A comparison of human lymphoid cells in antibody-dependent cellular cytotoxicity (ADCC)

CLINICAL

IMMUNOLOGY

AND

3,377-384

IMMUNOPAT’HOLOGY

A Comparison Antibody-Dependent

(19%)

of Human Lymphoid Cells in Cellular Cytotoxicity (ADCC)

ROBERT PETER GALE, JACOB ZIGHELBOIM, R. CLIFFORD OSSORIO, AND JOHN L. FAHEY Department

of Microbiology Uniuersity

and Immunology, Human lmmunobiology of California School of Medicine, Los Angeles, California 90024 Receioed

April

Group,

18, 1974

Human lymphoid cells from normal donors, hospitalized controls, patients with chronic lymphocytic leukemia (CLL), and lymphoblastoid cell lines (LCLs) were examined for their ability to act as effector cells in antibody-dependent eelhdar cytotoxicity (ADCC). These cells were also examined for the presence of a cell surface receptor for antigen-antibody complexes, i.e., an Fc receptor. Cells from normal and hospitalized controls exhibited comparable levels of ADCC activity and Fc receptors. Those from patients with CLL had decreased or absent ADCC activity and normal to increased percentages of cells with Fc receptors. Cells from LCL had no ADCC activity and no detectable cells with Fc receptors. The presence of an Fc receptor on lymphoid cells appears to be a necessary prerequisite of normal ADCC activity, but is in itself insufficient to insure normal ADCC activity.

The cytolysis of antibody-coated target cells by nonimmune effector cells, antibody-dependent cellular cytotoxicity (ADCC), has been described (for reviews, see l-3). In man, lymphocytes (1,2,4), polymorphonuclear leukocytes (5), monocytes (6), and macrophages (7) can function as effector cells in ADCC. This activity in lymphocytes is a function of thymus-independent cells (8). Thymocytes and circulating peripheral T cells are ineffective. Involvement of B lymphocytes is suggested by the observation that pretreatment of effector cells with anti-Ig antiserum inhibits ADCC activity and that effector cell activity can be depleted by anti-Ig-coated columns. Recent studies have suggested that ADCC effector cell activity may be a function of “null” or “K” cells, i.e., non-T lymphocytes which lack easily demonstrable surface Ig; however, this point remains unsettled (9-11). Normal ADCC activity requires a cell surface receptor for the Fc portion of the Ig molecule (12,13). Fc receptors are present on human B lymphocytes (14-16), as well as on other cell types, and can be demonstrated by several techniques including autoradiography, fluorescent aggregate binding, and rosette formation with antibody-coated erythrocytes. The current study was undertaken to determine the ability of four populations of human lymphoid cells to mediate ADCC. These included (1) lymphocytes from normal donors; (2) lymphocytes from patients with CLL; (3) lymphocytes from patients with nonhematologic diseases; and (4) cells obtained from established lymphoblastoid cell lines (LCLs). ADCC function was correlated with the presence of an Fc receptor on these cells. 377 Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

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MATERIALS

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METHODS

Study groups. Studies were undertaken in nine patients with CLL, none of whom was receiving chemotherapy at the time of study, Patient ages ranged from 40-65 yr. Peripheral white blood cell (WBC) counts ranged from 20,000 to lOO,OOO/~l with from 90 to 98% lymphocytes by standard morphological criteria. Nine normal donors, age range of 20-35 yr, were also studied. Additionally, four hospitalized patients with congestive heart failure with normal WBC counts, age range 45-95 yr, were studied. The latter were not receiving any known immunosuppressive therapy at the time of study. Cells from each source were tested at effector: target cell ratios of 100, 50, and 10: 1. Normal control cells were evaluated each time a patient’s cells were tested. Some individuals were tested on more than one occasion. Lymphocytes. Lymphocytes were obtained by isopycnic centrifugation oi 50 ml of heparinized peripheral blood on a Ficoll-Hypaque density gradient as described by Boyum (17). The mononuclear cell layer was harvested: washed twice, and resuspended in Eagle’s Minimum Essential Media (MEM; Flow Laboratories, Inglewood, CA) supplemented with 10% fetal calf serum (MEM-FCS). Cell preparations obtained by this method contained 299% mononuclear cells. May-Griinwald-Giemsa staining, latexparticle ingestion, and neutral red staining revealed 5-15% monocytes in the mononuclear cell preparations from normal individuals and ,i 1% in CLL patients. To eliminate monocytes in some instances, the mononuclear cell preparation was incubated with an equal volume of lymphocyte-separating reagent (Technicon Corp., Tarrytown, NY) containing iron filings for 30 min in a rocking incubator at 37°C. The cell suspension was passed through a 12-cm length of Tigon plastic tubing coiled around the poles of a magnet. Nonadherent cells were collected, washed, and resuspended in MEM-FCS. These preparations contained 5 1% monocytes. Lymphoblastoid cell lines (LCLs). Lymphoblastoid cells were obtained from five established cell lines (UCLA-62, 81,85, 91, and 101) initiated from normal donors. The cells were washed twice and resuspended in MEM-FCS. These cell lines have been shown to produce Ig in tiitro and are of B-cell origin. LCL cells were unable to form spontaneous rosettes with sheep red blood cells (SRBCs). Target cell. EL-4, a murine leukemia syngeneic to C57BL/6 mice, was obtained from Dr. R. Herberman and serially passaged in ascites form. Daily, 10’ cells (1 ml) were harvested, washed, incubated with 200 &i of “‘Cl (Na,CrO,; Amersham Searle Corp., Chicago, IL) for 30 min at 37°C and employed as target cells as described by Brunner et al. (18). Antiserum. Rabbits were inoculated with EL-4 at multiple subcutaneous sites. A total of 2 x 10fi cells were injected in complete Freund’s adjuvant. Animals were reinjected 3 wk later with a similar number of cells and bled 10 days after the second injection. This immune rabbit serum (IRS) was pooled and heat inactivated at 56°C for 30 min to remove heat-labile complement components. IRS with exogenous complement was cytolytic to EL-4

