CELLULAR IMMUNOLOGY 43, 160- 175 (1979)

Spontaneous

Human Lymphocyte-Mediated Tumor Target Cells VII. The Effect of lmmunodeficiency

Cytotoxicity

against

Disease

H. F. PROS,* S. GUPTA,~ R. A. GOOD,? AND M. G. BAINESS Departments of *Radiation Oncology, $*Microbiology and Immunology, and *Obstetrics Gynecology, Queen’s University, Kingston, Ontario, Canada, K7L 2V7, and TMemorial Sloan-Kettering Cancer Center, New York, New York 10021

and

Received April 19, 1978

Spontaneous lymphocyte-mediated cytotoxicity (SLMC) and antibody-dependent cellular cytotoxicity (ADCC) was assessed in 13 patients with immunodeficiency diseasesimmunodeficiency-thymoma syndrome (1), Bruton type agammaglobulinemia (3), and common variable hypogammaglobulinemia (9). SLMC and ADCC function were intact (and possibly enhanced) in the patient with immunodeficiency thymoma. Both ADCC and SLMC were detectable in the three patients with X-linked agammaglobulinemia, one of whom had lower than expected SLMC. In all of the immunodeficient patients, the relative inability of B lymphocytes to produce immunoglobulin in vivo or in vitro did not consistently affect the ability of (presumably) other lymphocytes to mediate SLMC and ADCC, although in three of the CVH patients this was lower than normal. In every case, removal of Fc receptor-bearing cells from the patients’ lymphocyte preparations severely depleted SLMC (and ADCC when tested), but cytotoxicity was either unchanged or enhanced by depletion of E rosette forming T cells. The effects of Fc receptor-positive cell depletion, T-cell depletion, culture serum variation, or the addition of antibody-coated erythrocytes to the assay were similar on both SLMC and ADCC effector cells (“NK” and “K” cells), and whether patients’ or normal lymphocytes were tested. The possible significance of the results with respect to surveillance against cancer is discussed.

INTRODUCTION The interrelationship between immunodeficiency and malignant disease has been the subject of considerable research in the last two decades, primarily in attempts to substantiate or negate the theory of immune surveillance against cancer (l-3). Several reviews have been written on this subject and, as with most biological phenomena, the problem is considerably more complicated than had originally been supposed. With the increasing accumulation of experimental data from both in vivo and in vitro models, the concept is gradually evolving that T-cell surveillance is only one aspect of the phenomenon, and that several systems may well play an important role in preventing the early development of malignancy. One such system is that of spontaneous lymphocyte-mediated cytotoxicity (SLMC) [reviewed in (4)], also known as “natural” killing. Although now well established as an in vitro phenomenon, the ability of lymphocytes from apparently normal donors to lyse 160 0008-8749/79/030160-16$02.00/O Copyright 0 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.

SLMC IN IMMUNODEFICIENCY

DISEASE

161

tumor cells has been viewed with scepticism as far as being a genuine in vivo event was concerned. To some extent this has been tempered by the considerable data which has been obtained in animal systems (5-7). In humans, however, the evidence for an in vivo role for spontaneous killer cells is more tenuous and is, by necessity, limited to indirect conclusions drawn from experiments such as those designed to demonstrate the relationship between SLMC function and the extent of malignant disease (8, 9), the nature of the target cells which are susceptible to SLMC (lo- 12), and the nature of the “antigen” which is recognized by the killer cell (13, 14). In the present work we have studied the SLMC activity of lymphocytes from patients with various immunodeficiency diseases. In this study three basic questions were considered: Is SLMC function intact in these patients (with the resultant implications with respect to surveillance)? Is the effector cell subpopulation similar to that of normal donors? Can our knowledge of the nature of the patient’s immunodeficiency be used to form conclusions as to the mechanism of SLMC? MATERIALS

