Immunology 1976 30 325
Cell-mediated cytotoxicity CHARACTERIZATION OF THE EFFECTOR CELLS
MARIA M. DE E. DE BRACCO, M. A. ISTURIZ & J. A. MANNIInstitutodeInvestigaciones Mtedicas, Universidad de Buenos Aires, Buenos Aires, Argentina
Received 7 July 1975; accepted for publication 17 July 1975
INTRODUCTION
Summary. Isolated human mononuclear cells were fractionated according to their membrane characteristics or physical properties. Adherent cells were depleted by filtration through glass columns; phagocytic cells were removed by iron treatment and cell subpopulations capable of forming rosettes with sheep erythrocytes (E), erythrocyte-antibody-complement (EAC) and chicken erythrocyte-antibody complexes (CEA) were separated by centrifugation of Ficoll-Hypaque gradients. The functional activity of the cell subpopulations obtained was assayed by testing PHA-induced cytotoxicity (PIC), antibodydependent cytotoxicity (ADCC) and blast transformation by PHA. The results of this study demonstrate that: (1) cells reacting in PIC and ADCC assays are different, adherent and phagocytic cells being necessary for full expression of PIC and not for ADCC; (2) PHA induces direct blast transformation of purified E-RFC in the absence of PIC cytotoxic cells; (3) cell populations specifically enriched in E or EAC rosette-forming cells are not cytotoxic neither in the PHA nor in antibody mediated cytotoxic assays; (4) cells participating in ADCC can be selectively purified by centrifugation of CEA
The ability of non-sensitized lymphocytes to destroy target cells combined with antibody to cell membrane antigens has been demonstrated in several species and systems (reviewed by Perlmann and Holm, 1969). Lymphoid cells activated by non-specific mitogens may also display cytotoxic activity against allogeneic, syngeneic and xenogeneic target cells (Perlmann and Holm, 1969; Holm, Perlmann and Werner, 1964). A series of studies have been performed in order to establish the nature of the effector cells involved in the different models of cytotoxicity (Harding, Pudifin, Gotch and MacLennan, 1971; Greenberg, Hudson, Shen and Roitt, 1973; Wisl0ff, Fr0land and Michaelsen, 1974a). While most evidence indicates that the cells mediating antibody-dependent cytotoxicity (ADCC) are thymus-independent (Perlmann and Holm, 1969; MacLennan, Loewi and Howard, 1969) it has been assumed that PHA-induced cytotoxicity (PIC) is a function of T cells (Sherwood and Blaese, 1973; Wisl0ff, Fr0land and Michaelsen, 1974b). However, in the latter system more than one class of cells appears to be involved (Britton, Perlmann and Perlmann, 1973; Wisl0ff et al., 1974b; Hallberg, 1974). In this study we have compared ADCC and PIC of normal human lymphoid cells after fractionation
rosettes. Correspondence: Dr Maria M. de E. de Bracco, Instituto de Investigaciones M6dicas, D. Alvarez 3000, Hospital Tornfl, Buenos Aires, Argentina. B
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Maria M. de E. de Bracco, M. A. Isturiz and J. A. Manni
procedures based on the ability of the different subclasses of human lymphocytes to form rosettes with sheep red cells (E), erythrocyte intermediates with antibody and complement (EAC) or chicken erythrocyte-antibody complexes (CEA). In addition, the effect of removing adherent and phagocytic cells on PHA-induced and antibody-mediated cytotoxicity was explored. MATERIALS AND METHODS Human lymphoid cells Lymphoid cells were isolated from heparinized or defibrinated venous blood from human volunteer donors by centrifugation on Ficoll-Hypaque gradients (B0yum, 1968). The pellet was washed with TC199 medium (Difco Laboratories, Detroit, Michigan) containing 100 u/ml penicillin 100 pg/ml of streptomycin and 2-5 per cent heat-inactivated foetal calf serum (FCS). The concentration of mononuclear cells was determined by counting the cell suspensions stained with Turk's solution. Ficollpurified cell suspensions containing 95-98 per cent mononuclear cells and 2-5 per cent granulocytes were utilized for further fractionation procedures. In some experiments the glass adherent cell population was depleted by filtration of gelatin sedimented leucocytes obtained from defibrinated blood (Coulson and Chambers, 1964) through glass bead columns (Rabinowitz, 1964). The resultant cell suspensions were centrifuged on Ficoll-Hypaque mixtures (1 -077 specific gravity), washed with TC199 and counted. Glass-filtered non-adherent cells were composed of 95-99 per cent small lymphocytes and 1-5 per cent
polymorphonuclear leucocytes. Depletion ofphagocytic cells with iron powder One millilitre of purified mononuclear cells (F), 10-20 x 106/ml were incubated with 0-2 ml of carbonyl iron powder suspension (10 mg/ml) for 1 h at 370, with occasional agitation. Cells containing iron and adhering to iron clumps were removed with a magnet. The resultant cell suspensions was composed of 95-98 per cent small lymphocytes.
Purification of E, EAC and CEA rosette-forming cells E rosettes were prepared as described by Jondal, Holm and Wigzell (1972) and EAC rosettes were obtained as described previously (Isturiz, de Bracco and Manni, 1975). When the reactions were performed for preparative purposes, the amount of the
reactants was scaled up and the concentration of E or EAC rosettes obtained was checked. Sensitized chicken erythrocyte rosettes (CEA rosettes) were prepared by incubating 1 ml of TC199 containing 5-10 x 106 lymphocytes with 1 ml of 0 5 per cent chicken erythrocytes (CE) sensitized with the maximum non-agglutinating dose of rabbit anti-CE antiserum (Manni, de Bracco and Patrucco, 1974) for 1 h at 4°. Two to three volumes (10-15 ml) of E, EAC or CEA rosettes were layered on top of a Ficoll-Hypaque gradient, centrifuged at 400 g for 20 min and both the interphase and pellet were recovered and washed three times with TC199. E and EAC were haemolysed with 0 02 M Tris buffer (pH 7 2) containing 0 75 per cent NH4CI. CEA were haemolysed by exposure to distilled water for 80 s at 40 and immediate addition of concentrated saline to restore isotonicity. Control gradient tubes were mixed again after centrifugation, washed with TC199 and E, EAC or CEA were haemolysed as described above. Viability checked by trypan blue exclusion was above 90 per cent. Cells isolated from gradient fractions were suspended in TC199 at 5 x 106/ml. All experiments were repeated three to five times.
Antibody-dependent cell cytotoxicity Antibody-dependent cytotoxicity (ADCC) was assayed as described previously utilizing 5'Cr-labelled antibody-coated CE target cells (Isturiz, de Bracco and Manni, 1975; Manni, de Bracco and Patrucco, 1974). Variable amounts of lymphocytes ranging from 0 3 to 2-5 x 106 cells/-5 ml were cultured with 2 x105 5'Cr-labelled CEA for 18 h at 37°. After centrifugation, both 1-ml aliquots of the supernatant and the residual fluid plus sediment were counted in an automatic Packard 3320 gamma counter. The percentage of s 'Cr released was calculated as follows: percentage 5'Cr released = [(activity of supernatant x 1 *5)/(total activity)] x 100. The highest sensitivity of the assay lies within 1-3 x 106 effector cells and 20-60 per cent 51Cr release (Isturiz, de Bracco and Manni, 1975).
