J. Cell Sci. 35, 355-3&6 (1979) 355Printed in Great Britain © Company of Biologists Limited 1979

THE MECHANISM OF K-CELL (ANTIBODY-

DEPENDENT) MEDIATED CYTOTOXICITY

III. THE ULTRASTRUCTURE OF K CELL PROJECTIONSAND THEIR POSSIBLE ROLE IN TARGET CELLKILLING

AUDREY M. GLAUERTStrangeways Research Laboratory, Worts' Causetoay,Cambridge CBi 4RZV, England

COLIN J. SANDERSONClinical Research Centre, Watford Road, Harrotu,Middlesex HAi 3UJ, England

SUMMARY

The cytotoxic interaction between lymphoid K cells from normal rat spleen and antibody-coated P815 mastocytoma cells has been studied in conditions under which the number ofcytolytic events occurring at the time of observation was at a maximum.

Electron micrographs of material fixed during the first 15 min after contact between thetarget and effector cells had been initiated by centrifugation showed that the K cells producelong projections which push deeply into the P815 cells, causing infoldings of the plasmamembrane and distortion of the nucleus. The plasma membranes of the effector and targetcells, and the nuclear membrane, remain intact. Subsequently the target cells undergo violentcytoplasmic blebbing (zeiosis) which is the first stage of cell lysis.

The evidence for the hypothesis that projections from lymphoid K cells develop as a resultof contact between receptors on the K cell surface and antibody bound to the target cell, andthat the projections are involved in the cytotoxic mechanism is discussed.

INTRODUCTION

The name K cell has been suggested for the effector cells in antibody-dependent,cell-mediated cytotoxicity (Anon, 1973). The term is often applied loosely to includeany antibody-dependent 'killer' cell, including granulocytes. In the present studythe term is restricted to lymphoid cells capable of 'killing' antibody-coated nucleatedmammalian cells (Sanderson & Taylor, 1976), and for convenience lymphoid cellsforming contacts with antibody-coated target cells are referred to as K cells in themorphological descriptions in this paper. The evidence associating these cells withcytotoxic activity is considered in the Discussion.

Although little is known about the origin or biological function of K cells, theirpossible role in defence mechanisms has led to considerable interest in their modeof action and in their distribution. It has been shown that the kinetics of release oftarget cell components is correlated with morphological changes observable by time-lapse cinematography (Sanderson & Thomas, 1977a, b), and it has been demonstrated

356 A. M. Glauert and C. J. Sanderson

that there are many similarities between K cells and cytotoxic T cells. For example,K cells share some physical and morphological properties of T cells, and in bothsystems the target cell dies in a burst of membrane blebbing (zeiosis) which is quitedifferent from the changes seen when a cell is lysed by antibody and complement.There are, however, important differences between the T and K cell systems. First,T cells appear as a result of antigenic stimulation and possess specific receptors forantigen, whereas K cells are present in normal animals and attach to target cells bymeans of receptors for the Fc pieces of immunoglobulins. Secondly, whereas withT cells target cell death occurs as a random event in time after contact, varying fromseconds to several hours (Sanderson, 1976a, b), K cells appear to bring about targetcell death within about 15 min of contact (Sanderson & Thomas, 1977a, b).

In a study of T cell cytotoxicity by electron microscopy, Sanderson & Glauert (1977)observed projections from T cells which had pushed into target cells. These pro-jections were only rarely seen and so it was not possible to come to any conclusions asto their role in the cytotoxic process. It seemed possible that the difficulty of findingthese projections in electron micrographs might be related to their short time span,and the study reported in this paper was carried out because the kinetics of K cellcytotoxicity indicated that there would be a better chance of observing the cytolyticevent, by fixing material during the first 15 min after contact, than there would be inthe T cell system. The results show that K cells do produce long projections and thepossibility that these are related to the mechanism of target cell killing is discussed.As a result of these findings we have been encouraged to return to the T cell systemand have found that projections are frequently observed in this system as well whenthe experimental parameters are adjusted to maximize the number of cytolytic eventsoccurring at a certain time. These findings have been reported fully elsewhere(Sanderson & Glauert, 1979).