AND METHODS

Patient selection. The results described in this study were obtained using purified lymphocytes from patients under investigation at the Memorial Sloan-Kettering Cancer Center for immunodeficiency disease. Thirteen patients were studied, with the following diagnoses: immunodeficiency-thymoma syndrome (one), Brutontype agammaglobulinemia (three), and common variable hypogammaglobulinemia (nine). The clinical and immunological profile of many of these patients has been reported (15-17) and these facts are summarized in Tables 1 and 2. None of the patients had received therapeutic human gammaglobulin within 3 weeks of our assessment. Control lymphocyte preparations were obtained from at least two normal healthy donors, one in New York and one in Kingston. Antisera. Anti-Ox-RBC antibodies were raised in rabbits by repeated weekly injections of 1 ml of 25% Ox-RBC (Woodlyn Farms, Guelph, Ontario). The hyperimmune serum was heat inactivated (56°C 30 min) and used at the dilution giving optimum EA-RFC with human peripheral blood lymphocytes (usually 1:100). To raise rabbit anti-P815 antibodies, a rabbit was immunized with a subcutaneous injection of 10’ cultured P815 cells in complete Freund’s adjuvant, followed by periodic intravenous booster injections of 1 ml of 5-10 x lo6 P815 cells/ml in RPMI. This antiserum was effective at dilutions of lo+ to lop5 and was used at a dilution of 10p3. Lymphocyte preparation. Mononuclear cells were isolated from peripheral venous blood on Ficoll-Hypaque (FH) or Ficoll-Isopaque (FIp) density gradients. Cells were washed three times in Hank’s balanced salt solution (HBSS) or RPM1 and resuspended in medium RPMI-1640 containing penicillin (100 IU/ml), streptomycin (100 i&ml), and 20% fetal calf strum (FCS), at a concentration of 2 x IO6cells/ml. Phagocytic cells were removed by incubation with carbonyl iron at 37°C for 30 min and separating on FH gradient, or by magnetism. Purified cells so obtained contained less than 1% peroxidase-positive cells and less than 1% phagocytic cells as determined by the uptake of antibody-coated Ox-RBC. After preparation, the lymphocytes were transported at ambient temperature by air

162

PROSS ET AL. TABLE 1 Lymphocyte Subpopulations in Immunodeficient Patients Rosette-forming cells (%) Name

T

Fc

C3

Surface Ig-positive cells (%)

R-Fcb MRFC

PVC

M

D

G

A

Thymoma and hypogammaglobulinemia ws Bruton-type agammaglobulinemia BL RM MR

75.5

23.0

3.0

22.0

0.5

0

0

0

0

0

91.0 81.0 88.0

17.0 25.0 11.0

9.0 18.0 10.5

9.0 20.0 5.0

0.5 0.5 0.0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

Common variable immunodeficiency TD CB MD cv ME EC MG RR JD

81.0 83.0 78.5 89.0 89.0 80.0 88.0 91.0 84.0

20.0 13.0 21.0 24.0 20.0 18.0 14.0 10.0 25.0

12.0 8.0 17.0 5.0 7.0 8.0 6.0 10.0 12.0

10.0 6.0 9.0 10.0 12.0 8.0 9.0 6.5 8.0

8.0 4.0 6.0 5.0 7.5 6.0 4.0 3.0 10.0

10.0 7.0 12.0 10.0 15.0 7.0 8.0 7.0 10.0

9 4 12 5 5 4 6 6.5 8.0

6 6 10 4 3 2 4 4 5

1 0.5 0 2 0 1 0.5 2 0

0.5 1 0.5 0 0.5 1 0 1 0.4

84.7 20.3 9.7 14.0 f 4.5 2 6.0 f 4.0 f 3.4

6.5 zi 2.5

Normal donors (50) Mean 2 SD

12.4 6.9 f 4.8 k 4.7

2.0 4.3 1.8 k 3.1 2 1.9 ?r 1.7

a Determined by the method described (15, 16). b Ripley Fc-RFC-(high aftinity Fc receptors)-human erythrocytes coated with Ripley type serum containing anti-RBC-IgG. c Fluorescein isothiocyanate conjugated F(ab’), antiserum against IgM, IgG, and IgA.