Phytohaemagglutinin-induced cytotoxicity (PIC) Cytotoxicity of human mononuclear cells was induced by stimulation with phytohaemagglutinin (PHA-P, Difco Laboratories, Detroit, Michigan) as described by Holm and Perlmann (1967) utilizing 5'Cr-labelled CE target cells. The optimal PHA/ lymphocyte ratio was found to be 10 ug PHA/106 cells. Reaction mixtures containing 1 25-7 5 x 106
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Effector cells in cell-mediated cytotoxicity
Ficoll-purified cells, 10 ug of PHA-P/106 lymphocytes and 2 x 105 5tCr-labelled CE suspended in 1 5 ml TC199 were cultured for 18 h at 37°. Control tubes without PHA were set up simultaneously. An example of a control PHA-induced cytotoxicity (PIC) curve at constant PHA/effector cell ratio is shown in Fig. 1. As reported by others (Wisl0ff, 70r
toxicity induced by PHA (PIC) or antibody (ADCC) was explored by removing the cells capable of ingesting carbonyl iron particles (Golstein, Schirrmacher, Rubin and Wigzell, 1973) or adhering to glass. The results presented in Table 1 demonstrate Table 1. Effect of depletion of adherent or phagocytic cells on antibody-dependent or PHA-induced cytotoxicity
60[Donor 50II-
1
_ 40 H
2
w In a)
-Y
a)g 3CDH
mT_ 2C IC I
12
3
4
5
6
8I8
7
Cells (X106) Figure 1. PHA-induced cell cytotoxicity (PIC). Variable numbers of Ficoll-purified mononuclear cells were incubated in 1'S ml TC199 containing 10 pg of PHA-P/106 cells and 2x 105 51 Cr-labelled CE for 18 h at 37°. PIC is expressed as the percentage of 5 Cr released corrected for spontaneous release in the absence of PHA.
Fr0land and Michaelsen, 1974b) there was considerable variation between different donors. The percentage of 51Cr liberated was measured and calculated as described in the preceding section. Lymphocyte stimulation with phytohaemagglutinin Duplicate or triplicate culture tubes containing 106 lymphocytes suspended in 1 ml of 20 per cent FCS-TC199 were incubated with 10 ug of PHA at 370 for 48-72 h. Lymphocytes were harvested, fixed in Carnoy's solution stained with May-GrunwaldGiemsa and observed by light microscopy. The percentage of lymphoblastoid cells in 500 cells was recorded. RESULTS
Effect of the depletion of phagocytic or adherent cells on cytotoxicity The role of phagocytic and adherent cells on cyto-
Treatment of cells* None Iron None Glass filtration
ADCC (per cent)
PIC (per cent)
43 44 47
42 9 30
45
17
* Ficoll-purified cells were incubated with carbonyl iron or filtered through glass columns. Antibody-dependent cytotoxicity (ADCC) was assayed before and after treatment by incubating 2x 106 cells with 2x 105 51Cr-labelled CEA at 370 for 18 h. ADCC is expressed as the percentage of 51Cr released, corrected for spontaneous release in the absence of anti-CE. PHA-induced cytotoxicity (PlC) was assayed by incubating 4 x 10" control and treated cells with 10 pg of PHA/106 cells and 2 x 105 51 Cr-labelled CE at 370 for 18 h. PIC is expressed as the percentage of 51Cr released corrected for spontaneous release in the absence of PHA.
that depletion of phagocytic or adherent cells diminished the cytotoxic response induced by PHA, while ADCC was not altered. Iron or glass treatment of Ficoll-purified lymphocytes yielded a final cell suspension composed of 97 per cent small lymphocytes. Thus, the participation of phagocytic or adherent cells appears to be important for the full expression of PIC and not for ADCC. Distribution of cytotoxic cells after centrifugation of E rosette-forming cells
Antibody-dependent and PHA-induced cytotoxicity were tested in cellular fractions obtained by gradient centrifugation of E-rosette-forming cells (E-RFC). It has been reported previously that T cells capable of forming E rosettes can be selectively depleted from the original cell suspensions by this method (Yata, Desgranges, Tachibana and de ThM, 1973; Wybran, Chantler and Fudenberg, 1973). The results shown in Table 2 indicate that most of the cells in
Maria M. de E. de Bracco, M. A. Isturiz and J. A. Manni
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Table 2. Fractionation of E rosette-forming cells on FicollHypaque gradients
EAC-RFC
E-RFC Type of cells
Before gradient Interphase Bottom
Number of cells (x 106)
Per cent
No. cells (x 106)
Per cent
No. cells (x 106)
43 5 5-2 38-0
70 0 50 87-0
30-2 0-26 33-1
n.d. 25 4-2
n.d. 1-25 1-52
E rosettes were obtained by reacting 10 ml of Ficollpurified, glass-filtered lymphocytes (5 x 106/ml) with 10 ml of a 1 per cent sheep erythrocyte (E) suspension for 15 min at 370, centrifuged for 5 min at 200 g and kept at 40 for 1 h. After gentle homogenization the reaction mixture was centrifuged on a Ficoll-Hypaque gradient for 20 min at 400 g. Fractions were obtained from the bottom and the interphase of the gradient and E were haemolysed with 0-02 M, pH 7-2 Tris buffer containing 0 75 per cent NH4Cl. The percentage and absolute number of rosette-forming cells (E-RFC and EAC-RFC) was determined in the original and post-gradient fractions. Experimental conditions are detailed in the text. n.d. = Not determined.
the bottom fractions were E-RFC. In contrast, around 70 per cent of the cells in the interphase did not form E or EAC rosettes. The overall recovery ranged from 80-100 per cent. (a)
60k
As shown in Fig. 2, cells recovered from the interphase (non-E-RFC) exhibited greater ADCC than the original cell suspensions and the activity of the bottom fraction was low. Similarly, very low PIC activity was recovered from the sedimented E-RFC and the interphase fractions displayed activity comparable to that of controls (Fig. 2). This activity was absent in iron-treated interphase fractions. Also, experiments performed utilizing mixtures of interphase and bottom fractions in different ratios failed to reveal an enhancement of inter-phase activity by E-RFC.
Distribution of PHA blast-forming and cytotoxic cells after centrifugation of E-RFC Contro Inon-fractionated cells and the interphase and bottom fractions of an E-RFC sedimentation gradient were assayed for PIC, E-rosette formation and the blastogenic response to optimum amounts of PHA. Table 3 summarizes the results of a representative experiment. Clearly, PHA cytotoxicity to CE is present in fractions unable to respond to the same mitogen by blast transformation. As was previously reported (Yata et al., 1973), the distribution of E-RFC parallels that of blast-forming cells.
r
~01'
( b)
0
(-) a) 40k
C-
.
201-
5I
I
2
3
4
5 Cel Is (x 106 )
2
3
4
5
6
7
Figure 2. Distribution of cytotoxic cells after centrifugation of E-RFC. E rosettes were obtained by reacting 10 ml of Ficollpurified cells (5x 106/ml) with 10 ml of I per cent sheep erythrocyte (E) suspension for 15 min at 370 followed by centrifugation for 5 min at 200 g and storage at 4° for 1 h. After gentle homogenization the reaction mixture was centrifuged on a Ficoll-Hypaque gradient at 400 g for 20 min. Fractions were obtained from the interphase and bottom of the gradient (a) antibody-dependent (ADCC) and (b) PHA-induced (PIC) cytotoxicity were assayed by incubating variable amounts of unfractionated (0); interphase (0); or bottom (A) cells with 2x 105 antibody-coated 51Cr-labelled chicken erythrocytes (51Cr-labelled CEA) or 5'Cr CE and 10 pg of PHA/106 effector cells respectively.
Effector cells in cell-mediated cytotoxicity Table 3. PHA-induced cytotoxicity and blast transformation after gradient centrifugation of E-rosette-forming cells
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rosettes. As shown in Table 4 the overall recovery of cells was 70 per cent (interphase plus bottom) and
the recovery of EAC and E-RFC was 60 and 62 per Type of cells
Before gradient Interphase Bottom
Blast Number E-RFC of cells (per transformation (x 106) cent) (per cent)
96-0 20-0 60-0
58 17 85
PIC (per cent)
28-6 33-5 8-7
33 0 40
E rosettes were obtained by reacting Ficoll-purified lymphocytes with sheep erythrocytes (E). Details of the experimental procedure are given in the text. The mixture of E rosettes and cells that did not form rosettes was centrifuged on a Ficoll-Hypaque gradient. Erythrocytes in the original cell mixture, interphase and bottom fractions were subjected to lysis with pH 7-2, 0-02 M Tris buffer, 0 75 per cent NH4Cl, and the total cell number was counted. The percentage of E rosette-forming cells (E-RFC) was measured by reacting 01 ml containing 5 x 106 lymphocytes with 0-1 ml, 1 per cent E at 370 for 15 min. After 5 min centrifugation at 200 g the mixture was kept at 40 for I h and the percentage of E-RFC was recorded. The percentage of transformed cells was counted in triplicate 1 ml cultures containing 106 cells and 10 pg/ml PHA, incubated at 370 for 48 h. The percentage transformation of control simultaneous cultures without PHA was subtracted. PHA-induced cytotoxicity (PIC) of control and post-gradient fractions was measured as described in Table 1.