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cells at a titer of 5 x lo-“. The ADCC assay was performed at a titer of lo-‘. Normal rabbit serum (NRS) was employed as a control. ADCC assay. The ADCC assay has been previously described (8). Effector cells (107-lo” cells, 0.1 ml), target cells (lo5 cells, 0.1 ml), and 0.1 ml of appropriate serum (IRS, NRS, or MEM-FCS) was added to the experimental wells of a Linbro plastic plate (Linbro Plastics, Los Angeles, CA). The plates were incubated for 60 min at 37”C, 1.0 ml of cold MEM added, and the contents of each transferred to a 10 x 75-mm test tube. These were centrifuged at 400g for 10 min at 4°C and the supematant transferred and counted in a gamma scintillation counter. The total releasable chromium was determined by incubation of lo5 target cells with 1 ml of distilled H,O and represented 60-70% of the total “‘Cr label present. Specific lysis was determined as follows: Specific lysis = E - C/T - C, where E is counts in experimental tube, C is the counts in the appropriate control, and T is the total releasable chromium. Ratios of effector cells to target cells varied from 10: 1 to 100: 1. Values for C ranged from 5 to 20% of the total label. Experiments in which C exceeded 20% were excluded. Target cell phagocytosis is not observed in this system. SRBC rosettes. Lymphocytes (2 x 10c) suspended in Hanks Balanced Salt Solution (HBSS; Grand Island Biologicals, Berkeley, CA) and 40 x 10” SRBC (Mission Laboratories, Rosemead, CA) in a total volume of 1 ml were centrifuged at 50g for 6 min. The pellet was gently resuspended. One tenth milliliter of the preparation was mixed with 0.1 ml crystal violet (1 mg/ml), allowed to remain at 4°C for 5 min, and then placed in a hemocytometer chamber. Two hundred lymphocytes were counted and those surrounded by 23 SRBC were scored as rosettes. The final calculations are expressed as percentage of rosettes. In man, SRBC formation under these conditions is a marker of T cells (19-21). SRBC-Ab rosettes. SRBCs were incubated with a subagglutinating dilution of a hyperimmune rabbit anti-SRBC antiserum at 37°C for 30 min, washed, and resuspended to 10H/ml. Four tenths milliliter was mixed with 0.2 ml of the lymphocyte preparation (107/ml) and 0.4 ml of HBSS. Tubes were centrifuged at 50g for 6 min at 4X, and resuspended on a rotating apparatus (Drummond Instrument Co., Bloomall, PA) (18 cycles/min) for 2 min. The preparations were allowed to remain at 37°C for 30 min, and were resuspended and counted in a manner similar to the SRBC rosettes. In man, SRBC-Ab rosette formation is a function of cells with an Ig receptor, i.e., polymorphonuclear leukocytes, B cells, and monocytes (10,11,22,23). Human thymocytes showed no SRBC-Ab formation. Parallel studies of SRBC-Ab rosette formation and fluorescent anti-human Ig staining have revealed a high level of concordance (24). Statistical analysis. Statistical analysis between groups was determined by Student’s t test.