freight to Kingston. On receipt the following day the cells were washed twice in RPM1 1640-10% FCS (hereafter referred to as RPMI) and resuspended at a concentration of 2 x lo6 cells/ml. Preliminary studies showed that SLMC function was unaffected by overnight storage at room temperature or at 4°C. Rosette formation. E, EA, and M rosettes, for the identification of T cells (18), Fc receptor bearing cells (19), and B cells (20), respectively, were prepared by centrifuging 2 x lo5 lymphocytes in 100 ~1 RPM1 with 100 ~1 of 1% of the appropriate indicator cells at 200g for 5 min. This was followed by immediate resuspension and enumeration (EA-RFC), incubation at 28°C for 60 min (M-RFC), or incubation at 37°C for 15 min and 4°C for 60 min (E-RFC). The indicator cells were as follows: EA, Ox-RBC coated with hyperimmune rabbit anti-Ox-RBC suspended in RPMI; E, sheep RBC suspended in FCS absorbed with SRBC, and MC57B1/6 mouse RBC suspended in FCS absorbed with MRBC. E-rosette assays reported in the original assessment of the patients (Table 1) were done using SRBC-absorbed human serum (AB +) with overnight incubation incubation at 4°C.

SLMC IN IMMUNODEFICIENCY

163

DISEASE

TABLE 2 Immunoglobulin Levels and in Vitro Ig Secretion by Peripheral Blood Mononuclear Cells from Immunodeficient Patients Serum immunoglobulin (mg/dl) Ig secretion M

Thymoma and hypogammaglobulinemia ws

580

540

280

- h

Bruton-type agammaglobulinemia BL RM MR

0 0 0

22 48 44

0 0 0

ND

Common variable immunodeficiency TD CB MD cv ME EC MC RR JD

30 40 328 21 3 30 40 20 55

230 650 195 1100 44 230 340 230 775

20 0 280 0 3 20 10 IO 32

140 60-280

1250 800- 1800

210 90-450

Normal Mean

G

A

Name

in vitro”

ND 2 _f

ND ND if

++

(1Determined by the method described (17). The values were converted to ng/mI. b (-) no synthesis and secretion of Ig; (ND) not done; (k) barely detectable Ig (100 r&IO6 cells); (+ +) normal Ig synthesis and secretion (- 1000 ng/ IO6cells).

Rosette depletion. EA and E rosettes were formed by mixing equal volumes of 2 x 10Yml lymphocytes and 1% indicator cells as described above. Control preparations consisted of lymphocytes plus 1% Ox-RBC. After centrifugation and incubation (above), all but 2 ml of the supernatant was removed, and the rosettes were gently resuspended. This suspension was carefully layered onto 2 ml of FIp in a 17 x IOO-mmplastic culture tube, and centrifuged at 20°C for 12min at 800g. The ring was removed, washed three times, and resuspended at 10Yml. The lymphocyte yields from this procedure, as a percentage of the number of cells placed on the FIp, were variable with the different patients and donors, and have therefore been tabulated with the Results. SLMC and ADCC. Allogeneic SLMC vs the chronic myelogenous leukemia cell line K562 (21) and xenogeneic SLMC vs the DBA/2 mouse mastocytoma P815x2 (22) were performed as previously described (10, 23). The reasons for using the K562-SLMC assay as the method of choice in assessing human SLMC have also been outlined in detail (4, 10, 14). Briefly the technique consists of an 18-h “‘Cr release assay using V-bottom microplates. Although cell lines are maintained in

164

PRO!% ET AL.

RPMI-1640 with 10% FCS, and the assay is performed in this medium, the results are independent of FCS-derived antigens or stimulating factors present in the FCS (14). ADCC, also evaluated in an 18-h assay, was determined by the incorporation of rabbit anti-P815 into the culture system at a final concentration of 10p3.This was compared with a similar concentration of normal rabbit serum (NRS). The percentage cell-mediated lysis (%CML) was calculated as follows %CML =

cpm test - cpm medium x 100%. cpm max - cpm medium

Although the tables presented show only the actual data obtained at a single lymphocyte to target (L/T) ratio, dose response curves were done for each control and patient using at least three L/T ratios on the linear part of the curve. To compare patient with control, the value for %CML caused by 5 x lo4 patient’s lymphocytes (vs lo4 K562) was calculated from the dose response curve. The dose response curve of the control was then used to calculate the number of normal lymphocytes required to cause an equivalent amount of lysis (equivalent control ratio, ECR) (8). The relative SLMC activity of the patient’s lymphocytes was then expressed as a proportion of the control lymphocyte SLMC activity. As an example, a patient lymphocyte preparation causing 25% lysis at 5/l has a relative SLMC activity of 50% or 0.5 when compared to a control preparation requiring a ratio of only 2.5/l to achieve equivalent lysis. This has previously been explained graphically (8). The method is similar to that using lytic units except that a fixed L/T ratio is chosen as the reference point instead of a fixed percentage cytotoxicity. This method was chosen because an L/T ratio of 5/l is more frequently on the linear part of the dose response curve in the K562 assay than any particular percentage cell-mediated lysis. Statistical evaluation. The standard error of the results from triplicate cultures was rarely greater than + 2%. Because of the small number of patients available for study, no attempt has been made to draw statistical conclusions, apart from comparisons with a previously obtained normal range. RESULTS Immunological