Distribution of cytotoxic cells after centrifugation of EAC rosette-forming cells ADCC and PIC activities were surveyed in cellular fractions obtained after centrifugation of EAC
cent respectively. The sum of EAC- + E-RFC
accounted for the total number of cells that sedimented to the bottom, whereas 56-5 per cent of the cells in the interphase were neither E-RFC nor EAC-RFC. EAC-RFC were enriched in the bottom and E-RFC were distributed between the interphase and the bottom fractions. The presence of E-RFC in the bottom suggests that T lymphocytes reacted with the E portion of EAC and sedimented during the fractionation procedure. While cytotoxicity was minimal in bottom fractions, both ADCC and PIC were present in the interphase. Distribution of cytotoxic cells after centrifugation of CEA rosette-forming cells Cells forming rosettes with antibody-coated chicken erythrocytes (CEA-RFC) were depleted from the original lymphocyte suspension by centrifugation of CEA-RFC on Ficoll-Hypaque gradients. As shown in Table 5, overall recovery of cells (54 per cent) was not as good as after fractionation of E- or EACRFC, possibly due to agglutination of lymphocytes with CE stroma and nuclei. However, no selective loss of E- or EAC-RFC could be detected (48 and 51 per cent recovery respectively). The total number of cells present in the interphase could be accounted for by E- and EAC-RFC whereas approximately
Table 4. Distribution of cytotoxic and E or EAC rosette-forming cells after gradient centrifugation of EAC rosettes EAC-RFC Type of cells
Unfractionated Interphase Bottom
E-RFC
Cells (x 106)
Per cent
Cells (x 106)
Per cent
Cells (x 106)
36-0 16-4 8-8
22 5 46
7-9 0-8 4-1
50 38 56
18 0 6-3 49
ADCC PIC (per cent) (per cent) 46 58-3 6-0
62-5 65 8
1V8
EAC rosettes were obtained by reacting Ficoll-purified lymphocytes with a suspension of sheep erythrocyte-antibody-complement intermediates (EAC). Details of the experimental procedure are given in the text. The mixtures of rosettes and non-reacting cells was centrifuged on a Ficoll-Hypaque gradient. Erythrocytes in the original cell mixture, interphase and bottom fractions were haemolysed with pH 7-2, 0-02 M Tris buffer, 0-75 per cent NH4Cl and the total cell number was counted. The percentage and absolute numbers of E and EAC rosette-forming cells (E- and EAC-RFC) was determined as described in the text. Antibody-dependent (ADCC) and PHA-induced cytotoxicity (PIC) were measured as described in Table 1.
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Table 5. Fractionation of CEA rosette-forming cells on Ficoll-Hypaque gradients EAC-RFC Type of cells
Unfractionated Interphase Bottom
E-RFC
Cells (x 106)
Recovery (per cent)
Per cent
Cells (x 106)
Per cent
Cells (x 106)
89-2 30 0 18-0
100 34 20-2
29 24 31
25 7 75 5-6
65 77 28
58 23-1 4-85
CEA rosettes were obtained by reacting 10 ml of purified lymphocytes with 10 ml of 0 5 per cent sensitized chicken erythrocytes (CEA) at 40 for I h. The suspension was layered on a Ficoll-Hypaque gradient and centrifuged at 400 g for 20 min. Fractions were obtained from the bottom and the interphase and CEA were osmotically lysed. Control cells were treated in the same way but gradient fractions were mixed again after centrifugation. The percentage and absolute numbers of EAC- or E-rosette-forming cells (EAC- or E-RFC) were determined in the original and post gradient fractions as described in Tables 2, 3 and 4.
40 per cent of the cells recovered in the bottom fractions were neither E- nor EAC-RFC. Antibody-dependent cell cytotoxicity, assayed in the interphase and sedimented cells could only be detected in the bottom fractions. As shown in Table 6, sedimented CEA-RFC were cytotoxic for nonsensitized CE. On the other hand, the specific cytotoxic response of bottom fractions for CEA was not increased to the expected levels. Similar results have Table 6. Antibody-dependent cell cytotoxicity after centrifugation of CEA-RFC
Cytotoxicity against: Type of cells
Unfractionated Interphase Bottom
Cells (x 106) 0 37 0-75 1-50 0-37 0-75 1-50 0-37 0-75 1-50
51Cr-labelled IICr-labelled CE (per cent) CEA (per cent) 13-4 8-9 14-0 9.7 99 11-6 16-2 32-5 47-2
39-1 42-2 68-0 15-2 15-2 17-2 19-9 39-8 45-0
Cytotoxicity of purified lymphoid cells or cell fractions obtained after gradient centrifugation of CEA rosetteforming cells (CEA-RFC) (performed as described in Table 5) was assayed utilizing uncoated 51Cr-labelled chicken erythrocytes (51Cr-labelled CE) or sensitized CE (5"Crlabelled CEA) as targets. The percentage of 5tCr released after 18 h of incubation at 370 was recorded.
been reported by Perlmann, Perlmann and Biberfeld (1972). These authors demonstrated that adsorbed immune complexes were responsible for the cytotoxic effect on non-sensitized targets while they inhibited the cytotoxic response for the same antibody-coated cells.