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RESULTS Effector cell function in ADCC. A summary of the results of 23 tests is presented in Fig. 1. Each patient and control was tested in all three effector cell : target cell ratios. Of nine patients with CLL, eight were tested once and one was tested on two separate occasions. The nine normal controls and the four patient controls were each tested once. No statistically significant difference was observed between healthy controls and hospitalized controls at any of the effector cell : target cell ratios tested (P > 0.30, n = 13). In contrast, lymphocytes from patients with CLL were significantly less effective than lymphocytes from both control populations at effector cell : target cell ratios of 100: 1, 50: 1, and 10: 1 (P < 0.001, tz = 18; P < 0.005, n = 14; P < 0.005, n = 14, respectively). This defect was most evident at the two lower ratios. In two patients studied using a range of IRS dilutions (lo-‘-lo-“), the defect was evident at all concentrations tested. Figure 2 presents studies in two patients with CLL in whom a significant defect in effector cell function could be detected only at ratios of 50: 1 and 10 : 1 (P < 0.025, n = 9; P < 0.001, n = 9, respectively). Cells from LCL were ineffective in mediating ADCC at all effector cell : target cell ratios. Rosette formation. Representative results of rosette formation with SRBC and antibody-coated SRBC (SRBC-Ab) using lymphocytes from patients with CLL are indicated in Table 1. The percentage of SRBC rosette-fomring cells was uniformly decreased from control values in patients with CLL (P < 0.001, n = 14). The percentage of SRBC-Ab rosette-forming cells was variable in CLL patients. In the majority of patients it was normal (patients 4,6,7), while in two patients the percentage was increased (8,9). Cells from LCLs were unable to form rosettes with either SRBC or SRBC-Ab. Comparison of ADCC activity und rosette,fornlution. A comparison of thth ability of cells from normal controls and CLL patients to form SRBC and SRBC-Ab rosettes and to function as effector cells in ADCC is presented in Table 1. CLL cells were invariably less effective as effector cells in ADCC than were control cells. This defect was present in all patients studied, regardless of whether the percentage of cells capable of forming SRBC-Ab rosettes was depressed (patients 4,6,7) or elevated (patients 8,9). The presence of a surface Fc receptor did not correlate with ADCC effector cell function in these patients.

EFFECTOR

FIG. 1. ADCC activity of lymphocytes trols (hatched bars), and patients with

CELL:

TARGET CELL

RATIO

from normal controls (diagonal CLL (striped bars). Bars represent

bars), hospitalized mean

-t SE.

con-

LYMPHOID

CELLS

9 3 it i! 8 FIG.

2. ADCC

activity of lymphocytes bars), and two patients

CELL:

from with

TARGf3

381

ADCC

SO:1 E!=FEClW

trols (hatched

IN

IO:1 CELL

RATIO

normal controls (diagonal bars), hospitalized CLL (striped bars). Bars represent mean

con+-SE.

A similar comparison of ADCC activity and rosette formation of lymphoblastoid cells was made (Table 1). None of the cells tested was able to mediate ADCC, nor were they able to form SRBC-Ab rosettes. DISCUSSION The proliferation of cells in CLL involves B lymphocytes as detected by surface markers of membrane-bound Ig (25-27), complement receptors (22,28,29), and aggregated Ig receptors (30,31). We have used the SRBC-Ab assay, which detects the presence of a surface receptor for the Fc portion of the Ig molecule, to document the B-cell origin of these cells. Similar assays using antibody-coated sheep or human erythrocytes have been developed by several investigators to identify B cells in man (10,11,22,23). Our findings are consistent with those of Dickler et al. (30) who demonstrated aggregate binding cells in all patients with CLL. The discrepancy between our own percentages and theirs probably relates to the relative insensitivity of the SRBC-Ab assay. Possibly the receptor density TABLE

1

HOSETTE FOHMATION ANU ADCC ACTIVITY OF LYMPHOCYTES FROM NORMAL CONTROLS, PATIENTS WITH CLL, AND LYMPHOBLASTOID CELL LINES Group Controls” Patient Patient Patient Patient Patient LCL

WBC”

4 6 7 8 9

7,000 60,000 45,000 62,000 43,000 76,000 -

Percent lymphocytes

Percent SRBC-RFC”

34 95 93 95 90 98 100

‘I WBC count/p1 to nearest 1000. * Rosette-forming cells. Mean of triplicates. c Effector cell : target cell ratio 100: 1, mean d Mean of controls studied (n = 9). p SD k 5.5, SE k 1.8. ‘SD k7.1. SE k2.4.

72” 3 1 0 1 2 0

of triplicates.