MATERIALS AND METHODS

Target cells

The mouse mastocytoma (P815) cell line of DBA/2 origin was used as target cells. The cellswere grown in stationary culture in RPMI-1640 medium supplemented with 10% foetal calfserum. In each experiment the target cells were labelled with "chromium so that cytotoxicitycould be estimated by measuring the release of isotope (Sanderson & Thomas, 19770).

Effector cellsAgus rat spleen was used as a source of K cells. The population of K cells in the spleen cell

suspensions was enriched by Ficoll-triosil separation (by taking the lymphoid cells at theinterface), followed by passage through columns of nylon wool (taking the non-adherent cells).Full details of the procedure for the preparation of suspensions enriched in K cells have beendescribed previously (Sanderson & Thomas, 19776).

Antisera

Antisera were obtained 14 days after intraperitoneal injection of 3 x io7 P815 cells into rats.The antisera were heat inactivated and used at a final dilution of 1:1000.

Projections from cytotoxic K cells 357

K cell-mediated cytotoxicity

The cytotoxic reactions were carried out in 075 ml polypropylene tubes with pointed bottoms(Walter Sarstedt Ltd, Leicester, England). 2-5 x 10' cells from the suspensions enriched inK cells were mixed with io5 P815 cells, in the presence or absence of anti-P8is antiserum. Theeffector and target cells were prewarmed to 37 °C and were centrifuged at 37 °C immediatelyafter mixing by bringing the centrifuge up to 400 r.c.f. and then applying the brake. The cellswere then resuspended on a vortex mixer and centrifuged a second time.

Electron microscopy

After incubation at 37 °C for 4-15 min from the commencement of the first centrifugation,the cell pellets were fixed by carefully removing the supernatant medium and replacing it withprewarmed fixative, consisting of 2-5 % glutaraldehyde in 0-09 M cacodylate buffer, pH 7-2,containing 2-5 mM calcium chloride. After 15 min at 37 CC, the pellets were kept at 22 °C fora further 45 min. The fixative was then removed and replaced with cacodylate buffer, and thefixed pellets were stored in buffer at 4 CC. Subsequently the pellets were removed from thecentrifuge tubes and each pellet was divided into a number of small pieces. These pieces werethen postfixed in 1 % osmium tetroxide in 0 1 M cacodylate buffer, pH 7 2 , containing 2-5 mMcalcium chloride, for 1 h, rinsed briefly in distilled water, stained with 0-5 % aqueous uranylacetate for 1 h, dehydrated in ethanol and embedded in Araldite by standard techniques.Pellets which showed a tendency to disperse in buffer after the primary fixation in glutaraldehydewere encapsulated in. a small drop of 2 % agar (Oxoid, London) before postfixation in osmiumtetroxide and subssquent processing.

The Araldite blocks were sectioned on an LKB Ultrotome III or a Cambridge HuxleyMark 2 ultramicrotome, and thin sections were stained with lead citrate and examined in anAEI-EM6B electron microscope operating at 60 kV with a 50-/HT1 objective aperture.

RESULTS

Isotope release

Cytotoxicity was tested by assaying the release of "chromium from labelled P815cells (Sanderson & Thomas, 1977a) after incubation with effector cells for 15 min at37 °C. Isotope release was determined in two separate, but similar, experiments inwhich the effector cell to target cell ratio was 25:1. In the first experiment 30%specific release of isotope was obtained, and in the second, 11%. This illustrates thelarge differences between cell preparations which is a common feature of this typeof experiment. The experiment with the highest levels of chromium release gave thegreatest number of effector-to-target cell interactions, and the majority of the micro-graphs illustrating this paper come from this experiment.

Types of cell contact

Control cultures, in the absence of antibody, showed no close contacts betweenlymphoid cells and P815 cells.