DISCUSSION The results of the present study indicate that phytohaemagglutinin (PHA) and antibody-dependent cytotoxicity for chicken erythrocytes (CE) are mediated by different cells. While PHA-induced cytotoxicity (PIC) is impaired by removal of adherent or phagocytic cells, antibody-dependent cell cytotoxicity (ADCC) is preserved by the same procedures. This is in agreement with the results of previous reports (Britton et al., 1973). Since PHA is a mitogenic stimulus for thymusderived lymphocytes, it has been assumed that PHA cytotoxicity is also a function of T cells. Recently, however, it has been suggested that in addition to T lymphocytes other cell types participate in mitogen-induced cytotoxicity for xenogeneic target cells (Britton et al., 1973; Wisl0ff and Fr0land, 1975; Hallberg, 1974). Indeed, the cell fractionation experiments performed in this work emphasize the important role of non-T cells in PIC (Table 2, Fig. 2). Additional information on the nature of the cells
involved in PHA-induced lysis was obtained by purification of lymphocyte subpopulations that form different rosettes. Thus, T lymphocytes were selec-
Effector cells in cell-mediated cytotoxicity tively depleted by gradient centrifugation of E-RFC (Wybran et al., 1973). Cells recovered from the bottom fractions were composed mainly of T lymphocytes. In contrast, approximately 70 per cent of the cells remaining in the interphase were unable to form E- or EAC-rosettes. Although sedimented T cells could be induced directly to blast transformation by PHA (Wybran et al., 1973; Yata et al., 1973), we found that they were not cytotoxic in the PIC assay. On the contrary, interphase cell fractions, unable to transform, exhibited cytotoxicity that could be readily abolished by iron treatment. This fact provides additional supporting evidence for the central role of phagocytic, adherent cells in this cytotoxic model. Nevertheless, the present data do not exclude that the small amount of E-RFC remaining in the interphase after fractionation are necessary for PIC, co-operating with other cell types to achieve cytotoxicity. In addition, Britton, Perlmann and Perlmann (1973), utilizing spleen effector cells from congenitally athymic mice, have demonstrated that PHA cytotoxicity for CE was as effective as in normal mice. In contrast, PIC activity in an allogeneic system seems to depend also on the presence of T cells. Thus, O'Toole, Stejskal, Perlmann and Karlsson (1974) have shown that in a system composed of human effector cells and Chang target cells, fractions enriched in T lymphocytes (E-RFC) were more effective than interphase fractions in yielding PHA-induced cytotoxicity, possibly through histocompatibility recognition mechanisms. Recently, Zeylemaker, Roos, Meyer, Schellekens and Eijsvoogel (1974), utilizing a similar experimental design, have shown that PHA-induced blast transformation and the ability to react to allogeneic stimulus (MLC) were both present in fractions of human cells depleted of E-RFC. These results contrast with ours and with those of Yata et al. (1973) and Wybran et al. (1973). While the reason for these discrepancies is not clear, they may be related to different technical conditions. It is noteworthy that the dose of PHA employed by Zeylemaker et al. (1974) is five times greater than the dose used in our studies and in those of Yata et al. (1973) The moderate decrease in the cytotoxic titre observed by anti-O treatment of mouse spleen cell suspensions (Britton et al., 1973) may be due to inhibition of the reactive cells by non-related immune complexes (0-anti-0). In fact, procedures that involve phagocytosis or activation of monocyte membranes (latex ingestion exposure to immune com-
331
plexes, glass or plastic surfaces, etc., reduced PIC activity of Ficoll-purified human cells (de Bracco, Isturiz and Manni, unpublished observations). As T cell-rich fractions were not induced to cytotoxicity by PHA, the participation of non-T cells in PIC was investigated. For this purpose EAC-RFC depleted and EAC-RFC rich fractions were obtained by centrifugation of EAC-RFC. Depletion of C receptor lymphocytes failed to reduce PIC below that of control values. Furthermore, the sedimented EAC-RFC were not cytotoxic in this assay. In a different cytotoxic model, ADCC, non-T cells appear to be the effector cells (Perlmann and Holm, 1969; MacLennan et al., 1969; Gelfand, Resch and Prester, 1972; Greenberg et al., 1973; Wisl0ff et al., 1974). We have studied human peripheral cells active in antibody-dependent cytotoxicity in comparison to PHA reactive cells. In contrast to PHA cytotoxic cells, ADCC-reactive cells were neither glass-adherent nor phagocytic. Antibody-dependent cytotoxicity was absent in E- or EAC-RFC-rich fractions. Furthermore, 50-70 per cent of the cells remaining in the interphase cytotoxic fractions after depletion of EAC- or E-RFC were not identifiable as T or B cells by means of some of their membrane markers (E and EAC receptors). These cells however, constituted 10-20 per cent of the original cell preparation. Antibody-dependent cytotoxic cells were specifically sedimented by centrifugation of CEA rosettes. In this case, cells bearing Fc receptors are selectively depleted from the interphase. Thus, while non-sedimented cells could form E- or EAC-rosettes (Table 5) more than 50 per cent of the bottom CEARFC did not react in these tests. Although 31 per cent of the fractionated CEA-RFC possessed C receptors, these cells are only a fraction of the total pool of EAC-RFC. This may explain why ADCC of fractions enriched in EAC-RFC was not enhanced (Table 4). It could also be likely that only a fraction of the Fc receptor cells can be cytotoxic for sensitized target cells. In a previous report we have shown that the percentage of complement receptor lymphocytes (CRL) was reduced by filtration through aggregated IgG-C columns, while ADCC remained unaffected (Isturiz et al., 1975). On the other hand, filtration through aggregated IgG columns diminished ADCC and not CRL. Recently however, Perlmann, Perlmann and MUller-Eberhard (1974) suggested that human effector cells having both the Fc and C3 receptors on their membrane were reactive in ADCC. The apparent discrepancies
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Maria M. de E. de Bracco, M. A. Isturiz and J. A. Manni
between our data and the results of these authors may derive from differences in the fractionation procedures and/or sensitivity of the CRL detection. Nevertheless, while the role of C receptors in ADCC is not fully understood, there is general agreement on the importance of the Fc receptor (Perlmann et al., 1974; Wisl0ff and Fr0land, 1974). In fact, the acquired cytotoxicity for non-sensitized CE that was present in CEA-RFC rich fractions (Table 6) may be due to the persistence of CE-anti-CE complexes bound to Fc receptors on the cell membrane (Perlmann et al., 1972). The experiments performed in this study, utilizing two different cytotoxic models provide an insight on the nature of human cells that participate in these processes. Clearly, PHA cytotoxicity to xenogeneic target cells and ADCC are fulfilled by different cell classes that can be separated by physical procedures based on their membrane properties. Currently we are utilizing these techniques for the in vitro assessment of cell function in various disease states and preliminary results suggest that these assays may be a helpful aid for the evaluation of immunosuppressive therapy or disease activity.
ACKNOWLEDGMENTS The authors gratefully acknowledge the generous aid of Dr E. Slepoy, Dr A. Castaie' and the staff of the Blood Bank, Instituto de Investigaciones Medicas. They also wish to thank Dr Rosalia Lelchuk and Dr Rita Cardoni for helpful suggestions. This work was supported by CONICET grants numbers 5840/73 and 5840/74. M.M. de E. de B. and J.A.M. are career investigators of the National Council for Scientific and Technical Investigation (CONICET). REFERENCES B0YUM A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, supplement 97, 77. BRirroN S., PERLMANN H. & PERLMANN P. (1973) Thymus dependent and thymus independent effector functions of mouse lymphoid cells. Comparison of cytotoxicity and primary antibody formation in vitro. Cell. Immunol. 8, 420. COULSON A.S. & CHAMBERS D.G. (1964) Separation of viable lymphocytes from human blood. Lancet, i, 468.
GELFAND E.W., RESCH K. & PRESTER M. (1972) Antibody mediated target cell lysis by non-immune cells. Characterization of the antibody and effector cell populations. Europ. J. Immunol. 2, 419. GOLSTEIN P., SCHIRRMACHER V., RUBIN B. & WIGZELL H. (1973) Cytotoxic immune cells with specificity for defined soluble antigens. II. Chasing the killing cells. Cell. Immunol. 9, 211. GREENBERG A.H., HUDSON L., SHEN L. & Rorrr I.M. (1973) Antibody dependent cytotoxicity due to a 'null' lymphoid cell. Nature: New Biol. 242, 111. HALLBERG T. (1974) In vitro cytotoxicity of human lymphocytes: reduction of the lymphocyte cytotoxicity induced by antibodies or phytohemagglutinin by removing immune complex binding cells. Scand. J. Immunol. 3, 645. HARDING B., PUDIFIN D.J., GOTCH F. & MACLENNAN I.C.M. (1971) Cytotoxic lymphocytes from rats depleted of thymus processed cells. Nature: New Biol. 232, 80. HOLM G. & PERLMANN P. (1967) Quantitative studies on phytohemagglutinin cytotoxicity by human lymphocytes against homologous cells in tissue culture. Immunology, 12, 525. HOLM G., PERLMANN P. & WERNER B. (1964) Phytohemagglutinin-induced cytotoxic action of normal lymphoid cells in tissue culture. Nature (Lond.) 203, 841. ISTURIZ M.A., BRACCO M.M.E. DE & MANNI J.A. (1975) Reaction of human lymphocytes with aggregated IgG columns. Effect on antibody dependent cytotoxicity and EAC rosette formation. Cell. Immunol. 16, 82. JONDAL M., HOLM G. & WIGZELL H. (1972) Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming non-immune rosettes with SRBC. J. exp. Med. 136, 207. MACLENNAN I.C.M., LOEwi G. & HOWARD A. (1969) A human serum immunoglobulin with specificity for certain homologous target cells, which induces cell damage by normal human lymphocytes. Immunology, 17, 897. MANNi J.A., BRACCO M.M.E. DE & PATRUCcO A. (1974) Citotoxicidad de linfocitos humanos inducida por anticuerop: respuesta en pacientes con lupus eritematosos sistemico y efecto del tratamiento inmunodepresor. Medicina (Buenos Aires), 34, 185. O'TOOLE C., STEJSKAL V., PERLMANN P. & KARLSSON M. (1974) Lymphoid cells mediating tumor specific cytotoxicity to carcinoma of the urinary bladder. J. exp. Med.
