Clin. exp. Immunol. (1979) 38, 549-560.
Characterization of the human peripheral effector cells mediating antibody dependent cellular cytotoxicity against allogenic cells MIREILLE DONNER, * COLETTE RAFFOUXt & F. STREIFFt * Research Unit ofExperimental Cancerology and Radiobiology, U.95 INSERM, Plateau de Brabois, 54500 Vandoeuvre-lis-Nancy and t The Transfusion Center-19, rue Lionnois, 54000 Nancy, France.
(Accepted for publication 9 April 1979)
SUMMARY
The effector cell populations in human peripheral blood responsible for antibody-dependent cellular cytotoxicity against allogenic cells coated with HLA polyspecific antibodies were investigated using several separation techniques including preparative electrophoresis. Electrophoresis produced a marked effector cells enrichment in a range of 2-5 fractions which exhibited an intermediary electrophoretic mobility. Monocytic cells do not contribute an effector mechanism but minor subsets ofpolymorphonuclear cells and nylon wool non-adherent non-phagocytic lymphocytes displayed ADCC. Both effector cell populations were found to exhibit a similar electrical charge of cell surface centered around - 1 05 jim sec 1 V-i cm. These observations provided a precise biophysical basis for the identification of effector cells in ADCC. -
INTRODUCTION The cytotoxic potential of non-sensitized cells against target cells coated with anti-target cell IgG has been extensively studied over the past years but it is not completely understood. In order to display antibody-dependent cellular cytotoxicity (ADCC), the effector cells must have receptors for structures in the Fc fragment of homologous and heterologous IgG. However, Fc-bearing cells are heterogeneous and the nature of cells effecting ADCC is still a central issue. An important observation is that the type of target cells and the source of antibody appear to dictate the nature of the effector cells. In some target systems, these effector cells include polymorphonuclear leucocytes (Clark & Klebanoff, 1977; Gill, Waller & MacLennan, 1977), monocytes (Poplack et al., 1976; Nyholm & Currie, 1978), and macrophages (Mantovani et al., 1977). In other systems, undefined lymphocyte like cells, known as 'killer cells' (K cells) are effector cells in ADCC. The nature of human K cells is controversial and some groups have claimed that they appear to be of T cell origin (Kay et al., 1977; Pape, Troye & Perlmann, 1977; Saal et al., 1977), whereas others attribute the antibody-dependent cellular cytotoxicity to B cells at some stages of their differentiation (Chess & Schlossman, 1977) or to non-T lymphocytes possessing a low concentration of surface immunoglobulin (Eremin et al., 1977). In contrast, recent studies indicated that K cells are 'null cells' which lack easily detectable T and B lymphocyte markers (Horwitz & Garrett, 1977; Bakacs, Gergely & Klein, 1977). Great caution is in order when considering these results. Studies have shown a variable expression of surface markers and receptors on lymphocyte subpopulations. Cell separation techniques involving Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; K cells, killer cells; EPM, electrophoretic mobility; FCS, foetal calf serum; ANAE, a-naphthyl acetate esterase; E-RFC: E-rosette forming cells. Correspondence: Dr Mireille Donner, Research Unit of Experimental Cancerology and Radiobiology, U.95 INSERM, Plateau de Brabois, 54500 Vandoeuvre-les-Nancy, France. 0099-9104/79/1200-0549$02.00 (0 1979 Blackwell Scientific Publications
549
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Mireille Donner, Colette Raffoux & F. Streiff
rosette sedimentation with SRBC have limitations. Recent observations suggest that some discrepancies resulted from the use of different procedures for separating rosetting from non-rosetting cells (West et al., 1977). Moreover, a cell subset combines the surface markers of both T and B cell populations (Chiao, Pantic & Good, 1974; Dickler, Adkinson & Terry, 1974). Apart from classical markers, other means of identification of cell subpopulations involve biophysical properties of cells, including electrical charge of cell surface or cell density. Thus, cell electrophoresis has been useful for the identification of cell subsets in human system (Stein, 1975; Kaplan & Uzgiris, 1976; Chassagne et aL., 1977; Chollet et aL., 1977; Hanjan et aL., 1977; Chollet et aL., 1978). These previous results led us to raise the question whether K cells might be unambiguously defined using the electrical charge of cell surface. The advantage of characterizing cells on this biophysical basis is that, as long as a cell is living and intact, the ionizable groups of the surface macromolecules will give rise to an electrical charge. The present report describes studies designated to characterize human K cells by combining preparative and analytical cell electrophoresis. As K cells might play an important role in graft rejection, antitumour responses and autoimmune diseases, we considered allogenic cells as the source of target cells. In our assay system, we present evidence for the identification of human K cells as subsets of polymorphonuclear cells and nylon wool non-adherent non-phagocytic lymphocytes exhibiting an intermediate electrophoretic mobility (EPM) of -105 + 002 pm sect v -i cm. -
MATERIALS AND METHODS Cell collection. Blood was drawn from healthy adult donors of blood group 0 and defibrinated by rotary shaking on glass beads. Preparation of polymorph-enriched leucocyte suspensions. Defibrinated blood was sedimented at room temperature for 45 min with one half its volume of 6% dextran in 0.9% saline. The leucocyte-rich plasma was removed, centrifuged at 200 g for 8 min and the cell pellet washed in MEM medium, and finally suspended in a low ionic strength buffer suitable for freeflow electrophoresis (Zeiller, 1972). Polymorphonuclear cells represented 80-90% of the total cell population, as revealed by cytocentrifuged cell smears. Preparation of mononuclear cells. Mononuclear cells were obtained by separation on Ficoll-Hypaque (S.P. 1.077) and then washing twice in MEM medium. Differential counting of cytocentrifuge preparations stained to detect cells containing nonspecific esterase revealed that the percentage of monocytes ranged from 10 to 25%. In some series of experiments, phagocytic cells were removed from cell suspensions after ingestion of carbonyl iron (2 mg carbonyl iron/ 106 nucleated cells). The cell mixture was incubated at 370C for 20 min, then exposed to a strong magnetic field for 10 min. Cells not attracted to the magnet were harvested and washed twice in MEM medium+ 10% FCS. Following this treatment, mononuclear preparations contained less than 4% monocytes. Cellfractionation on nylon wool columns. In some series of experiments, nylon wool separation of Ficoll-Hypaque purified and carbonyl iron treated cells was performed as described by Julius, Simpson & Herzenberg (1973). After the recovery of non-adherent cells, nylon wool columns were rapidly washed with 100 ml MEM medium+ 10% FCS. Cells in the second effluent were referred to as 'weakly adherent cells'. Finally, the adherent cells were recovered by strongly compressing the nylon wool with a syringe plunger as described by Handwerger & Schwartz (1974). Preparative electrophoresis. Cells were fractionated in a free-flow preparative electrophoresis Model FF5 (Bender Hobein, Munich, Germany). The buffer used in the separation chamber had the following composition: 0 04 M potassium acetate; 0-015 M triethanolamine; 0-24 M glycine made isoosmotic with glucose. The pH was adjusted to 7-2-7-4 with acetic acid. The electrode chambers contained buffer with 0.075 M triethanolamine, 0 004 M potassium acetate. For fractionation of cells a field strength of approximately 87-90 V/cm and a current of 210 mA were used. Just before electrophoretic fractionation, cells were suspended in the weak ionic strength buffer and filtered on nylon mesh to remove cell clumps. Separations were performed at 6VC. To achieve satisfactory separation, the buffer flow rate was adjusted to about 450 ml/hr, each cell remaining in the electrical field for about 240 sec. Cell suspensions at a concentration of 8-12 x 106 cells/ml were injected into the electrophoresis chamber at a flow rate of 5 ml/hr. The fractions were collected into test tubes containing 1 ml of MEM medium with 10% foetal calf serum. To minimize cell injury, test tubes were harvested each hour. Cells were immediately washed and suspended in MEM medium with 10% FCS: cells in each fraction were then counted in a haemocytometer. Analytical cell electrophoresis. The electrophoretic mobility was determined by the use of an electrophoresis chamber of a circular cross section and a small volume (0-8 ml) equipped with reversible silver-silver chloride-potassium chloride (1.0 M) electrodes at 25°C (Mehrishi, 1972). Cells were scored in 0-145 M NaCl adjusted to pH 7-2+ 0-1 with NaHCO3. The electrophoretic mobility is expressed in pm-sec- V-1 cm. The reliable performance of the apparatus was monitored by deter-
Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
551