In the presence of antibody, survey electron micrographs showed that some P815cells are surrounded by a number of lymphoid cells (Fig. 1), while elsewhere a singlecell had made contact with more than one tumour cell (Fig. 2). The cells in contactwith P815 cells have the typical morphology of lymphoid cells, with large, indentednuclei surrounded by a thin layer of cytoplasm (Figs. 1, 2), and will be referred toas K cells.

358 A. M. Glauert and C. J. Sanderson

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Projections from cytotoxic K cells 359

Two types of initial contact could be distinguished between K cells and P815 cells.In the first, and less frequent type, there were large areas in which the membranes ofthe effector and target cells were in close contact (Fig. 3). In the second, and mostfrequent type, there were only point contacts separated by areas in which there wasa wide space of varying width between the two cells (Fig. 4). The projections fromthe K cells which are described below appear to develop from these point contacts.

Formation of projections from K cells

Many of the K cells in contact with P815 cells had surface projections. Theproduction of projections did not appear to be restricted to any particular part of theK cell membrane. For example, projections have developed in all three areas of contactof the K cell with tumour cells in Fig. 2. Furthermore, projections are mainly seenin these areas of contact and not on other regions of the K cell surface (Figs. 1, 2),suggesting that the formation of the projections may be initiated by localized contactbetween the K and P815 cells. This suggestion is supported by the observation thatthe tips of the projections are often in close contact with the target cell membrane(Fig, 5). The formation of projections is an early event in the interaction with targetcells, since they were most frequently observed in preparations fixed 4-5 min aftercontact between the effector and target cells had been initiated by centrifugation.

Morphology of the projections

The projections from K cells in contact with P815 cells vary in shape; some arestraight (Figs. 2, arrow, and 6), while others are curved (Fig. 5) or branched withcomplex outlines. The tips of the projections are usually pointed, although a few haveblunt ends (Fig. 2, double arrow). When seen in cross-section (Fig. 5, arrow) theprojections appear approximately round and, except at the tip, there is a space betweenthe limiting membrane of the projection and the tumour cell membrane (Fig. 5). Theprojections contain a meshwork of filamentous material (Fig. 6) to the exclusion ofribosomes and other organelles.

In regions the surfaces of the K cell and the target cell appear to have interdigitated(Fig. 6). It is noticeable that the tumour cell interdigitations contain ribosomes, whilethose from the K cell do not.

Some of the projections have pushed deeply into the cytoplasm of a P815 cell. Thefull depth of these projections is difficult to determine because of the unlikelihood of

All figures are electron micrographs of thin sections of antibody-coated P815mastocytoma cells incubated with lymphoid cells from normal rat spleen.Fig. 1. A survey electron micrograph illustrating the general appearance of the P815cells (m). The cell is surrounded by a number of cells which have the typical morph-ology of lymphoid cells, with large, indented nuclei and a thin layer of cytoplasm (4 minincubation).

Fig. 2. A single K cell (fc) is in contact with three P815 cells (m). Cytoplasmic pro-jections from the K cell are only present in regions of contact with the target cells. Theprojections vary in shape; some are pointed (arrow), while others have blunt ends(double arrow) (4 min. incubation).

The bars represent 1 fim.

A. M. Glauert and C. J. Sanderson

Fig. 3. A P815 (m) and K cell (A) are in close parallel contact over a large area(4 min incubation).Fig. 4. A K cell has made point contacts with a P815 cell (m) (4 min incubation).Fig. 5. A curved projection from a K cell has pushed into the cytoplasm of a targetcell (m). The tip of the projection is in close contact with the target cell plasmamembrane. Another projection is seen in cross-section (arrow) and appears approxi-mately round (4 min incubation).

The bars represent 1 fim.

Projections from cytotoxic K cells 361

sectioning a projection along its full length, but cross-sections of what appear to beparts of projections are observed deep in the cytoplasm of some target cells (Fig. 7,arrows). The plasma membranes of both cells have remained intact.