139,457. PERLMANN P. & HOLM G. (1969) Cytotoxic effects of lymphoid cells in vitro. Advanc. Immunol. 11, 117. PERLMANN P., PERLMANN H. & BIBERFELD P. (1972) Specifically cytotoxic lymphocytes produced by preincubation with antibody-complexed target cells. J. Immunol. 108, 558. RABINOWITZ Y. (1964) Separation of lymphocytes, polymorphonuclear leukocytes and monocytes on glass columns, including tissue culture observations. Blood, 23, 811. SHERWOOD G. & BLAESE R.M. (1973) Phytohemagglutinininduced cytotoxic effector lymphocyte function in patients with the Wiskott-Aldrich syndrome (WAS). Clin. exp. Immunol. 13, 515. WISL0FF F. & FR0LAND S.S. (1975) VII Meeting of the
Effector cells in cell-mediated cytotoxicity Argentine Society of Immunology. Medicina (Buenos Aires) 34, 355. WISL0FF F., FR0LAND S.S. & MICHAELSEN T.E. (1974a) Antibody dependent cytotoxicity mediated by human Fc-receptor bearing cells lacking markers for B and T lymphocytes. Int. Arch. Allergy appl. Immunol. 47, 139. WISL0FF F., FR0LAND S.S. & MICHAELSEN T.E. (1974b) Characterization of human lymphoid cells participating in PHA and Concanavalin A induced cytotoxicity. Int. Arch. Allergy appl. Immunol. 47, 488. WYBRAN J., CHANTLER S. & FUDENBERG H.H. (1973) Human
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blood T cells: response to phytohemagglutinin. J. Immunol. 110, 1157. YATA J., DESGRANGES C., TACHIBANA T. & THE G. DE (1973) Separation of human lymphocytes forming spontaneous rosettes with sheep erythrocytes. Biomedicine Express, 19, 475. ZEYLEMAKER W.P., Roos M.T.L., MEYER C.J.L.M., SCHELLEKENS P.T.A. & EIJSVOOGEL V.P. (1974) Separation of human lymphocyte subpopulations. I. Transformation characteristics of rosette-forming cells. Cell. Immunol. 14, 346.
Cell-mediated cytotoxicity CHARACTERIZATION OF THE EFFECTOR CELLS
MARIA M. DE E. DE BRACCO, M. A. ISTURIZ & J. A. MANNIInstitutodeInvestigaciones Mtedicas, Universidad de Buenos Aires, Buenos Aires, Argentina
Received 7 July 1975; accepted for publication 17 July 1975
INTRODUCTION
Summary. Isolated human mononuclear cells were fractionated according to their membrane characteristics or physical properties. Adherent cells were depleted by filtration through glass columns; phagocytic cells were removed by iron treatment and cell subpopulations capable of forming rosettes with sheep erythrocytes (E), erythrocyte-antibody-complement (EAC) and chicken erythrocyte-antibody complexes (CEA) were separated by centrifugation of Ficoll-Hypaque gradients. The functional activity of the cell subpopulations obtained was assayed by testing PHA-induced cytotoxicity (PIC), antibodydependent cytotoxicity (ADCC) and blast transformation by PHA. The results of this study demonstrate that: (1) cells reacting in PIC and ADCC assays are different, adherent and phagocytic cells being necessary for full expression of PIC and not for ADCC; (2) PHA induces direct blast transformation of purified E-RFC in the absence of PIC cytotoxic cells; (3) cell populations specifically enriched in E or EAC rosette-forming cells are not cytotoxic neither in the PHA nor in antibody mediated cytotoxic assays; (4) cells participating in ADCC can be selectively purified by centrifugation of CEA
The ability of non-sensitized lymphocytes to destroy target cells combined with antibody to cell membrane antigens has been demonstrated in several species and systems (reviewed by Perlmann and Holm, 1969). Lymphoid cells activated by non-specific mitogens may also display cytotoxic activity against allogeneic, syngeneic and xenogeneic target cells (Perlmann and Holm, 1969; Holm, Perlmann and Werner, 1964). A series of studies have been performed in order to establish the nature of the effector cells involved in the different models of cytotoxicity (Harding, Pudifin, Gotch and MacLennan, 1971; Greenberg, Hudson, Shen and Roitt, 1973; Wisl0ff, Fr0land and Michaelsen, 1974a). While most evidence indicates that the cells mediating antibody-dependent cytotoxicity (ADCC) are thymus-independent (Perlmann and Holm, 1969; MacLennan, Loewi and Howard, 1969) it has been assumed that PHA-induced cytotoxicity (PIC) is a function of T cells (Sherwood and Blaese, 1973; Wisl0ff, Fr0land and Michaelsen, 1974b). However, in the latter system more than one class of cells appears to be involved (Britton, Perlmann and Perlmann, 1973; Wisl0ff et al., 1974b; Hallberg, 1974). In this study we have compared ADCC and PIC of normal human lymphoid cells after fractionation
rosettes. Correspondence: Dr Maria M. de E. de Bracco, Instituto de Investigaciones M6dicas, D. Alvarez 3000, Hospital Tornfl, Buenos Aires, Argentina. B
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Maria M. de E. de Bracco, M. A. Isturiz and J. A. Manni
procedures based on the ability of the different subclasses of human lymphocytes to form rosettes with sheep red cells (E), erythrocyte intermediates with antibody and complement (EAC) or chicken erythrocyte-antibody complexes (CEA). In addition, the effect of removing adherent and phagocytic cells on PHA-induced and antibody-mediated cytotoxicity was explored. MATERIALS AND METHODS Human lymphoid cells Lymphoid cells were isolated from heparinized or defibrinated venous blood from human volunteer donors by centrifugation on Ficoll-Hypaque gradients (B0yum, 1968). The pellet was washed with TC199 medium (Difco Laboratories, Detroit, Michigan) containing 100 u/ml penicillin 100 pg/ml of streptomycin and 2-5 per cent heat-inactivated foetal calf serum (FCS). The concentration of mononuclear cells was determined by counting the cell suspensions stained with Turk's solution. Ficollpurified cell suspensions containing 95-98 per cent mononuclear cells and 2-5 per cent granulocytes were utilized for further fractionation procedures. In some experiments the glass adherent cell population was depleted by filtration of gelatin sedimented leucocytes obtained from defibrinated blood (Coulson and Chambers, 1964) through glass bead columns (Rabinowitz, 1964). The resultant cell suspensions were centrifuged on Ficoll-Hypaque mixtures (1 -077 specific gravity), washed with TC199 and counted. Glass-filtered non-adherent cells were composed of 95-99 per cent small lymphocytes and 1-5 per cent
polymorphonuclear leucocytes. Depletion ofphagocytic cells with iron powder One millilitre of purified mononuclear cells (F), 10-20 x 106/ml were incubated with 0-2 ml of carbonyl iron powder suspension (10 mg/ml) for 1 h at 370, with occasional agitation. Cells containing iron and adhering to iron clumps were removed with a magnet. The resultant cell suspensions was composed of 95-98 per cent small lymphocytes.
Purification of E, EAC and CEA rosette-forming cells E rosettes were prepared as described by Jondal, Holm and Wigzell (1972) and EAC rosettes were obtained as described previously (Isturiz, de Bracco and Manni, 1975). When the reactions were performed for preparative purposes, the amount of the
reactants was scaled up and the concentration of E or EAC rosettes obtained was checked. Sensitized chicken erythrocyte rosettes (CEA rosettes) were prepared by incubating 1 ml of TC199 containing 5-10 x 106 lymphocytes with 1 ml of 0 5 per cent chicken erythrocytes (CE) sensitized with the maximum non-agglutinating dose of rabbit anti-CE antiserum (Manni, de Bracco and Patrucco, 1974) for 1 h at 4°. Two to three volumes (10-15 ml) of E, EAC or CEA rosettes were layered on top of a Ficoll-Hypaque gradient, centrifuged at 400 g for 20 min and both the interphase and pellet were recovered and washed three times with TC199. E and EAC were haemolysed with 0 02 M Tris buffer (pH 7 2) containing 0 75 per cent NH4CI. CEA were haemolysed by exposure to distilled water for 80 s at 40 and immediate addition of concentrated saline to restore isotonicity. Control gradient tubes were mixed again after centrifugation, washed with TC199 and E, EAC or CEA were haemolysed as described above. Viability checked by trypan blue exclusion was above 90 per cent. Cells isolated from gradient fractions were suspended in TC199 at 5 x 106/ml. All experiments were repeated three to five times.