mining the EPM of washed human erythrocytes which present a reproducible electrophoretic mobility of - 108 + 0 03 pum sect1 V-' cm. Cytotoxicity assay. Human leucocytes of peripheral blood obtained from a panel of healthy adult donors were used as target cells. Leucocytes were prepared from 10 ml heparinized whole blood using a Ficoll-Hypaque gradient. The cells in the interface were collected, washed once in autologous plasma, then in Hanks' BSS and resuspended in the same medium at a concentration of 2 x 106 nucleated cells/ml. The use of this type of target cells led us to take into consideration the recent observation that monocytes, T and non-T human lymphocytes take up and release different amounts of 51Cr (Kovithavongs & Dossetor, 1977). It could be the origin of artefacts in ADCC and we have therefore chosen trypan blue exclusion test instead of 51Cr as a means of testing ADCC. Cell fractions obtained after electrophoretic separation were washed, suspended in Hanks' BSS at a concentration of 2 x 106 cells/ml and used as effectors. Purified IgG antibodies were prepared by Sephadex G 200 fractionation of polyspecific anti-HLA sera. IgG were lyophilized and reconstituted aliquots always used within 2 weeks. Cytotoxicity assay was adapted from a standard assay routinely used in Blood Transfusion Center (Terasaki, 1969). Target cells in 1 pl were dispensed into each well of a Microtest I plate (Falcon 3034). One microlitre of polyspecific antiHLA IgG was added in appropriate dilutions (undiluted or diluted 1: 2, 1:3, before addition to the assay) and the mixures target cell-IgG were incubated at room temperature for 30 min. Effector cells in 1 p1 were then added and a further incuba-tion was carried out at room temperature. Preliminary studies of lytic activity of effector cells at different times of incubation (1-18 hr) showed that a cytotoxicity in the control preparations was apparent after 3 hr incubation period and a 2 hr incubation was therefore chosen for the subsequent experiments. Moreover, previous observations that electrophoretic separation introduced itself an enrichment of effector cells in a ratio ranging from 15 to 25 led us to take an effector-target cell ratio of 1:1. After the incubation, supernatants were withdrawn with a Pasteur pipette and 1 pl of trypan blue was dispensed into each well. Readings were undertaken with an inverted microscope 10 min after adding the dye. The ADCC was estimated by the percentage of stained cells/total number of cells in several microscope fields. A cytotoxicity scale was established as following: + if less than 30% dead cells, + if 30-50% dead cells, + + if 50-75% dead cells. Since microscope readings cannot differentiate effector cells from target cells, some experiments were undertaken with PHA-lymphoblasts as targets to determine if lytic activity was directed to target cells. Ficoll-Hypaque purified blood cells were stimulated with 0 5 mg/ml PHA (Wellcome) at 370C for 5 days. After the incubation period, most of the cultures contained 90% of blast cells which were used as targets and could be easily distinguished from effector cells by their size. These experiments showed that target cells were killed and effector cells excluded trypan blue. Several controls were included in each assay: (1) target cells along with AB serum, (2) effector cells with AB serum, (3) effector and target cells with AB serum to verify that results were not owing to a natural cytotoxicity of effectors, (4) target cells with polyspecific anti-HLA IgG to exclude a complement-independent cytotoxic action of IgG. Finally, each assay included additional wells where rabbit normal serum was added as source of complement to verify the cytotoxic potential of anti-HLA IgG against target cells. Staining for acid a-naphthyl acetate esterase (ANAE). Aliquots of cell suspensions and different fractions obtained after electrophoretic separation were stained for non-specific esterase as described by Mueller et al. (1975) and Hayry, Totterman & Ranki (1977). Non-specific esterase staining applied to peripheral blood cell suspensions is a valuable marker of monocytic cells. Activity is very strong in monocytes and cells were easily distinguishable by the pre sence of multiple intensely redstained granules in the cytoplasm.
RESULTS
Evidence that electrophoretic fractionation of peripheral blood cells induces an enrichment of etfector cells
in ADCC Ficoll-Hypaque purified cells were fractionated in free-flow electrophoresis and the distribution profiles were established by calculating the relative proportions of nucleated cells in each fraction. To compare the results of separate experiments required great caution. The separation depends on numerous parameters (buffer flow rate, temperature, current) which may undergo weak day-to-day variations even under carefully controlled technical conditions. The use of an electrophoretic 'marker' was therefore necessary. Red blood cells which exhibit a highly reproducible surface electrical charge provided such a marker. In each experiment the fraction containing the red cell peak was designated as zero. Fractions with higher EPMs were numbered + 1, + 2, + 3,. . ., and the fractions with lower EPMs were numbered - 1, -2, -3,... Ficoll-Hypaque purified were usually found in 30-35 fractions (fractions -25 to + 10). However, the shapes of profiles presented important individual variations and in more than 35 experiments three different types of profile were recognized (Fig. 1). Type A corresponded to a profile where nucleated cells had a peak in fractions -6 to -8. In the profile B, the peak is broader and was found in fractions 0 to -5. In the profile C, most of the nucleated cells were found in fraction 0. The relative proportions of profiles A, B and C were 600/, 300 0 and 100 0, respectively.
552
Mireille Donner, Colette Raffoux & F. Streif (a)
5
(b) 00
0
.C5 0)
( c) 10
5
-20
0 -10 Standard fractions
FIG. 1. Electrophoretic mobility distribution of human peripheral blood lymphocytes. Abscissa: standard fractions according to the peak of erythrocytes; the fraction with the peak of erythrocytes is designated 0.
In each experiment, the fractions of extreme ranges corresponding to the low EPM cells and the high EPM cells were separately pooled to get a sufficiently large number of cells for the cytotoxicity assay. Fig. 2 shows the results of ADCC assays of fifteen representative experiments. In our experimental conditions, no cytotoxicity was observed when Ficoll-Hypaque purified and electrophoretically unseparated cells were used as effectors. In contrast, cytotoxicity was detected with fractionated samples. Fig. 2 shows that effector cells distributed in intermediary fractions between fractions containing slow and fast moving cells. Although the number of cytotoxic fractions varied according to the experiment, it appeared from Fig. 2 that effector cells were found in nearly similar fractions.
Electrokinetic surface properties of cells responsible for ADCC The identification of effector cells in ADCC required an unambiguous characterization of cells on a
Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
553
15 14
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FIG. 2. Comparison of the fractions positive in antibody-dependent cellular cytotoxicity from peripheral blood fractionated with preparative electrophoresis. Each area represents number of cellular fractions which were recovered within each experiment and shaded area, fractions positive in ADCC.
precise biophysical basis: the EPM or the zeta potential. However, direct calculations of absolute values of EPMs from free-flow electrophoretic separation may lead to artefacts. The use of a weak ionic strength buffer in free-flow electrophoresis markedly modify the spatial distribution of electrical charges near the cell surface and consequently the hydrodynamic plane of shear (Larcan, Stoltz & Streiff, 1974). Moreover, a high uniform electric field resulted in a redistribution of membrane receptors and it might be suggested that alterations in membrane electrical characteristic occur when cells migrate in a high electric field (Poo, Poo & Lames, 1978). To eliminate possible artefacts, cells contained in fractions after electrophoretic fractionation were investigated by the use of the classical microscope method of cell electrophoresis to determine the EPMs in physiological saline under a low electric field as described in 'Methods' section. The mean EPMs of cells contained in cytotoxic fractions are summarized in Table 1. As it can be seen the EPMs of cells were centered around -105 + 0-02 pm sec 1 V'- cm. -
The nature of effector cells Since monocytes and polymorphs have been shown to act as effector cells in some ADCC systems TABLE 1. Mean electrophoretic mobility values of cells in fractions positive in antibody-dependent cellular cytotoxicity Experiment number
Mean electrophoretic mobility in pm sec- I V-1 cm+ s.e.*
Number of scored cells
1 2 3 4 5 6 7
- 103+002t - 1-08+0-01
74 57 92 52 43 37 52
-
-1-03+0-02 - 1-07+ 0*02
-1-07+0*01 - 1-06+ 0-02 - 1-07+ 0-02
* Electrophoretic mobility was determined at
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in physiological
serum 0-145 M adjusted to pH 7-2.
f EPM values centimeter+ s.e.
were
expressed in micron per second and per Volt
Mireille Donner, Colette Raffoux & F. Streiff
554
-5 0
+-----++
Cytotoxicity
~
scale
03
20
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10 -20
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FIG. 3. Electrophoretic distribution profiles of Ficoll-purified cells. Repartition of monocytes and ADCC activity in each fraction. The percentage of monocytes in a fraction refers to the number of nucleated cells contained in this fraction.