Involvement of the tumour cell nucleus

Observations by time-lapse cinematography have shown that K cells are alwaysclose to the target cell nucleus immediately before the onset of changes which lead totarget cell death (Sanderson & Thomas, 19776). More recently (Sanderson, un-published observations) K cells have been observed to push repeatedly into thenuclear regions of target cells and to distort the outline of the nucleus. It was thereforeof interest to note that some of the long cytoplasmic projections from K cells seen inelectron micrographs had pushed through the cytoplasm of the target cell and hadnearly reached the nucleus (Figs. 6, 8). The nucleus was often indented at the tip ofsuch projections (Fig. 6), although the nuclear membrane, as well as the plasmamembranes of the K and target cells, were still intact.

Zeiosis and cell lysis

Following K cell contact and interaction with a target cell in the region of the targetcell nucleus, the target cell undergoes violent blebbing, or zeiosis (Sanderson &Thomas, 1977 ft), during which the cell takes on very bizarre shapes. Cells in zeiosiswere easily identified in electron micrographs (Figs. 9, 10), and some of these cellsstill had K cells attached by means of short projections from the K cell (Fig. 9).

At the final stage of lysis the target cell returns to a normal spherical shape(Sanderson & Thomas, 1977ft), ^ u t becomes swollen and loses most of its cytoplasmiccontents (Fig. 11). Cell lysis was observed in material fixed only 15 min after cellcontact had been initiated by centrifugation, illustrating the rapidity of the cytotoxicprocess.

DISCUSSION

While many of the characteristics of T cell-mediated cytotoxicity have beendescribed (see reviews by Berke, 1977, and Kimura & Wigzell, 1977), the K cellsystem has not been examined in the same detail. While cytotoxic systems witherythrocyte targets probably involve granulocyte effector cells (for example, seeGreenberg, Shen & Roitt, 1973; Frye & Friou, 1975; Golstein & Gomperts, 1975), insystems with nucleated mammalian target cells the most efficient effector cell (K cell)is a low density, non-adherent cell (Sanderson, Clark & Taylor, 1975; Sanderson &Thomas, 1978) and in the present study a cell separation technique has been used toenrich this population of cells. Although these procedures result in a considerableincrease in cytotoxic activity (Sanderson & Thomas, 1977 a), the cell suspension is byno means a pure preparation of K cells. It is important therefore to consider thepossibility that the cells observed in contact with target cells in the present study areunrelated to the cytotoxicity observed in 61chromium release assays.

Various observations suggest that the lymphoid cells in contact with target cells in

A. M. Glanert and C. J. Sanderson

Projections from cytotoxic K cells 363

our preparations are K cells. Cell contacts are not observed in the absence ofantibody, indicating that the initial interaction is antibody-dependent, and most ofthe other cell types known to have Fc receptors (granulocytes and B cells, for example)are removed by the cell separation procedures used. In addition, analysis of time-lapse films of similar preparations has indicated that contacts that do not result intarget cell death are rare (Sanderson & Thomas, 19776), and that the cells responsiblefor target cell lysis have the morphology typical of lymphoid cells. Furthermore, cellsof similar morphology are observed attached to target cells undergoing zeiosis(e.g. Fig. 9).

Characteristics of the interaction between K cells and target cells

A few K cells and target cells formed contacts in which the membranes of the twocells were in close, parallel contact with each other over a wide area (Fig. 3), asoriginally described by Biberfeld & Johannson (1975). More frequently pointcontacts, separated by regions with a considerable intercellular space, were observed(Fig. 4). These point contacts appeared to be the precursors to the projections fromK cells, and it is not clear whether the larger areas of close, parallel contact play anessential role in the cytotoxic process.

The most striking feature of the interaction between K cells and target cells observedin the present study was the formation of long projections from the K cells whichpushed into the target cells. These projections were only seen frequently in prep-arations fixed within 5 min of the initiation of contact between the effector and targetcells and could thus be easily missed. In addition, it is important to fix the prep-arations at the temperature of the incubation (37 °C), since a reduction in temperaturemay well lead to a retraction of such slender structures.