Antibody-dependent cell cytotoxicity Antibody-dependent cytotoxicity (ADCC) was assayed as described previously utilizing 5'Cr-labelled antibody-coated CE target cells (Isturiz, de Bracco and Manni, 1975; Manni, de Bracco and Patrucco, 1974). Variable amounts of lymphocytes ranging from 0 3 to 2-5 x 106 cells/-5 ml were cultured with 2 x105 5'Cr-labelled CEA for 18 h at 37°. After centrifugation, both 1-ml aliquots of the supernatant and the residual fluid plus sediment were counted in an automatic Packard 3320 gamma counter. The percentage of s 'Cr released was calculated as follows: percentage 5'Cr released = [(activity of supernatant x 1 *5)/(total activity)] x 100. The highest sensitivity of the assay lies within 1-3 x 106 effector cells and 20-60 per cent 51Cr release (Isturiz, de Bracco and Manni, 1975).
Phytohaemagglutinin-induced cytotoxicity (PIC) Cytotoxicity of human mononuclear cells was induced by stimulation with phytohaemagglutinin (PHA-P, Difco Laboratories, Detroit, Michigan) as described by Holm and Perlmann (1967) utilizing 5'Cr-labelled CE target cells. The optimal PHA/ lymphocyte ratio was found to be 10 ug PHA/106 cells. Reaction mixtures containing 1 25-7 5 x 106
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Effector cells in cell-mediated cytotoxicity
Ficoll-purified cells, 10 ug of PHA-P/106 lymphocytes and 2 x 105 5tCr-labelled CE suspended in 1 5 ml TC199 were cultured for 18 h at 37°. Control tubes without PHA were set up simultaneously. An example of a control PHA-induced cytotoxicity (PIC) curve at constant PHA/effector cell ratio is shown in Fig. 1. As reported by others (Wisl0ff, 70r
toxicity induced by PHA (PIC) or antibody (ADCC) was explored by removing the cells capable of ingesting carbonyl iron particles (Golstein, Schirrmacher, Rubin and Wigzell, 1973) or adhering to glass. The results presented in Table 1 demonstrate Table 1. Effect of depletion of adherent or phagocytic cells on antibody-dependent or PHA-induced cytotoxicity
60[Donor 50II-
1
_ 40 H
2
w In a)
-Y
a)g 3CDH
mT_ 2C IC I
12
3
4
5
6
8I8
7
Cells (X106) Figure 1. PHA-induced cell cytotoxicity (PIC). Variable numbers of Ficoll-purified mononuclear cells were incubated in 1'S ml TC199 containing 10 pg of PHA-P/106 cells and 2x 105 51 Cr-labelled CE for 18 h at 37°. PIC is expressed as the percentage of 5 Cr released corrected for spontaneous release in the absence of PHA.
Fr0land and Michaelsen, 1974b) there was considerable variation between different donors. The percentage of 51Cr liberated was measured and calculated as described in the preceding section. Lymphocyte stimulation with phytohaemagglutinin Duplicate or triplicate culture tubes containing 106 lymphocytes suspended in 1 ml of 20 per cent FCS-TC199 were incubated with 10 ug of PHA at 370 for 48-72 h. Lymphocytes were harvested, fixed in Carnoy's solution stained with May-GrunwaldGiemsa and observed by light microscopy. The percentage of lymphoblastoid cells in 500 cells was recorded. RESULTS
Effect of the depletion of phagocytic or adherent cells on cytotoxicity The role of phagocytic and adherent cells on cyto-
Treatment of cells* None Iron None Glass filtration
ADCC (per cent)
PIC (per cent)
43 44 47
42 9 30
45
17
* Ficoll-purified cells were incubated with carbonyl iron or filtered through glass columns. Antibody-dependent cytotoxicity (ADCC) was assayed before and after treatment by incubating 2x 106 cells with 2x 105 51Cr-labelled CEA at 370 for 18 h. ADCC is expressed as the percentage of 51Cr released, corrected for spontaneous release in the absence of anti-CE. PHA-induced cytotoxicity (PlC) was assayed by incubating 4 x 10" control and treated cells with 10 pg of PHA/106 cells and 2 x 105 51 Cr-labelled CE at 370 for 18 h. PIC is expressed as the percentage of 51Cr released corrected for spontaneous release in the absence of PHA.
that depletion of phagocytic or adherent cells diminished the cytotoxic response induced by PHA, while ADCC was not altered. Iron or glass treatment of Ficoll-purified lymphocytes yielded a final cell suspension composed of 97 per cent small lymphocytes. Thus, the participation of phagocytic or adherent cells appears to be important for the full expression of PIC and not for ADCC. Distribution of cytotoxic cells after centrifugation of E rosette-forming cells
Antibody-dependent and PHA-induced cytotoxicity were tested in cellular fractions obtained by gradient centrifugation of E-rosette-forming cells (E-RFC). It has been reported previously that T cells capable of forming E rosettes can be selectively depleted from the original cell suspensions by this method (Yata, Desgranges, Tachibana and de ThM, 1973; Wybran, Chantler and Fudenberg, 1973). The results shown in Table 2 indicate that most of the cells in
Maria M. de E. de Bracco, M. A. Isturiz and J. A. Manni
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Table 2. Fractionation of E rosette-forming cells on FicollHypaque gradients
EAC-RFC
E-RFC Type of cells
Before gradient Interphase Bottom
Number of cells (x 106)
Per cent
No. cells (x 106)
Per cent
No. cells (x 106)
43 5 5-2 38-0
70 0 50 87-0
30-2 0-26 33-1
n.d. 25 4-2
n.d. 1-25 1-52
E rosettes were obtained by reacting 10 ml of Ficollpurified, glass-filtered lymphocytes (5 x 106/ml) with 10 ml of a 1 per cent sheep erythrocyte (E) suspension for 15 min at 370, centrifuged for 5 min at 200 g and kept at 40 for 1 h. After gentle homogenization the reaction mixture was centrifuged on a Ficoll-Hypaque gradient for 20 min at 400 g. Fractions were obtained from the bottom and the interphase of the gradient and E were haemolysed with 0-02 M, pH 7-2 Tris buffer containing 0 75 per cent NH4Cl. The percentage and absolute number of rosette-forming cells (E-RFC and EAC-RFC) was determined in the original and post-gradient fractions. Experimental conditions are detailed in the text. n.d. = Not determined.
the bottom fractions were E-RFC. In contrast, around 70 per cent of the cells in the interphase did not form E or EAC rosettes. The overall recovery ranged from 80-100 per cent. (a)
60k
As shown in Fig. 2, cells recovered from the interphase (non-E-RFC) exhibited greater ADCC than the original cell suspensions and the activity of the bottom fraction was low. Similarly, very low PIC activity was recovered from the sedimented E-RFC and the interphase fractions displayed activity comparable to that of controls (Fig. 2). This activity was absent in iron-treated interphase fractions. Also, experiments performed utilizing mixtures of interphase and bottom fractions in different ratios failed to reveal an enhancement of inter-phase activity by E-RFC.