Function

Studies

The results of the clinical and immunological assessment of the patients studied in this paper have been reported elsewhere (15- 17), and will not be repeated in detail here. A summary of the findings is presented in Tables 1 and 2. The principle defect in these patients was in the ability of their B cells to produce immunoglobulin in vitro upon stimulation with pokeweed mitogen. For the most part this was reflected in the serum immunoglobulin levels, which ranged from the lower limits of detection in patients with Bruton-type agammaglobulinemia to being severely to moderately depressed in the patients with common-variable immunodeficiency and immunodeficiency-thymoma syndrome. In all cases except the thymoma-immunodeficiency patient, E-rosette proportions and T-cell functions were normal. In the case of the thymoma patient, normal T-cell levels were associated with anergy and a gross defect in mitogen responsiveness. The analyses shown in Tables 1 and 2 were not necessarily done at the same time as the experiments described in the text, mainly because of the number of

SLMC IN IMMUNODEFICIENCY

165

DISEASE

100, .-u) m 2-Y -I -cl 5

go60r”60-

$

so-

5

40-

jj

30-

E

zo-

k8

IO20

IO

5

125

2.5

Lymphocyte

40

IO

20

5

to Target Ratio

FIG. 1. Dose-response curve of immunodeficiency-thymoma lymphocytes in SLMC. (A) vs KS62 (allogeneic SLMC) (B) vs P815 (xenogeneic SLMC). (0) Immunodeficiency-thymoma, (0) control (New York), (A) control (Kingston). Standard errors about the means did not exceed 2%.

procedures involved. This accounts to some extent for the discrepancies observed between rosette counts tabulated in Table 1 and those in Tables 3-5, especially with respect to E rosettes. This may also be due to the effects of transport and storage overnight, or to minor differences in techniques of rosette formation. SLMC and ADCC in Immunodeficiency

-Thymoma

Syndrome

The effect of a severe deficiency in T- and B-cell function on spontaneous and antibody-dependent cytotoxicity is illustrated in Fig. 1 and Table 3. SLMC was assessed in both the allogeneic and xenogeneic assays, and ADCC was evaluated using antibody against P815. In Fig. 1 the complete dose response curve is shown for both types of SLMC assays. It can be seen in this figure that SLMC function was markedly enhanced in this patient, and that this observation was consistent using TABLE 3 SLMC and ADCC by Unfractionated and RFC-Depleted Lymphocytes Immunodeficiency-Thymoma Syndrome Cell-mediated lysis (%)b RFC (%)

Cell recovery’ (%I

Preparation

Donor”

E

EA

KS62

NRSP815

Unfractionated

Control L Control A Patient WS

60 68 71

33 36 23

25 22 60

19 16 43

57 58 98

-

EA-RFC depleted

Control L Control A Patient WS

78 84 88

2 3 1

3 4 6

-

-

45 31 42

AbP815

a Controls Land A were normal donors whose lymphocytes were obtained and separated at Kingston and New York, respectively. b Data are shown for lymphocyte target ratios of 5/l (vs K562) and 20/l (vs P815). The %CML against antibody (ab)-coated P815 is shown without correction for “background” SLMC (NRSP815). r The number of cells recovered at the interface expressed as a percentage of those put on the gradient.