(Clark & Klebanoff, 1977; Gill et al., 1977; Poplack et al., 1976; Nyholm & Currie, 1978), the question raised whether these cell types could be effectors in our ADCC assay. Lytic activity of monocytes Monocytes represented 10-25% of Ficoll-Hypaque purified cells. To determine the possible role of monocytic cells in the lysis of target cells, the content of monocytes was measured in each fraction after electrophoretic separation. As it can be seen in Fig. 3, there was no correlation between ADCC activity and the content of esterase-positive cells. Some no cytotoxic fractions contained more monocytes than cytotoxic ones. To examine further the relationships between cytotoxic activity and monocyte number, mononuclear cell preparations were depleted of monocytes by the iron-filing magnetic separation before free-flow electrophoresis. Table 2 shows that iron treatment failed to reduce lytic activity of electrophoretically fractionated cells.
Lytic activity ofpolymorph-enriched leucocyte preparations The potential involvement of polymorphonuclear cells in ADCC was evaluated after electrophoretic fractionation of polymorph-enriched leucocyte preparations. As depicted in Fig. 4, polymorph--rich populations were moderately slower than Ficoll-purified mononuclear cells. The cytological analysis of cells contained in fractions showed that polymorphs represented more than 95%4 of cells except in fractions of high electrophoretic mobility which were significantly contaminated with lymphoid cells. Cells with killer activity were confined in some fractions where contaminating lymphoid cells do not account for a high percentage. Although 15-20 fractions were recovered, the lytic activity was localized in a range of 2-5 fractions suggesting that only a polymorph subset is involved in ADCC. It should be also noticed that these cytotoxic fractions nearly corresponded to those found in Ficoll-purified and electrophoretically fractionated cells.
Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
555
TABLE 2. Failure of monocyte depletion to suppress ADCC activity of electrophoretically fractionated blood cells
Experiment
Carbonyl iron test
Cell recovery after treatment (%)
Monocytes in unfractionated cells (%)
1 2 3 1 2 3
None None None Yes Yes Yes
100* 100 100 55 52 54
12t 21 11 2 4 3
Monocytes in
cytotoxic
Cytotoxicity
fractions (%)
scale
8t
++ + + + ++ +
29 24 5 5 4
* Cell suspensions which were not treated with carbonyl iron refer to 100% of recovery.
tFigures represent percentage of monocytes in cell suspensions before electrophoretic separation.
Effect offractionation on nylon wool columns To further characterize the cells responsible for ADCC, we combined the fractionations by nylon wool columns and free-flow electrophoresis. The passage of Ficoll-purified human peripheral blood over nylon wool columns in our experiment revealed three subpopulations: non-adherent, weakly adherent, and adherent which about represented 60%, 5%0, and 10%, respectively, of the cell population loaded on the columns. When these populations were investigated by the use of classical particle/
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FIG. 4. Comparison between electrophoretic distribution profiles of polymorph enriched cell suspensions (---) and Ficoll-purified mononuclear cells (- --).
556
Streiff
Mireille Donner, Colette Raffoux & F.
cell electrophoresis to determine the EPM, it was found that non-adherent cells had EPMs higher than - 10 pm * sec1 V-i cm (Fig. 5). In contrast, weakly adherent cells contained a mixture of cells which exhibited low and high mobilities. As regards adherent cells, although most cells had an EPM lower - V-i cm, a small percentage of high mobility cells was found. than -10 pm sec' These findings suggest that the fractionation by nylon wool columns before electrophoretic separation
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FIG. 5. Electrophoretic mobilities of non-adherent, weakly adherent and adherent cells. Each point represents the EPM of a single cell. Relative proportions of non-adherent, weakly adherent and adherent cells are not respected.
20 l 20
15
5
-15
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FIG. 6. Electrophoretic distribution profile of non-phagocytic, nylon wool non-adherent cells from human peripheral blood.
Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
557
might be meaningful to characterize cells responsible for ADCC. Therefore, Ficoll-purified cells and treated with carbonyl iron were fractionated by passage over nylon wool columns and subsequently by free-flow electrophoresis. In all experiments, a lytic activity was observed in some fractions of non-adherent cells. Fig. 6 shows an electrophoretic distribution profile of nylon wool non-adherent cells where the lytic cells could be recovered from fractions -5-8. It should be noted that lytic activity of Ficoll-purified and nylon wool non-adherent cells distributed in identical fractions. The cytological analysis of effector cells contained in lytic fractions of electrophoretically fractionated nylon wool non-adherent cells revealed that most of the cells appeared to be lymphocytes with esterase activity. It should be noticed that a high percentage of cells presented esterase activity distributed in several spots near the plasma membrane (Fig. 7). .
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FIG. 7. Demonstration of ANAE activity in smears of nylon wool non-adherent non-phagocytic effector cells in ADCC. Most of the cells presented small esterase positive spots near the plasma membrane (indicated by an arrow).