Distribution of PHA blast-forming and cytotoxic cells after centrifugation of E-RFC Contro Inon-fractionated cells and the interphase and bottom fractions of an E-RFC sedimentation gradient were assayed for PIC, E-rosette formation and the blastogenic response to optimum amounts of PHA. Table 3 summarizes the results of a representative experiment. Clearly, PHA cytotoxicity to CE is present in fractions unable to respond to the same mitogen by blast transformation. As was previously reported (Yata et al., 1973), the distribution of E-RFC parallels that of blast-forming cells.
r
~01'
( b)
0
(-) a) 40k
C-
.
201-
5I
I
2
3
4
5 Cel Is (x 106 )
2
3
4
5
6
7
Figure 2. Distribution of cytotoxic cells after centrifugation of E-RFC. E rosettes were obtained by reacting 10 ml of Ficollpurified cells (5x 106/ml) with 10 ml of I per cent sheep erythrocyte (E) suspension for 15 min at 370 followed by centrifugation for 5 min at 200 g and storage at 4° for 1 h. After gentle homogenization the reaction mixture was centrifuged on a Ficoll-Hypaque gradient at 400 g for 20 min. Fractions were obtained from the interphase and bottom of the gradient (a) antibody-dependent (ADCC) and (b) PHA-induced (PIC) cytotoxicity were assayed by incubating variable amounts of unfractionated (0); interphase (0); or bottom (A) cells with 2x 105 antibody-coated 51Cr-labelled chicken erythrocytes (51Cr-labelled CEA) or 5'Cr CE and 10 pg of PHA/106 effector cells respectively.
Effector cells in cell-mediated cytotoxicity Table 3. PHA-induced cytotoxicity and blast transformation after gradient centrifugation of E-rosette-forming cells
329
rosettes. As shown in Table 4 the overall recovery of cells was 70 per cent (interphase plus bottom) and
the recovery of EAC and E-RFC was 60 and 62 per Type of cells
Before gradient Interphase Bottom
Blast Number E-RFC of cells (per transformation (x 106) cent) (per cent)
96-0 20-0 60-0
58 17 85
PIC (per cent)
28-6 33-5 8-7
33 0 40
E rosettes were obtained by reacting Ficoll-purified lymphocytes with sheep erythrocytes (E). Details of the experimental procedure are given in the text. The mixture of E rosettes and cells that did not form rosettes was centrifuged on a Ficoll-Hypaque gradient. Erythrocytes in the original cell mixture, interphase and bottom fractions were subjected to lysis with pH 7-2, 0-02 M Tris buffer, 0 75 per cent NH4Cl, and the total cell number was counted. The percentage of E rosette-forming cells (E-RFC) was measured by reacting 01 ml containing 5 x 106 lymphocytes with 0-1 ml, 1 per cent E at 370 for 15 min. After 5 min centrifugation at 200 g the mixture was kept at 40 for I h and the percentage of E-RFC was recorded. The percentage of transformed cells was counted in triplicate 1 ml cultures containing 106 cells and 10 pg/ml PHA, incubated at 370 for 48 h. The percentage transformation of control simultaneous cultures without PHA was subtracted. PHA-induced cytotoxicity (PIC) of control and post-gradient fractions was measured as described in Table 1.
Distribution of cytotoxic cells after centrifugation of EAC rosette-forming cells ADCC and PIC activities were surveyed in cellular fractions obtained after centrifugation of EAC
cent respectively. The sum of EAC- + E-RFC
accounted for the total number of cells that sedimented to the bottom, whereas 56-5 per cent of the cells in the interphase were neither E-RFC nor EAC-RFC. EAC-RFC were enriched in the bottom and E-RFC were distributed between the interphase and the bottom fractions. The presence of E-RFC in the bottom suggests that T lymphocytes reacted with the E portion of EAC and sedimented during the fractionation procedure. While cytotoxicity was minimal in bottom fractions, both ADCC and PIC were present in the interphase. Distribution of cytotoxic cells after centrifugation of CEA rosette-forming cells Cells forming rosettes with antibody-coated chicken erythrocytes (CEA-RFC) were depleted from the original lymphocyte suspension by centrifugation of CEA-RFC on Ficoll-Hypaque gradients. As shown in Table 5, overall recovery of cells (54 per cent) was not as good as after fractionation of E- or EACRFC, possibly due to agglutination of lymphocytes with CE stroma and nuclei. However, no selective loss of E- or EAC-RFC could be detected (48 and 51 per cent recovery respectively). The total number of cells present in the interphase could be accounted for by E- and EAC-RFC whereas approximately
Table 4. Distribution of cytotoxic and E or EAC rosette-forming cells after gradient centrifugation of EAC rosettes EAC-RFC Type of cells
Unfractionated Interphase Bottom
E-RFC
Cells (x 106)
Per cent
Cells (x 106)
Per cent
Cells (x 106)
36-0 16-4 8-8
22 5 46
7-9 0-8 4-1
50 38 56
18 0 6-3 49
ADCC PIC (per cent) (per cent) 46 58-3 6-0
62-5 65 8
1V8
EAC rosettes were obtained by reacting Ficoll-purified lymphocytes with a suspension of sheep erythrocyte-antibody-complement intermediates (EAC). Details of the experimental procedure are given in the text. The mixtures of rosettes and non-reacting cells was centrifuged on a Ficoll-Hypaque gradient. Erythrocytes in the original cell mixture, interphase and bottom fractions were haemolysed with pH 7-2, 0-02 M Tris buffer, 0-75 per cent NH4Cl and the total cell number was counted. The percentage and absolute numbers of E and EAC rosette-forming cells (E- and EAC-RFC) was determined as described in the text. Antibody-dependent (ADCC) and PHA-induced cytotoxicity (PIC) were measured as described in Table 1.
Maria M. de E. de Bracco, M. A. Isturiz and J. A. Manni
330
Table 5. Fractionation of CEA rosette-forming cells on Ficoll-Hypaque gradients EAC-RFC Type of cells
Unfractionated Interphase Bottom
E-RFC
Cells (x 106)
Recovery (per cent)
Per cent
Cells (x 106)
Per cent
Cells (x 106)
89-2 30 0 18-0
100 34 20-2
29 24 31
25 7 75 5-6
65 77 28
58 23-1 4-85
CEA rosettes were obtained by reacting 10 ml of purified lymphocytes with 10 ml of 0 5 per cent sensitized chicken erythrocytes (CEA) at 40 for I h. The suspension was layered on a Ficoll-Hypaque gradient and centrifuged at 400 g for 20 min. Fractions were obtained from the bottom and the interphase and CEA were osmotically lysed. Control cells were treated in the same way but gradient fractions were mixed again after centrifugation. The percentage and absolute numbers of EAC- or E-rosette-forming cells (EAC- or E-RFC) were determined in the original and post gradient fractions as described in Tables 2, 3 and 4.
40 per cent of the cells recovered in the bottom fractions were neither E- nor EAC-RFC. Antibody-dependent cell cytotoxicity, assayed in the interphase and sedimented cells could only be detected in the bottom fractions. As shown in Table 6, sedimented CEA-RFC were cytotoxic for nonsensitized CE. On the other hand, the specific cytotoxic response of bottom fractions for CEA was not increased to the expected levels. Similar results have Table 6. Antibody-dependent cell cytotoxicity after centrifugation of CEA-RFC
Cytotoxicity against: Type of cells
Unfractionated Interphase Bottom
Cells (x 106) 0 37 0-75 1-50 0-37 0-75 1-50 0-37 0-75 1-50
51Cr-labelled IICr-labelled CE (per cent) CEA (per cent) 13-4 8-9 14-0 9.7 99 11-6 16-2 32-5 47-2
39-1 42-2 68-0 15-2 15-2 17-2 19-9 39-8 45-0
Cytotoxicity of purified lymphoid cells or cell fractions obtained after gradient centrifugation of CEA rosetteforming cells (CEA-RFC) (performed as described in Table 5) was assayed utilizing uncoated 51Cr-labelled chicken erythrocytes (51Cr-labelled CE) or sensitized CE (5"Crlabelled CEA) as targets. The percentage of 5tCr released after 18 h of incubation at 370 was recorded.
been reported by Perlmann, Perlmann and Biberfeld (1972). These authors demonstrated that adsorbed immune complexes were responsible for the cytotoxic effect on non-sensitized targets while they inhibited the cytotoxic response for the same antibody-coated cells.
