Nikon 40x /1.3NA through tubing surrounding brought the of various CTL forward here. Additionally, analysis 50 of cell was added to the chamber lens. objective Approximately Al suspension clones different efficiencies for the same the After the cells had settled and adhered exhibiting killing target containing polylysine-coated coverslip. unattached cells were washed off with HBSS-3% FCS. After the cell will provide information as to which activation step (CTL collecting measurements a second cell was then added. baseline type hit is the of or lethal delivery) limiting process killing. Fluorescence ratio 32 hues to the range images using pseudocolor represent ratios were obtained as described et of fluorescence al., 1986). (Poenie previously have been normalized to that of a thin film of solu- All fluorescence ratio images 120 mM 10 mM 20 mM 7.5 and tion KCI, Hepes pH containing K2H2EDTA, Materials and methods The 10 mM fura-2 salt Probes, OR). (Molecular City, pentapotassium Oregon to account for the effect of and cells: and culture conditions ratios and have been Effector target origin viscosity Rmin Rmax adjusted and in (M.Poenie R.Y.Tsien, BM 3.3 is an alloreactive H-2Kb T cell clone derived from CBA/J mice preparation). specific and values The were (Hua et al., This line was maintained as described Hua et al. (1986) intensity 1986). by images analyzed by taking average [Ca2+]i each cell in the field. The mean within the area of a over spot placed intensity stimulation with 2500 rad irradiated cells from H-2b mice and by weekly spleen in the various formats in were then used and graph display presented from the EL4.C16 cell. The tumor cell [Ca2+]i IL-2 containing supematant thymoma the text. as were the H-2b EL4.BU and the H-2k lines used targets lymphoma thymoma line RDM-4. Cells were cultured in RPMI 1640 8% fetal calf serum containing and as described et Acknowledgements (FCS), supplemented (Hua al., 1986). for his her sabbatical 5ICr release thanks Dr Weissman Measure of cytotoxic activity by A.-M.S.-V. hospitality during Irving Henkart for his of in his We also thank Pierre leave classical 51Cr release cells cells/ml RPMI-5% FCS) gift cytolytic In the assay, target (107 laboratory. Henkart for their discussions. and Pierre Henkart and were labelled with 0.1 mCi Na2 1CrO4 for 1 h at 370C followed 3 washes helpful by granules Maryanna from NIH the Searle Scholars This work was cells cells (GM31004), FCS. of 51Cr-loaded well) in RPMI-5% Triplicate samples (104 per supported by grants of Califor- and the Cancer Research numbers of effector cells in 200 of RPMI-5% Committee, were then incubated with various University yd Program, Coordinating was undertaken the tenure of an nia. The work in this FCS at 37°C in microtitre after a 2-min cen- reported during V-shaped plates (Greiner, FRG) paper -Eleanore Roosevelt International award- American Cancer 200 At the time in Fellowship of the at Society trifugation microplate g. appropriate points (given Union Cancer to ed the International were then harvested and counted on a A.-M.S.-V. by Against the tables) supernatants (0.1 ml) gamma 51Cr release was determined as follows: The % counter. specific [experimental - release (1 N - medium References medium HCI) c.p.m.] c.p.m. c.p.m./maximum c.p.m. of the BM 3.3 effector cells also Hua et al., 1986) x 100. The specificity (see and Schmitt-Verhulst,A.-M. (1985) Hua,C., Truneh,A., Pierres,M. Albert,F., of 5'Cr release from the H-2k RDM4 tumor the absence target is indicated by 3649-3655. J. Immunol., 134, of BM 3.3 cells was not altered the cells. The effectiveness by target lysis by J. 8746-8759. Biol. and Chem., 260, (1985) Albert,P.R. 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(1986) Im- J. 377-382. munol., 136, Zagury,D. (1982) Adv. Exp. Med. Biol., 146, 149-165. Received on February 20, 1987; revised on May 19, 1987

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The EMBO Journal – Springer Journals

Published: Aug 1, 1987

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