DISCUSSION The present study clearly suggests that free-flow electrophoresis of leucocytes from human peripheral blood represents a reliable and convenient method to obtain enriched cell subpopulations exhibiting functional properties. Ficoll-Hypaque purified and unfractionated cells were unable to mediate ADCC
558
Mireille Donner, Colette Raffoux & F. Streiff
in our experiment, while some fractions of electrophoretically separated cells were effective in ADCC. Thus, free-flow electrophoresis allowed a 15-20 fold enrichment of effector cells. It should be appreciated that this method is useful to fractionate a complex population of viable cells into component subpopulations without manipulations of cells involving fixation of immunological reagent on cells. Moreover, the fractions are directly recovered. Our data also show that the effector cells of ADCC may be characterized by their electrical charge of cell surface. The determination of EPM at the single cell level by classical cell/particle microelectrophoresis showed that Ficoll-Hypaque purified cells from human peripheral blood distributed between -0f60um sec' V` cm and - 1-50 um sec1 V` cm. In our assay system, killer activity was found in fractions exhibiting EPMs centered around - 105 um * sec1 V- cm. The findings that killer activity was not found in strictly identical fractions and that the number of cytotoxic fractions varied according to the experiments is presumably attributable to individual variations registered in distribution profiles of EPM. These fluctuations likely reflect individual differences in relative proportions of cell subsets and cannot be explained by technical factors such as lymphocyte separation methods (Mookerge, 1976), since we always used the same technique with well defined conditions. The possibility that the low ionic strength buffer used in electrophoretic separation may be responsible for these variations has been also excluded. Vassar, Levy & Brook (1976) reported similar observations with a high ionic strength buffer. Rather, there are some reasons to suppose that 'physiological' factors could account for these variations, as reported previously (Yu, Clements & Pearson, 1977; Raptopoulou & Goulis, 1977; Pudifin et al., 1978). In characterizing the cells responsible for lytic activity against allogenic lymphocytes coated with polyspecific anti-HLA IgG, several facts are clear. Our results showed a lack of correlation between K cell activity and levels of monocytes in different fractions after electrophoretic separation. Furthermore, the results observed after the 'iron and magnet' technique are not in favour of monocytes as lytic cells despite the fact that killer activity is weakly diminished after removal of iron-phagocytozing cells. Since the yield of cells removed after carbonyl iron treatment was nearly 4000 and the monocyte percentage was not so high, there is possibility that other cell types perhaps mediating ADCC, are eliminated with monocytes. It seems therefore reasonable to assume that, in our assay system, monocytes do not constitute an effector mechanism. In contrast, both polymorphonuclear cells and nylon wool non-adherent non-phagocytic lymphoid cells were able to lyse efficiently target cells. Since killer activity was found in a range of 2-5 fractions, it is clear that lytic cells constitute minor subsets of both cell types which is possible to characterize on the basis of the electrical charge of cell surface with the cell/particle analytical microelectrophoresis. The observation that the mean EPM of nylon wool non-adherent non-phagocytic cells is centered around - 1-05 um * sec1 V-' cm is interesting. West, Boozer & Herberman (1978) recently suggested that effector cells of ADCC could be classified as 'low affinity' E-RFC. Combining free-flow electrophoresis and analytical electrophoresis, Chollet et al. (1978) described three T cell subpopulations characterized by their EPM: -1 10, - 1-20 and - 1-35 jm * sec 1 V 1 cm. The 'high affinity' E-RFC were identified as the -1I 20 and -1I35 gum sec' V'- cm cell populations. These authors suggested that the Hm * sec 1 V-1 cm subpopulation contained the 'low affinity' E-RFC. This population is very -1-10 close to that centered around - 1-05 jim sec' V-1 cm and containing the lytic cells in our assay system. Our data are therefore compatible with the hypothesis that effector cells of ADCC could be the 'low affinity' E-RFC. The additional observation that in our hands the mean EPM of nylon wool 'weakly adherent' cells is close to - 1 05 pm * sec - 1 V-1 cm seems to be particularly important. Although this cell subset had surface charge similar to that of cells responsible for ADCC in our assay system it exhibited no lytic activity. It is at present unknown whether this minor cell subset contains adherent T cells responsible for suppressor activity (Folch & Waksman, 1974; Basten, Miller & Johnson, 1975; Hodes & Hatchcock, 1976). However, we have no reason to reject this possibility since suppressor cells have a mobility value in the range of -1-05 to -1I10 pm sec1 V-1 cm (Dr S.N.S. Hanjan, personal communication). Whatever it may be, the combination of both cell fractionations on the basis of nylon wool adhesiveness -
-
Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
559
and free-flow electrophoresis appeared to be a powerful tool for elucidating the relationships between cell subsets with different functional properties. An important finding of the present study is that subsets of polymorphonuclear cells and lymphocytes which acted as effectors in ADCC exhibited similar electrical surface charge. This biophysical characteristic of cell membrane is dependent upon the chemical nature and the topographical distribution of ionized chemical groups of the surface macromolecular components (Mehrishi, 1972). Thus, striking differences have been reported in the chemical composition of the surface membrane of T and B murine lymphocyte populations (Mehrishi & Zeiller, 1974a, b). In ADCC, the Fc portion of an IgG molecule, which had previously combined with antigens on target cells, interacts with Fc receptors on effector cells. In view of the identical surface charge of polymorph and lymphocyte effectors in ADCC, it might be argued that either a defined chemical composition of cell surface or some spatial arrangement of Fc receptors in the plasma membrane are required to initiate cytotoxicity. Thus, the detailed investigation of cell surface topochemistry might be essential to characterize definitely K cells and to elucidate the mechanism of cell interactions in ADCC. We are grateful to Dr C. Vigneron for help in purification of polyspecific anti-HLA IgG. The excellent technical assistance of M. Batoz, S. Droesch and M. Th. Lenarduzzi is greatly appreciated. We wish to thank J. Bara for typing the manuscript. The study was supported by a grant from INSERM, no. 79-5-018-2.
REFERENCES BASTEN, A., MILLER, J.F.A.P. & JOHNSON, P. (1975) T celldependent suppression of an anti-hapten antibody response. Transplant Rev. 26, 130. BAKACS, T., GERGELY, P. & KLEIN, E. (1977) Characterization of cytotoxic human lymphocyte subpopulations: the role of Fc-receptor-carrying cells. Cell. Immunol. 32, 317. CHASSAGNE, J., CHOLLET, P., VUILLAUME, C. & PLAGNE, R. (1977) Immunologic status of cancer patients: correlation between number of active rosettes and analytical electrophoresis of lymphocytes. Biomedicine, 27, 93. CHESS, L. & SCHLOSSMAN, F. (1977) Human lymphocyte subpopulations. Advances in Immunology (ed. by H.G. Kunkel & F.J. Dixon), 25, 213. CHIAO, J.W., PANTic, V.S. & GOOD, R.A. (1974) Human
peripheral lymphocytes bearing both B-cell complement and T cell characteristics for sheep erythrocytes detected by a mixed rosette method. ClGn. exp. Immunol. 18, 483. CHOLLET, P., CHASSAGNE, J., THIERRY, C., SAUVEZIE, B., SERROU, B. & PLAGNE, R. (1977) Isolation and electrophoretic mobility of three lymphoid populations in normal human blood. Europ. J. Cancer, 13, 333. CHOLLET, P., HERVE, P., CHASSAGNE, J., MASSE, M., PLAGNE, R. & PETERS, A. (1978) Applications of cell electrophoretic methods (preparative and analytical) in demonstrating the surface heterogeneity of T-lymphocytes in man. Biomedicine, 28, 119. CLARK, R.A. & KLEBANOFF, S.J. (1977) Studies on the mechanism of antibody-dependent polymorphonuclear leukocyte-mediated cytotoxicity. 7. Immunol. 119, 1413. DICKLER, H.B., ADKINSON, N.F. & TERRY, W.D. (1974) Evidence for individual human peripheral blood lymphocytes bearing both B and T cell markers. Nature, 247, 213. ERmIN, O., KRAFT, D., CooMBs, R.R.A., FRANKS, D., ASHBY, J. & PLUMB, D. (1977) Surface characteristics of the human K (Killer) lymphocytes. Int. Archs. Allergy and Appl. Immunol. 55, 112. FOLCH, H. & WAKSMAN, B.H. (1974) The splenic suppressor cell. I. Activity of thymus-dependent adherent cells: change with age and stress. A. Immunol. 113, 127. GILL, P.G., WALLER, C.A. & MACLENNAN, I.C.M. (1977) receptors
Relationships between different functional properties of human monocytes. Immunology, 33, 873. HANDWERGER, B.S. & SCHWARTZ, R.H. (1974) Separation of murine lymphoid cells using nylon wool columns. Recovery of the B cell-enriched population. Transplantation, 18, 544. HANJAN, S.N.S., TALWAR, G.P., KIDWAI, Z. & NATH, I. (1977) Delineation and quantitation of human peripheral blood lymphocyte subpopulations by electrophoretic mobility and role of surface charge in cell to cell interaction. J. Immunol. 118, 235. HAYRY, P., TOTTERMAN, T.H. & RANIU, A. (1977) Lack of subclass selection during density purification of human lymphocytes. Clin. exp. Immunol. 28, 341. HODES, R.J. & HATCHCOCK, S. (1976) In vitro generation of suppressor cell activity; suppression of in vitro induction of cell mediated cytotoxicity. A. Immunol. 116, 167. HORWITZ, D.A. & GARRETT, M.A. (1977) Distinctive functional properties of human blood lymphocytes: a comparison with T lymphocytes, B lymphocytes and monocytes. J. Immunol. 118, 1712.
JuLIUS, M.H., SIMPSON, E. & HERZENBERG, L.A. (1973) A
rapid method for the isolation of functional thymusderived murine lymphocytes. Europ. J. Immunol. 3, 645. KAPLAN, J.H. & UZGIRIS, E.E. (1976) Identification of T and B cell subpopulations in human peripheral blood: electrophoretic mobility distributions associated with surface marker characteristics. J. Immunol. 117, 115. KAY, H.D., BONNARD, G.D., WEST, W.H. & HERBERmAN, R.B. (1977) A functional comparison of human Fcreceptor bearing lymphocytes active in natural cytotoxicity and antibody-dependent cellular cytotoxicity. J. Immunol. 118, 2058. KOvITHAVONGS, T. & DOSSETOR, J.B. (1977) Difference in 51Cr uptake and release by T and non-T lymphocytes and its significance in interpreting some discrepant results of 5'Cr release cytotoxic assays. Transplantation, 23, 505. LARCAN, A., STOLTZ, J.F. & STREIFF, F. (Eds.) (1974) Introduction al'atude des potentiels 6lectriques aux interfaces solide-liquide. Considerations generales sur la double couche. In: La Charge Llectrique des leiments Figures du Sang, p. 14, Doin.