166

PROSS ET AL.

either the allogeneic (vs K562) or the xenogeneic (vs P815) assay. The addition of antibody against P815 resulted in a further increase in cytotoxicity by lymphocytes from both the controls and the patients (Table 3). It can also be seen in Table 3 that removal of Fc receptor-bearing lymphocytes from the effector cell population obliterated spontaneous cytotoxicity by both patient and normal control lymphocytes (as expected). SLMC and ADCC in Patients with Defects in Immunoglobulin Production To assess the role of B cells in SLMC, as well as to determine whether or not normal immunoglobulin synthesis is required for spontaneous cytotoxicity, a number of patients having antibody deficiency syndromes were examined with respect to ability of their lymphocytes to mediate SLMC and ADCC. The results of those studies are shown in Fig. 2 and Table 4 (Bruton-type agammaglobulinemia), and Table 5 (common variable hypogammaglobulinemia). The data obtained from these patients indicated that both spontaneous and antibody-dependent cytotoxic function remained intact in these patients, in spite of negligible immunoglobulin synthesis. As far as could be determined, the cytotoxic cell types in the patients’ lymphocyte preparations were identical to those of the controls, since the cytotoxicity was removed in every case by removal of Fc receptor-bearing lymphocytes. Reduction of E rosette-forming cells either had no effect, or enhanced cytotoxicity by both control and patient lymphocytes, a result which is similar to our previous observations on normal lymphocytes (10,23,24) but which is at variance with those of others (25,26). It can also be seen from these data that none of the experimental manipulations of the normal or the patients’ lymphocyte preparations lead to results indicating that the SLMC effector cell is different from the ADCC effector cell. Quantitative Differences in Spontaneous Cytotoxic Function Our primary aim in the studies described in this paper was to determine whether spontaneous cytotoxic function was still present in patients with defects in B-cell

IO

a

5

Lymphocyte

2.5

o- ........ .........._..0 .___......___.,_.___.. n I I 1

IO

to Target

5

2.5

Ratio

FIG. 2. Dose-response curves of Bruton-type agammaglobulinemia lymphocytes in SLMC. (A) vs KS62 (allogeneic SLMC). (B) vs P815 (xenogeneic SLMC). (0) Bruton type agammaglobulinemia, (0) controls (New York). ( ~ ) Patient BL, Control H; (---) Patient RM, Control N; ( . . . ) Patient MR, Control C. Standard errors about the means did not exceed 2%.

SLMC IN IMMUNODEFICIENCY

167

DISEASE

TABLE 4 SLMC and ADCC by Unfractionated and RFC-Depleted Lymphocytes Bruton-Type Agammaglobulinemia Cell-mediated lysis, 511(76) RFC (%) M

K562

NRSP815

AbP815

Cell i-ecovery @:-)

Preparation

Donor

E

EA

1. Unfractionated

Control H Patient L

57 73

28 11

7 1

44 51

32 25

42 34

EA-RFC depleted

Control H Patient BL

73 88

3 1

15 1

8 11

5 9

6 7

18 50

Ox-RBC depleted

Control H Patient BL

54 75

33 4

9 1

46 51

27 21

49 32

38 44

E-RFC depleted

Control H Patient BL

3 5

52 22

22 1

65 52

52 32

56 50

18 9

2. Unfractionated

Control H Control N Patient RM

60 71 71

24 21 18

10 11 2

50 48 24

20 14 8

50 37 26

-

EA-RFC depleted

Control H Control N Patient RM

72 68 77

1 2 1

2 6 1

3 5 6

2 3 I

6 4 1

60 51 52

Ox-RBC depleted

Control H Control N Patient RM

65 72 70

32 24 11

8 5 1

63 39 29

22 12 6

52 39 27

80 60 48

3. Unfractionated

Control P Control C Patient MR

62 75 86

25 35 30

-

28 3 12

-

-

-

EA(SRBC) depleted

Control P Control C Patient MR

67 79 86

2 4 3

-

1 0 3

-

-

23 25 33

F. Ip Only

Control P Control C Patient MR

57 66 79

23 28 27

-

21 2 12

-

-

66 63 50

-

number and/or function. Because of the small numbers of patients involved, and the variability of the assay from day to day even with normal healthy donors, it is difficult to assess whether there are any quantitative differences in cytotoxicity by lymphocytes from these patients. In Table 6 we have expressed the data obtained as a proportion of either of the two controls run at the same time. The method of expressing the relative SLMC between patients and control has been described previously (8), and utilizes the lymphocyte to target-cell dose response curve to calculate how many (or how few) normal lymphocytes are required to lyse the same number of target cells as are lysed by those of the patient at an arbitrarily chosen L/T ratio (5/l). In previous work (8) we found a wide range of R-SLMC comparing