Percent SRBC-AbRFC 15’ 13 12 12 43 62 0

Percent specific lysis in ADCC’ 100 57 73 22 0 18 0

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required for aggregate staining is less than that required for binding of an antibody-coated SRBC and this would explain the relatively low percentage (mean 28, range 12-62) of SRBC-Ab-binding cells in the patients studied. We selected the SRBC-Ab assay rather than fluorescent aggregate binding because of its functional relevance to the binding step of ADCC. The finding of a normal to high percentage of SRBC-Ab binding cells in CLL contrasts with the report of Shevach et al. (22) in which no rosette formation could be demonstrated in six patients with CLL, and a low percentage (4%) of rosette-forming cells in one patient. The two assays differ. In the present study a hyperimmune rabbit anti-SRBC antiserum was used, while Shevach et al. used a fractionated rabbit IgG with predominantly anti-For+ mann activity. Different quantities and subclasses of antibodies may be involved, and there is evidence to suggest that both factors may be important in the demonstration of SRBC-Ab binding cells (32-35). The observed absence of SRBC and SRBC-Ab rosette formation with cells obtained from lymphoblastoid cell lines (LCLs) is in agreement with reported studies (22). LCLs, with rare exceptions, have been demonstrated to be of B-cell origin as defined by surface Ig staining (36), in vitro Ig production (37), presence of the Ebstein-Barr virus (EBV) genome, and expression of viral products on the cell surface (38). Shevach et al. (22,39) found two of three LCLs derived from normal individuals to be devoid of cells capable of binding SRBC-Ab. One objective of the current investigation was to delineate the steps involved in the mediation of ADCC activity. Using homogeneous populations of B cells from patients with CLL, we were able to evaluate the relative importance of the presence of an Fc receptor to ADCC activity. In patients with normal or high levels of SRBC-Ab binding, decreased or absent ADCC activity was observed, suggesting that binding per se is insufficient to cause target cell lysis and that a subsequent step is involved. Cells from LCL which were unable to bind SRBC-Ab were ineffective in mediating ADCC activity. We interpret these observations to indicate that while an Fc receptor may be a necessary prerequisite for ADCC effector cell function, it is insufficient in itself for normal activity. Lymphocytes from patients with CLL exhibit a defect in a subsequent step in mediating target cell cytolysis. These observations contrast to those in normal controls in which the level of ADCC activity correlates with the absolute number of Fc receptor-bearing cells in the effector cell population (24). The exact subpopulation in which cells normally function as effector cells in the ADCC assay is not settled, In some CLL patients with virtually all B lymphocytes it appears that these effector cells are B cells. In normal individuals, we cannot exclude the possibility that in addition to B cells, “null” or “K” cells, i.e., non-T surface-fluorescent Ig-negative staining cells, which appear to possess either an Fc receptor, a C’3b receptor, or both, can act as effector cells (10,ll). Monocytes, which possess an Fc receptor and have been demonstrated to be active in ADCC, have been eliminated by removal of phagocytic cells. Recently, Greenberg et al. (40,41) have demonstrated

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ADCC activity in mice by nonphagocytic monocytes. We have not eliminated this possibility from our experiments with normal individuals as we used phagocytosis as the basis for monocyte depletion. It is difficult to conceive of these cells, if present in man, as being able to effect ADCC activity at low effector cell :target cell ratios. We have previously demonstrated ADCC activity at ratios as low as 1: 100 (24). Perlmann and Perlmann (42) have described a single patient with CLL whose lymphocytes demonstrated a relative defect in ADCC activity. This defect was present at low antiserum dilutions. The cells were tested at only one effector cell : target cell ratio and showed increased activity when stimulated with phytohemagglutinin. Campbell et al. have reported a selective defect in ADCC activity in a patient with macroglobulinemia (43), in patients with inflammatory bowel disease treated with azothioprine (44), and in children with leukemia treated with chemotherapy and radiotherapy (45). In summary, the present study documents a defect in the ability of lymphocytes from patients with CLL and in cells obtained from LCL to function as effector cells in ADCC. The defect in CLL is not related to a loss of Fc receptors by these cells and probably represents a defect in a subsequent step involved in cell-mediated cytolysis. ACKNOWLEDGMENTS The authors thank Ms. Leane Watson, Ms. Joanne Feldhaus, and Ms. Marilyn Jobin for their valuable technical assistance; and the physicians of the Divison of Hematology of the Department of Medicine, UCLA Medical Center, Los Angeles, California, for their cooperation. This work was supported by Grant CA 12800 from the National Cancer Institute. The authors also thank Dr. Martin J. Cline for his stimulating suggestions. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

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