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MANTOVANI, A., CAPIOLI, V., GRITTI, P. & SPREAFICO, F. (1977) Human mature macrophages mediate antibodydependent cellular cytotoxicity on tumour cells. Transplantation, 24, 291. MEHRISHI, J.N. (1972) Molecular aspects of the mammalian cell surface. Progr. Biophys. Mol. Biol. (Ed. by J.A.V. Butler and D. Noble), 25, 1. MEHRISHI, J.N. & ZEILLER, K. (1974a) T and B lymphocytes: striking differences in surface membrane. Brit. Med. 7. i, 360. MEHRISHI, J.N. & ZEILLER, K. (1974b) Surface molecular components of T and B lymphocytes. Europ. J. Immunol. 4, 474. MOOKERGE, B.K. (1976) Influence of separation techniques on the distribution and function of lymphocyte subpopulations. A comparison of three techniques. Transplantation, 22, 101. MUELLER, J., BRUN DEL RE, G., BUERKI, H., KELLER, H.U., HESS, M.W. & COTTIER, H. (1975) Non specific acid esterase activity: a criterion for differentiation of T and B lymphocytes in mouse lymph nodes. Europ. jr. Immunol. 5, 270. NYHOLM, R.E. & CURRIE, G.A. (1978) Monocytes and macrophages in malignant melanoma. II. Lysis of antibody coated human erythrocytes as an assay of monocyte function. Brit. 7. Cancer, 37, 337. PAPE, G.R., TROYE, M. & PERLMANN, P. (1977) Characterization of cytolytic effector cells in peripheral blood of healthy individuals and cancer patients. I. Surface markers and K cell activity after separation of B cells and lymphocytes with Fc-receptors by column fractionation. 7. Immunol. 118, 1919. Poo, M.M., Poo, W.J.H. & LAMES, J.W. (1978) Lateral electrophoresis and diffusion of Concanavalin A receptors in the membrane of embryonic muscle cell. J. Cell Biol. 76, 483.
S)reiff
POPLACK, D.G., BONNARD, G.D., HOLIMAN, B.J. & BLAISE, R.M. (1976) Monocyte-mediated antibody-dependent cellular cytotoxicity: a clinical test of monocyte function. Blood, 48, 809. PUDIFIN, D.J., DUURSMA, J., FORMO, R. & BRAIN, P. (1978) Double marker lymphocytes in patients receiving tetracycline. C/in. exp. Immunol. 34, 143. RAPTOPOULOU, G. & GoULIs, G. (1977) Physiological variations of T cells during the menstrual cycle. Clin. exp. Immunol. 28, 458. SAAL, J.G., RIEBER, E.P., HADAM, M. & RIETHMULLER, G. (1977) Lymphocytes with T-cell markers cooperate with IgG antibodies in the lysis of human tumour cells. Nature (Lond.), 265, 158. STEIN, G. (1975) Separation of human lymphoid cells by preparative cell electrophoresis. Z. Immun. Forsch. Bd. 150S, 68. TERASAKI, P.I. (1969) Manual of Tissue Typing. N.I.H., Bethesda Med., PB195, 24. VASSAR, P.S., LEVY, E.M. & BROOK, E.D. (1976) Studies on the electrophoretic separability of B and T human lymphocytes. Cell. Immunol. 21, 257. WEST, W.H., CANNON, G.B., KAY, H.D., BONNARD, G.D. & HERBERMAN, R.B. (1977) Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells. 7. Immunol. 118, 335. WEST, W.H., BOOZER, R.B. & HERBERMAN, R.B. (1978) Low affinity E-rosette formation by the human K cell. 7. Immunol. 120, 90. Yu, D.T.Y., CLEMENTS, P.J. & PEARSON, C.M. (1977) Effect of corticosteroids on exercise-induced lymphocytosis. C/in. exp. Immunol. 28, 326. ZEILLER, K. (1972) Separation of lymphocyte subpopulation by use of free-flow electrophoresis. A comprehensive survey of recent research. Behring Inst. Mitt. 52, 11.
Characterization of the human peripheral effector cells mediating antibody dependent cellular cytotoxicity against allogenic cells MIREILLE DONNER, * COLETTE RAFFOUXt & F. STREIFFt * Research Unit ofExperimental Cancerology and Radiobiology, U.95 INSERM, Plateau de Brabois, 54500 Vandoeuvre-lis-Nancy and t The Transfusion Center-19, rue Lionnois, 54000 Nancy, France.
(Accepted for publication 9 April 1979)
SUMMARY
The effector cell populations in human peripheral blood responsible for antibody-dependent cellular cytotoxicity against allogenic cells coated with HLA polyspecific antibodies were investigated using several separation techniques including preparative electrophoresis. Electrophoresis produced a marked effector cells enrichment in a range of 2-5 fractions which exhibited an intermediary electrophoretic mobility. Monocytic cells do not contribute an effector mechanism but minor subsets ofpolymorphonuclear cells and nylon wool non-adherent non-phagocytic lymphocytes displayed ADCC. Both effector cell populations were found to exhibit a similar electrical charge of cell surface centered around - 1 05 jim sec 1 V-i cm. These observations provided a precise biophysical basis for the identification of effector cells in ADCC. -
INTRODUCTION The cytotoxic potential of non-sensitized cells against target cells coated with anti-target cell IgG has been extensively studied over the past years but it is not completely understood. In order to display antibody-dependent cellular cytotoxicity (ADCC), the effector cells must have receptors for structures in the Fc fragment of homologous and heterologous IgG. However, Fc-bearing cells are heterogeneous and the nature of cells effecting ADCC is still a central issue. An important observation is that the type of target cells and the source of antibody appear to dictate the nature of the effector cells. In some target systems, these effector cells include polymorphonuclear leucocytes (Clark & Klebanoff, 1977; Gill, Waller & MacLennan, 1977), monocytes (Poplack et al., 1976; Nyholm & Currie, 1978), and macrophages (Mantovani et al., 1977). In other systems, undefined lymphocyte like cells, known as 'killer cells' (K cells) are effector cells in ADCC. The nature of human K cells is controversial and some groups have claimed that they appear to be of T cell origin (Kay et al., 1977; Pape, Troye & Perlmann, 1977; Saal et al., 1977), whereas others attribute the antibody-dependent cellular cytotoxicity to B cells at some stages of their differentiation (Chess & Schlossman, 1977) or to non-T lymphocytes possessing a low concentration of surface immunoglobulin (Eremin et al., 1977). In contrast, recent studies indicated that K cells are 'null cells' which lack easily detectable T and B lymphocyte markers (Horwitz & Garrett, 1977; Bakacs, Gergely & Klein, 1977). Great caution is in order when considering these results. Studies have shown a variable expression of surface markers and receptors on lymphocyte subpopulations. Cell separation techniques involving Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; K cells, killer cells; EPM, electrophoretic mobility; FCS, foetal calf serum; ANAE, a-naphthyl acetate esterase; E-RFC: E-rosette forming cells. Correspondence: Dr Mireille Donner, Research Unit of Experimental Cancerology and Radiobiology, U.95 INSERM, Plateau de Brabois, 54500 Vandoeuvre-les-Nancy, France. 0099-9104/79/1200-0549$02.00 (0 1979 Blackwell Scientific Publications
549
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Mireille Donner, Colette Raffoux & F. Streiff
rosette sedimentation with SRBC have limitations. Recent observations suggest that some discrepancies resulted from the use of different procedures for separating rosetting from non-rosetting cells (West et al., 1977). Moreover, a cell subset combines the surface markers of both T and B cell populations (Chiao, Pantic & Good, 1974; Dickler, Adkinson & Terry, 1974). Apart from classical markers, other means of identification of cell subpopulations involve biophysical properties of cells, including electrical charge of cell surface or cell density. Thus, cell electrophoresis has been useful for the identification of cell subsets in human system (Stein, 1975; Kaplan & Uzgiris, 1976; Chassagne et aL., 1977; Chollet et aL., 1977; Hanjan et aL., 1977; Chollet et aL., 1978). These previous results led us to raise the question whether K cells might be unambiguously defined using the electrical charge of cell surface. The advantage of characterizing cells on this biophysical basis is that, as long as a cell is living and intact, the ionizable groups of the surface macromolecules will give rise to an electrical charge. The present report describes studies designated to characterize human K cells by combining preparative and analytical cell electrophoresis. As K cells might play an important role in graft rejection, antitumour responses and autoimmune diseases, we considered allogenic cells as the source of target cells. In our assay system, we present evidence for the identification of human K cells as subsets of polymorphonuclear cells and nylon wool non-adherent non-phagocytic lymphocytes exhibiting an intermediate electrophoretic mobility (EPM) of -105 + 002 pm sect v -i cm. -