The long projections from lymphoid K cells which pushed into P185 cells causeddeep infoldings of the plasma membrane of the target cell and distortions of thenucleus, but there were no other detectable changes in the target cell, and the nuclearand plasma membranes remained intact. There is thus no direct proof that theprojections are the cause of target cell death, although two types of evidence suggestthat they do play an important role in the cytotoxic process. The rapid movement ofK cells near the target cell nucleus just before the onset of cell lysis (Sanderson &Thomas, 19776) and the accompanying cytoplasmic and nuclear distortions of thetarget cell are observed under the same conditions under which the projections aremost readily found in electron micrographs. In addition, similar projections have

Fig. 6. A long projection from a K cell has pushed through the cytoplasm of a P185cell (m) and appears to have caused an indentation in the outline of the target cellnucleus. The projection contains a network of filamentous material to the exclusionof ribosomes and other organelles. The plasma membranes of the K and target celland the nuclear membranes of the target cell all appear to be intact (4 min incubation).The bar represents o-i /im.Fig. 7. Cross-sections (arrows) of long curved projections from a K cell are visibledeep in the cytoplasm of a target cell (m) (15 min incubation). The bar represents 1 fim.

A. M. Glauert and C. J. Sanderson

Fig. 8. A curved projection from a K cell, which passes out of the plane of the sectionin one region, appears to have caused an indentation in the nucleus of a target cell (m)(4 min incubation).Fig. 9. A K cell is attached by a short projection to a complex bleb on the surface ofa P815 cell (m) which is undergoing zeiosis (15 min incubation).Fig. 10. A target cell at a late stage of zeiosis contains many vacuoles (15 min incubation).Fig. 11. A lysed P815 cell has lost most of its cytoplasmic contents and has returned toa rounded shape (15 min incubation).

The bars represent 1 fim.

Projections from cytotoxic K cells 365

been observed during interaction of cytotoxic T cells from immunized mice withtarget cells (Sanderson, 19776; Sanderson & Thomas, 19776).

The formation and function of K cell projections

The formation of projections from K cells as a result of contact with antibody-coated target cells provides an analogy with the membrane movements accompanyingthe engulfment of particles by phagocytic cells. In both systems the movement of themembrane of the effector cell is stimulated by contact of its receptors with a surface.Although the type of membrane movement is different, it appears that in bothsystems the receptors provided a signal which stimulates changes in the underlyingcytoskeletal system of the cell and the consequent production of projections in theform of filopodia or lamellipodia which contain filamentous material to the exclusionof other cytoplasmic organelles (Hartwig, Davies & Stossel, 1977). In phagocyticcells, projections are formed which move over the surface of the particle that is beingengulfed, while cytotoxic cells push out projections into the substance of the targetcell with sufficient force to distort the target cell membrane and to dent the nucleus.

The mechanism by which these projections might cause target cell death is not yetclear. On the one hand, the fact that zeiosis occurs simultaneously over the wholecell surface suggests that some type of widespread change has occurred within thecell, and not just local membrane damage. On the other hand, there are no obviouschanges in the cytoplasmic or nuclear structure of the target cell, even in the earlystages of zeiosis. The possibility cannot be ruled out, however, that the projectionsproduce lesions in the target cell which are too small to be visible in electron micro-graphs of thin sections, but which are sufficient to allow changes in the distributionof soluble factors and ions and which are lethal to the cell. It seems likely that theselesions occur in the nuclear area of the cell and perhaps in the nuclear membraneitself. Observations which provide an experimental analogy to the possible effect ofthe projections were reported by Munro & Daniel (1965). These authors found thatcells recovered rapidly from major disruption of their plasma membranes, but thata micro-electrode introduced into the nuclear region led to cell death, preceded byviolent zeiosis.

We acknowledge the support of the Sir Halley Stewart Trust (to A.M.G.) and we are verygrateful to R. A. Parker and Janet Atherton for skilled technical assistance and to the WellcomeTrust for the loan of the AEI-EM6B electron microscope.

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(Received 12 May 1978)