DISCUSSION The results of the present study indicate that phytohaemagglutinin (PHA) and antibody-dependent cytotoxicity for chicken erythrocytes (CE) are mediated by different cells. While PHA-induced cytotoxicity (PIC) is impaired by removal of adherent or phagocytic cells, antibody-dependent cell cytotoxicity (ADCC) is preserved by the same procedures. This is in agreement with the results of previous reports (Britton et al., 1973). Since PHA is a mitogenic stimulus for thymusderived lymphocytes, it has been assumed that PHA cytotoxicity is also a function of T cells. Recently, however, it has been suggested that in addition to T lymphocytes other cell types participate in mitogen-induced cytotoxicity for xenogeneic target cells (Britton et al., 1973; Wisl0ff and Fr0land, 1975; Hallberg, 1974). Indeed, the cell fractionation experiments performed in this work emphasize the important role of non-T cells in PIC (Table 2, Fig. 2). Additional information on the nature of the cells
involved in PHA-induced lysis was obtained by purification of lymphocyte subpopulations that form different rosettes. Thus, T lymphocytes were selec-
Effector cells in cell-mediated cytotoxicity tively depleted by gradient centrifugation of E-RFC (Wybran et al., 1973). Cells recovered from the bottom fractions were composed mainly of T lymphocytes. In contrast, approximately 70 per cent of the cells remaining in the interphase were unable to form E- or EAC-rosettes. Although sedimented T cells could be induced directly to blast transformation by PHA (Wybran et al., 1973; Yata et al., 1973), we found that they were not cytotoxic in the PIC assay. On the contrary, interphase cell fractions, unable to transform, exhibited cytotoxicity that could be readily abolished by iron treatment. This fact provides additional supporting evidence for the central role of phagocytic, adherent cells in this cytotoxic model. Nevertheless, the present data do not exclude that the small amount of E-RFC remaining in the interphase after fractionation are necessary for PIC, co-operating with other cell types to achieve cytotoxicity. In addition, Britton, Perlmann and Perlmann (1973), utilizing spleen effector cells from congenitally athymic mice, have demonstrated that PHA cytotoxicity for CE was as effective as in normal mice. In contrast, PIC activity in an allogeneic system seems to depend also on the presence of T cells. Thus, O'Toole, Stejskal, Perlmann and Karlsson (1974) have shown that in a system composed of human effector cells and Chang target cells, fractions enriched in T lymphocytes (E-RFC) were more effective than interphase fractions in yielding PHA-induced cytotoxicity, possibly through histocompatibility recognition mechanisms. Recently, Zeylemaker, Roos, Meyer, Schellekens and Eijsvoogel (1974), utilizing a similar experimental design, have shown that PHA-induced blast transformation and the ability to react to allogeneic stimulus (MLC) were both present in fractions of human cells depleted of E-RFC. These results contrast with ours and with those of Yata et al. (1973) and Wybran et al. (1973). While the reason for these discrepancies is not clear, they may be related to different technical conditions. It is noteworthy that the dose of PHA employed by Zeylemaker et al. (1974) is five times greater than the dose used in our studies and in those of Yata et al. (1973) The moderate decrease in the cytotoxic titre observed by anti-O treatment of mouse spleen cell suspensions (Britton et al., 1973) may be due to inhibition of the reactive cells by non-related immune complexes (0-anti-0). In fact, procedures that involve phagocytosis or activation of monocyte membranes (latex ingestion exposure to immune com-
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plexes, glass or plastic surfaces, etc., reduced PIC activity of Ficoll-purified human cells (de Bracco, Isturiz and Manni, unpublished observations). As T cell-rich fractions were not induced to cytotoxicity by PHA, the participation of non-T cells in PIC was investigated. For this purpose EAC-RFC depleted and EAC-RFC rich fractions were obtained by centrifugation of EAC-RFC. Depletion of C receptor lymphocytes failed to reduce PIC below that of control values. Furthermore, the sedimented EAC-RFC were not cytotoxic in this assay. In a different cytotoxic model, ADCC, non-T cells appear to be the effector cells (Perlmann and Holm, 1969; MacLennan et al., 1969; Gelfand, Resch and Prester, 1972; Greenberg et al., 1973; Wisl0ff et al., 1974). We have studied human peripheral cells active in antibody-dependent cytotoxicity in comparison to PHA reactive cells. In contrast to PHA cytotoxic cells, ADCC-reactive cells were neither glass-adherent nor phagocytic. Antibody-dependent cytotoxicity was absent in E- or EAC-RFC-rich fractions. Furthermore, 50-70 per cent of the cells remaining in the interphase cytotoxic fractions after depletion of EAC- or E-RFC were not identifiable as T or B cells by means of some of their membrane markers (E and EAC receptors). These cells however, constituted 10-20 per cent of the original cell preparation. Antibody-dependent cytotoxic cells were specifically sedimented by centrifugation of CEA rosettes. In this case, cells bearing Fc receptors are selectively depleted from the interphase. Thus, while non-sedimented cells could form E- or EAC-rosettes (Table 5) more than 50 per cent of the bottom CEARFC did not react in these tests. Although 31 per cent of the fractionated CEA-RFC possessed C receptors, these cells are only a fraction of the total pool of EAC-RFC. This may explain why ADCC of fractions enriched in EAC-RFC was not enhanced (Table 4). It could also be likely that only a fraction of the Fc receptor cells can be cytotoxic for sensitized target cells. In a previous report we have shown that the percentage of complement receptor lymphocytes (CRL) was reduced by filtration through aggregated IgG-C columns, while ADCC remained unaffected (Isturiz et al., 1975). On the other hand, filtration through aggregated IgG columns diminished ADCC and not CRL. Recently however, Perlmann, Perlmann and MUller-Eberhard (1974) suggested that human effector cells having both the Fc and C3 receptors on their membrane were reactive in ADCC. The apparent discrepancies
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Maria M. de E. de Bracco, M. A. Isturiz and J. A. Manni
between our data and the results of these authors may derive from differences in the fractionation procedures and/or sensitivity of the CRL detection. Nevertheless, while the role of C receptors in ADCC is not fully understood, there is general agreement on the importance of the Fc receptor (Perlmann et al., 1974; Wisl0ff and Fr0land, 1974). In fact, the acquired cytotoxicity for non-sensitized CE that was present in CEA-RFC rich fractions (Table 6) may be due to the persistence of CE-anti-CE complexes bound to Fc receptors on the cell membrane (Perlmann et al., 1972). The experiments performed in this study, utilizing two different cytotoxic models provide an insight on the nature of human cells that participate in these processes. Clearly, PHA cytotoxicity to xenogeneic target cells and ADCC are fulfilled by different cell classes that can be separated by physical procedures based on their membrane properties. Currently we are utilizing these techniques for the in vitro assessment of cell function in various disease states and preliminary results suggest that these assays may be a helpful aid for the evaluation of immunosuppressive therapy or disease activity.
ACKNOWLEDGMENTS The authors gratefully acknowledge the generous aid of Dr E. Slepoy, Dr A. Castaie' and the staff of the Blood Bank, Instituto de Investigaciones Medicas. They also wish to thank Dr Rosalia Lelchuk and Dr Rita Cardoni for helpful suggestions. This work was supported by CONICET grants numbers 5840/73 and 5840/74. M.M. de E. de B. and J.A.M. are career investigators of the National Council for Scientific and Technical Investigation (CONICET). REFERENCES B0YUM A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, supplement 97, 77. BRirroN S., PERLMANN H. & PERLMANN P. (1973) Thymus dependent and thymus independent effector functions of mouse lymphoid cells. Comparison of cytotoxicity and primary antibody formation in vitro. Cell. Immunol. 8, 420. COULSON A.S. & CHAMBERS D.G. (1964) Separation of viable lymphocytes from human blood. Lancet, i, 468.
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