MATERIALS AND METHODS Cell collection. Blood was drawn from healthy adult donors of blood group 0 and defibrinated by rotary shaking on glass beads. Preparation of polymorph-enriched leucocyte suspensions. Defibrinated blood was sedimented at room temperature for 45 min with one half its volume of 6% dextran in 0.9% saline. The leucocyte-rich plasma was removed, centrifuged at 200 g for 8 min and the cell pellet washed in MEM medium, and finally suspended in a low ionic strength buffer suitable for freeflow electrophoresis (Zeiller, 1972). Polymorphonuclear cells represented 80-90% of the total cell population, as revealed by cytocentrifuged cell smears. Preparation of mononuclear cells. Mononuclear cells were obtained by separation on Ficoll-Hypaque (S.P. 1.077) and then washing twice in MEM medium. Differential counting of cytocentrifuge preparations stained to detect cells containing nonspecific esterase revealed that the percentage of monocytes ranged from 10 to 25%. In some series of experiments, phagocytic cells were removed from cell suspensions after ingestion of carbonyl iron (2 mg carbonyl iron/ 106 nucleated cells). The cell mixture was incubated at 370C for 20 min, then exposed to a strong magnetic field for 10 min. Cells not attracted to the magnet were harvested and washed twice in MEM medium+ 10% FCS. Following this treatment, mononuclear preparations contained less than 4% monocytes. Cellfractionation on nylon wool columns. In some series of experiments, nylon wool separation of Ficoll-Hypaque purified and carbonyl iron treated cells was performed as described by Julius, Simpson & Herzenberg (1973). After the recovery of non-adherent cells, nylon wool columns were rapidly washed with 100 ml MEM medium+ 10% FCS. Cells in the second effluent were referred to as 'weakly adherent cells'. Finally, the adherent cells were recovered by strongly compressing the nylon wool with a syringe plunger as described by Handwerger & Schwartz (1974). Preparative electrophoresis. Cells were fractionated in a free-flow preparative electrophoresis Model FF5 (Bender Hobein, Munich, Germany). The buffer used in the separation chamber had the following composition: 0 04 M potassium acetate; 0-015 M triethanolamine; 0-24 M glycine made isoosmotic with glucose. The pH was adjusted to 7-2-7-4 with acetic acid. The electrode chambers contained buffer with 0.075 M triethanolamine, 0 004 M potassium acetate. For fractionation of cells a field strength of approximately 87-90 V/cm and a current of 210 mA were used. Just before electrophoretic fractionation, cells were suspended in the weak ionic strength buffer and filtered on nylon mesh to remove cell clumps. Separations were performed at 6VC. To achieve satisfactory separation, the buffer flow rate was adjusted to about 450 ml/hr, each cell remaining in the electrical field for about 240 sec. Cell suspensions at a concentration of 8-12 x 106 cells/ml were injected into the electrophoresis chamber at a flow rate of 5 ml/hr. The fractions were collected into test tubes containing 1 ml of MEM medium with 10% foetal calf serum. To minimize cell injury, test tubes were harvested each hour. Cells were immediately washed and suspended in MEM medium with 10% FCS: cells in each fraction were then counted in a haemocytometer. Analytical cell electrophoresis. The electrophoretic mobility was determined by the use of an electrophoresis chamber of a circular cross section and a small volume (0-8 ml) equipped with reversible silver-silver chloride-potassium chloride (1.0 M) electrodes at 25°C (Mehrishi, 1972). Cells were scored in 0-145 M NaCl adjusted to pH 7-2+ 0-1 with NaHCO3. The electrophoretic mobility is expressed in pm-sec- V-1 cm. The reliable performance of the apparatus was monitored by deter-
Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
551
mining the EPM of washed human erythrocytes which present a reproducible electrophoretic mobility of - 108 + 0 03 pum sect1 V-' cm. Cytotoxicity assay. Human leucocytes of peripheral blood obtained from a panel of healthy adult donors were used as target cells. Leucocytes were prepared from 10 ml heparinized whole blood using a Ficoll-Hypaque gradient. The cells in the interface were collected, washed once in autologous plasma, then in Hanks' BSS and resuspended in the same medium at a concentration of 2 x 106 nucleated cells/ml. The use of this type of target cells led us to take into consideration the recent observation that monocytes, T and non-T human lymphocytes take up and release different amounts of 51Cr (Kovithavongs & Dossetor, 1977). It could be the origin of artefacts in ADCC and we have therefore chosen trypan blue exclusion test instead of 51Cr as a means of testing ADCC. Cell fractions obtained after electrophoretic separation were washed, suspended in Hanks' BSS at a concentration of 2 x 106 cells/ml and used as effectors. Purified IgG antibodies were prepared by Sephadex G 200 fractionation of polyspecific anti-HLA sera. IgG were lyophilized and reconstituted aliquots always used within 2 weeks. Cytotoxicity assay was adapted from a standard assay routinely used in Blood Transfusion Center (Terasaki, 1969). Target cells in 1 pl were dispensed into each well of a Microtest I plate (Falcon 3034). One microlitre of polyspecific antiHLA IgG was added in appropriate dilutions (undiluted or diluted 1: 2, 1:3, before addition to the assay) and the mixures target cell-IgG were incubated at room temperature for 30 min. Effector cells in 1 p1 were then added and a further incuba-tion was carried out at room temperature. Preliminary studies of lytic activity of effector cells at different times of incubation (1-18 hr) showed that a cytotoxicity in the control preparations was apparent after 3 hr incubation period and a 2 hr incubation was therefore chosen for the subsequent experiments. Moreover, previous observations that electrophoretic separation introduced itself an enrichment of effector cells in a ratio ranging from 15 to 25 led us to take an effector-target cell ratio of 1:1. After the incubation, supernatants were withdrawn with a Pasteur pipette and 1 pl of trypan blue was dispensed into each well. Readings were undertaken with an inverted microscope 10 min after adding the dye. The ADCC was estimated by the percentage of stained cells/total number of cells in several microscope fields. A cytotoxicity scale was established as following: + if less than 30% dead cells, + if 30-50% dead cells, + + if 50-75% dead cells. Since microscope readings cannot differentiate effector cells from target cells, some experiments were undertaken with PHA-lymphoblasts as targets to determine if lytic activity was directed to target cells. Ficoll-Hypaque purified blood cells were stimulated with 0 5 mg/ml PHA (Wellcome) at 370C for 5 days. After the incubation period, most of the cultures contained 90% of blast cells which were used as targets and could be easily distinguished from effector cells by their size. These experiments showed that target cells were killed and effector cells excluded trypan blue. Several controls were included in each assay: (1) target cells along with AB serum, (2) effector cells with AB serum, (3) effector and target cells with AB serum to verify that results were not owing to a natural cytotoxicity of effectors, (4) target cells with polyspecific anti-HLA IgG to exclude a complement-independent cytotoxic action of IgG. Finally, each assay included additional wells where rabbit normal serum was added as source of complement to verify the cytotoxic potential of anti-HLA IgG against target cells. Staining for acid a-naphthyl acetate esterase (ANAE). Aliquots of cell suspensions and different fractions obtained after electrophoretic separation were stained for non-specific esterase as described by Mueller et al. (1975) and Hayry, Totterman & Ranki (1977). Non-specific esterase staining applied to peripheral blood cell suspensions is a valuable marker of monocytic cells. Activity is very strong in monocytes and cells were easily distinguishable by the pre sence of multiple intensely redstained granules in the cytoplasm.
RESULTS
Evidence that electrophoretic fractionation of peripheral blood cells induces an enrichment of etfector cells
in ADCC Ficoll-Hypaque purified cells were fractionated in free-flow electrophoresis and the distribution profiles were established by calculating the relative proportions of nucleated cells in each fraction. To compare the results of separate experiments required great caution. The separation depends on numerous parameters (buffer flow rate, temperature, current) which may undergo weak day-to-day variations even under carefully controlled technical conditions. The use of an electrophoretic 'marker' was therefore necessary. Red blood cells which exhibit a highly reproducible surface electrical charge provided such a marker. In each experiment the fraction containing the red cell peak was designated as zero. Fractions with higher EPMs were numbered + 1, + 2, + 3,. . ., and the fractions with lower EPMs were numbered - 1, -2, -3,... Ficoll-Hypaque purified were usually found in 30-35 fractions (fractions -25 to + 10). However, the shapes of profiles presented important individual variations and in more than 35 experiments three different types of profile were recognized (Fig. 1). Type A corresponded to a profile where nucleated cells had a peak in fractions -6 to -8. In the profile B, the peak is broader and was found in fractions 0 to -5. In the profile C, most of the nucleated cells were found in fraction 0. The relative proportions of profiles A, B and C were 600/, 300 0 and 100 0, respectively.
552
Mireille Donner, Colette Raffoux & F. Streif (a)
5
(b) 00
0
.C5 0)
( c) 10
5
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0 -10 Standard fractions
FIG. 1. Electrophoretic mobility distribution of human peripheral blood lymphocytes. Abscissa: standard fractions according to the peak of erythrocytes; the fraction with the peak of erythrocytes is designated 0.
In each experiment, the fractions of extreme ranges corresponding to the low EPM cells and the high EPM cells were separately pooled to get a sufficiently large number of cells for the cytotoxicity assay. Fig. 2 shows the results of ADCC assays of fifteen representative experiments. In our experimental conditions, no cytotoxicity was observed when Ficoll-Hypaque purified and electrophoretically unseparated cells were used as effectors. In contrast, cytotoxicity was detected with fractionated samples. Fig. 2 shows that effector cells distributed in intermediary fractions between fractions containing slow and fast moving cells. Although the number of cytotoxic fractions varied according to the experiment, it appeared from Fig. 2 that effector cells were found in nearly similar fractions.
Electrokinetic surface properties of cells responsible for ADCC The identification of effector cells in ADCC required an unambiguous characterization of cells on a
Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
553
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FIG. 2. Comparison of the fractions positive in antibody-dependent cellular cytotoxicity from peripheral blood fractionated with preparative electrophoresis. Each area represents number of cellular fractions which were recovered within each experiment and shaded area, fractions positive in ADCC.
precise biophysical basis: the EPM or the zeta potential. However, direct calculations of absolute values of EPMs from free-flow electrophoretic separation may lead to artefacts. The use of a weak ionic strength buffer in free-flow electrophoresis markedly modify the spatial distribution of electrical charges near the cell surface and consequently the hydrodynamic plane of shear (Larcan, Stoltz & Streiff, 1974). Moreover, a high uniform electric field resulted in a redistribution of membrane receptors and it might be suggested that alterations in membrane electrical characteristic occur when cells migrate in a high electric field (Poo, Poo & Lames, 1978). To eliminate possible artefacts, cells contained in fractions after electrophoretic fractionation were investigated by the use of the classical microscope method of cell electrophoresis to determine the EPMs in physiological saline under a low electric field as described in 'Methods' section. The mean EPMs of cells contained in cytotoxic fractions are summarized in Table 1. As it can be seen the EPMs of cells were centered around -105 + 0-02 pm sec 1 V'- cm. -
The nature of effector cells Since monocytes and polymorphs have been shown to act as effector cells in some ADCC systems TABLE 1. Mean electrophoretic mobility values of cells in fractions positive in antibody-dependent cellular cytotoxicity Experiment number
Mean electrophoretic mobility in pm sec- I V-1 cm+ s.e.*
Number of scored cells
1 2 3 4 5 6 7
- 103+002t - 1-08+0-01
74 57 92 52 43 37 52
-
-1-03+0-02 - 1-07+ 0*02
-1-07+0*01 - 1-06+ 0-02 - 1-07+ 0-02
* Electrophoretic mobility was determined at
250C
in physiological
serum 0-145 M adjusted to pH 7-2.
f EPM values centimeter+ s.e.
were
expressed in micron per second and per Volt
Mireille Donner, Colette Raffoux & F. Streiff
554
-5 0
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~
scale
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FIG. 3. Electrophoretic distribution profiles of Ficoll-purified cells. Repartition of monocytes and ADCC activity in each fraction. The percentage of monocytes in a fraction refers to the number of nucleated cells contained in this fraction.
(Clark & Klebanoff, 1977; Gill et al., 1977; Poplack et al., 1976; Nyholm & Currie, 1978), the question raised whether these cell types could be effectors in our ADCC assay. Lytic activity of monocytes Monocytes represented 10-25% of Ficoll-Hypaque purified cells. To determine the possible role of monocytic cells in the lysis of target cells, the content of monocytes was measured in each fraction after electrophoretic separation. As it can be seen in Fig. 3, there was no correlation between ADCC activity and the content of esterase-positive cells. Some no cytotoxic fractions contained more monocytes than cytotoxic ones. To examine further the relationships between cytotoxic activity and monocyte number, mononuclear cell preparations were depleted of monocytes by the iron-filing magnetic separation before free-flow electrophoresis. Table 2 shows that iron treatment failed to reduce lytic activity of electrophoretically fractionated cells.
Lytic activity ofpolymorph-enriched leucocyte preparations The potential involvement of polymorphonuclear cells in ADCC was evaluated after electrophoretic fractionation of polymorph-enriched leucocyte preparations. As depicted in Fig. 4, polymorph--rich populations were moderately slower than Ficoll-purified mononuclear cells. The cytological analysis of cells contained in fractions showed that polymorphs represented more than 95%4 of cells except in fractions of high electrophoretic mobility which were significantly contaminated with lymphoid cells. Cells with killer activity were confined in some fractions where contaminating lymphoid cells do not account for a high percentage. Although 15-20 fractions were recovered, the lytic activity was localized in a range of 2-5 fractions suggesting that only a polymorph subset is involved in ADCC. It should be also noticed that these cytotoxic fractions nearly corresponded to those found in Ficoll-purified and electrophoretically fractionated cells.
Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
555
TABLE 2. Failure of monocyte depletion to suppress ADCC activity of electrophoretically fractionated blood cells
Experiment
Carbonyl iron test
Cell recovery after treatment (%)
Monocytes in unfractionated cells (%)
1 2 3 1 2 3
None None None Yes Yes Yes
100* 100 100 55 52 54
12t 21 11 2 4 3
Monocytes in
cytotoxic
Cytotoxicity
fractions (%)
scale
8t
++ + + + ++ +
29 24 5 5 4
* Cell suspensions which were not treated with carbonyl iron refer to 100% of recovery.
tFigures represent percentage of monocytes in cell suspensions before electrophoretic separation.
Effect offractionation on nylon wool columns To further characterize the cells responsible for ADCC, we combined the fractionations by nylon wool columns and free-flow electrophoresis. The passage of Ficoll-purified human peripheral blood over nylon wool columns in our experiment revealed three subpopulations: non-adherent, weakly adherent, and adherent which about represented 60%, 5%0, and 10%, respectively, of the cell population loaded on the columns. When these populations were investigated by the use of classical particle/
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FIG. 4. Comparison between electrophoretic distribution profiles of polymorph enriched cell suspensions (---) and Ficoll-purified mononuclear cells (- --).
556
Streiff
Mireille Donner, Colette Raffoux & F.
cell electrophoresis to determine the EPM, it was found that non-adherent cells had EPMs higher than - 10 pm * sec1 V-i cm (Fig. 5). In contrast, weakly adherent cells contained a mixture of cells which exhibited low and high mobilities. As regards adherent cells, although most cells had an EPM lower - V-i cm, a small percentage of high mobility cells was found. than -10 pm sec' These findings suggest that the fractionation by nylon wool columns before electrophoretic separation
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FIG. 5. Electrophoretic mobilities of non-adherent, weakly adherent and adherent cells. Each point represents the EPM of a single cell. Relative proportions of non-adherent, weakly adherent and adherent cells are not respected.
20 l 20
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5
-15
-10
-5
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FIG. 6. Electrophoretic distribution profile of non-phagocytic, nylon wool non-adherent cells from human peripheral blood.
Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
557
might be meaningful to characterize cells responsible for ADCC. Therefore, Ficoll-purified cells and treated with carbonyl iron were fractionated by passage over nylon wool columns and subsequently by free-flow electrophoresis. In all experiments, a lytic activity was observed in some fractions of non-adherent cells. Fig. 6 shows an electrophoretic distribution profile of nylon wool non-adherent cells where the lytic cells could be recovered from fractions -5-8. It should be noted that lytic activity of Ficoll-purified and nylon wool non-adherent cells distributed in identical fractions. The cytological analysis of effector cells contained in lytic fractions of electrophoretically fractionated nylon wool non-adherent cells revealed that most of the cells appeared to be lymphocytes with esterase activity. It should be noticed that a high percentage of cells presented esterase activity distributed in several spots near the plasma membrane (Fig. 7). .
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FIG. 7. Demonstration of ANAE activity in smears of nylon wool non-adherent non-phagocytic effector cells in ADCC. Most of the cells presented small esterase positive spots near the plasma membrane (indicated by an arrow).
DISCUSSION The present study clearly suggests that free-flow electrophoresis of leucocytes from human peripheral blood represents a reliable and convenient method to obtain enriched cell subpopulations exhibiting functional properties. Ficoll-Hypaque purified and unfractionated cells were unable to mediate ADCC
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Mireille Donner, Colette Raffoux & F. Streiff
in our experiment, while some fractions of electrophoretically separated cells were effective in ADCC. Thus, free-flow electrophoresis allowed a 15-20 fold enrichment of effector cells. It should be appreciated that this method is useful to fractionate a complex population of viable cells into component subpopulations without manipulations of cells involving fixation of immunological reagent on cells. Moreover, the fractions are directly recovered. Our data also show that the effector cells of ADCC may be characterized by their electrical charge of cell surface. The determination of EPM at the single cell level by classical cell/particle microelectrophoresis showed that Ficoll-Hypaque purified cells from human peripheral blood distributed between -0f60um sec' V` cm and - 1-50 um sec1 V` cm. In our assay system, killer activity was found in fractions exhibiting EPMs centered around - 105 um * sec1 V- cm. The findings that killer activity was not found in strictly identical fractions and that the number of cytotoxic fractions varied according to the experiments is presumably attributable to individual variations registered in distribution profiles of EPM. These fluctuations likely reflect individual differences in relative proportions of cell subsets and cannot be explained by technical factors such as lymphocyte separation methods (Mookerge, 1976), since we always used the same technique with well defined conditions. The possibility that the low ionic strength buffer used in electrophoretic separation may be responsible for these variations has been also excluded. Vassar, Levy & Brook (1976) reported similar observations with a high ionic strength buffer. Rather, there are some reasons to suppose that 'physiological' factors could account for these variations, as reported previously (Yu, Clements & Pearson, 1977; Raptopoulou & Goulis, 1977; Pudifin et al., 1978). In characterizing the cells responsible for lytic activity against allogenic lymphocytes coated with polyspecific anti-HLA IgG, several facts are clear. Our results showed a lack of correlation between K cell activity and levels of monocytes in different fractions after electrophoretic separation. Furthermore, the results observed after the 'iron and magnet' technique are not in favour of monocytes as lytic cells despite the fact that killer activity is weakly diminished after removal of iron-phagocytozing cells. Since the yield of cells removed after carbonyl iron treatment was nearly 4000 and the monocyte percentage was not so high, there is possibility that other cell types perhaps mediating ADCC, are eliminated with monocytes. It seems therefore reasonable to assume that, in our assay system, monocytes do not constitute an effector mechanism. In contrast, both polymorphonuclear cells and nylon wool non-adherent non-phagocytic lymphoid cells were able to lyse efficiently target cells. Since killer activity was found in a range of 2-5 fractions, it is clear that lytic cells constitute minor subsets of both cell types which is possible to characterize on the basis of the electrical charge of cell surface with the cell/particle analytical microelectrophoresis. The observation that the mean EPM of nylon wool non-adherent non-phagocytic cells is centered around - 1-05 um * sec1 V-' cm is interesting. West, Boozer & Herberman (1978) recently suggested that effector cells of ADCC could be classified as 'low affinity' E-RFC. Combining free-flow electrophoresis and analytical electrophoresis, Chollet et al. (1978) described three T cell subpopulations characterized by their EPM: -1 10, - 1-20 and - 1-35 jm * sec 1 V 1 cm. The 'high affinity' E-RFC were identified as the -1I 20 and -1I35 gum sec' V'- cm cell populations. These authors suggested that the Hm * sec 1 V-1 cm subpopulation contained the 'low affinity' E-RFC. This population is very -1-10 close to that centered around - 1-05 jim sec' V-1 cm and containing the lytic cells in our assay system. Our data are therefore compatible with the hypothesis that effector cells of ADCC could be the 'low affinity' E-RFC. The additional observation that in our hands the mean EPM of nylon wool 'weakly adherent' cells is close to - 1 05 pm * sec - 1 V-1 cm seems to be particularly important. Although this cell subset had surface charge similar to that of cells responsible for ADCC in our assay system it exhibited no lytic activity. It is at present unknown whether this minor cell subset contains adherent T cells responsible for suppressor activity (Folch & Waksman, 1974; Basten, Miller & Johnson, 1975; Hodes & Hatchcock, 1976). However, we have no reason to reject this possibility since suppressor cells have a mobility value in the range of -1-05 to -1I10 pm sec1 V-1 cm (Dr S.N.S. Hanjan, personal communication). Whatever it may be, the combination of both cell fractionations on the basis of nylon wool adhesiveness -
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Antibody-dependent cellular cytotoxicity to allogenic lymphocytes
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and free-flow electrophoresis appeared to be a powerful tool for elucidating the relationships between cell subsets with different functional properties. An important finding of the present study is that subsets of polymorphonuclear cells and lymphocytes which acted as effectors in ADCC exhibited similar electrical surface charge. This biophysical characteristic of cell membrane is dependent upon the chemical nature and the topographical distribution of ionized chemical groups of the surface macromolecular components (Mehrishi, 1972). Thus, striking differences have been reported in the chemical composition of the surface membrane of T and B murine lymphocyte populations (Mehrishi & Zeiller, 1974a, b). In ADCC, the Fc portion of an IgG molecule, which had previously combined with antigens on target cells, interacts with Fc receptors on effector cells. In view of the identical surface charge of polymorph and lymphocyte effectors in ADCC, it might be argued that either a defined chemical composition of cell surface or some spatial arrangement of Fc receptors in the plasma membrane are required to initiate cytotoxicity. Thus, the detailed investigation of cell surface topochemistry might be essential to characterize definitely K cells and to elucidate the mechanism of cell interactions in ADCC. We are grateful to Dr C. Vigneron for help in purification of polyspecific anti-HLA IgG. The excellent technical assistance of M. Batoz, S. Droesch and M. Th. Lenarduzzi is greatly appreciated. We wish to thank J. Bara for typing the manuscript. The study was supported by a grant from INSERM, no. 79-5-018-2.
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