PROSS ET AL. TABLE 5 SLMC and ADCC by Unfractionated and RFC-Depleted Lymphocytes Common Variable Hypoagammaglobulinemia Cell-mediated lysis, 511(%) RFC (%)

Cell recovery m

Preparation

Donor

E

EA

M

K562

NRSP815

1. Unfractionated

Control S Patient TD

63 68

19 29

-

27 26

7 12

32 29

EA-RFC depleted

Control S Patient TD

67 65

1 2

-

2 3

1 2

4 4

50 38

Ox-RBC depleted

Control S Patient TD

65 53

6 20

-

21 20

10 15

36 34

50 50

E-RFC depleted

Control S Patient TD

2 0

56 47

-

36 22

14 16

42 37

5 18

Control N Patient CB Patient MD Patient CV

72 75 60 64

33 16 19 28

10 5 14 3

26 12 5 10

-

-

-

EA-RFC depleted

Control Patient Patient Patient

N CB MD CV

73 85 68 76

3 1 2 3

10 10 17 5

2 4 6 2

-

-

35 71 55 61

Ox-RBC depleted

Control Patient Patient Patient

N CB MD CV

64 84 68 70

35 13 15 26

6 5 11 3

27 9 19 18

-

-

80 180 25 42

E-RFC depleted

Control Patient Patient Patient

N CB MD CV

12 18 14 7

42 26 18 28

8 8 12 6

32 16 21 19

-

-

25 20 26 20

3. Unfractionated

Control B Patient ME

68 82

35 24

7 2

61 63

28 42

47 55

-

EA-RFC depleted

Control B Patient ME

90 83

2 1

5 3

2 8

-

-

45 35

Ox-RBC depleted

Control B Patient ME

73 85

27 20

6 4

38 44

-

-

100 70

4. Unfractionated

Control B Patient EC

65 62

26 21

9 5

34 56

11 15

37 52

-

EA-RFC depleted

Control B Patient EC

74 67

1 0

5 2

1 1

0 2

2 7

43 70

Ox-RBC depleted

Control B Patient EC

62 50

26 23

2 3

34 60

12 17

34 56

57 96

2. Unfractionated

AbP815

-

SLMC IN IMMUNODEFICIENCY

169

DISEASE

TABLE 5 (Continued) Cell-mediated lysis, 5/l (%) RFC (%) AbP815

Cell recovery (So)

Preparation

Donor

E

EA

M

K562

NRSP815

5. Unfractionated

Control N Patient MC

80 81

19 20

8 3

18 25

15 12

27 31

EA-RFC depleted

Control N Patient MG

82 86

3 1

5 2

1

0

2 0

6 1

67 57

Ox-RBC depleted

Control N Patient MG

82 72

18 30

9 4

18 25

13 15

31 35

58 64

6. Unfractionated

Control N Patient RR

71 83

26 23

-

16 24

2 4

19 28

-

EA-RFC depleted

Control N Patient RR

85 78

5 5

-

1 7

-

-

33 30

Ox-RBC depleted

Control N Patient RR

74 76

24 20

-

21 27

-

-

80 75

7. Unfractionated

Control H Patient JD

57 76

28 10

7 3

44 18

32 15

42 25

-

EA-RFC depleted

Control H Patient JD

73 83

3 1

15 2

8 4

5 4

6 1

18 46

Ox-RBC depleted

Control H Patient JD

54 70

33 5

9 2

46 15

27 7

49 18

38 75

E-RFC depleted

Control H Patient JD

3 7

52 17

22 7

65 40

52 26

56 30

18 9

-

normals with other normals as “controls” and the 95% confidence limits in that study were 0.38-2.8. It can be seen in Table 6 that several of the patients fall outside of this range. In most cases the patient’s relative cytotoxic function was similar no matter which control was used, and yielded the same conclusion as to whether or not it was decreased, normal or enhanced, The greatest discrepancy between the two controls occurred with Patient MR (Bruton-type agammaglobulinemia) and in this case the New York control was unusually low (see Fig. 2) and should perhaps be ignored. If this is done, Table 6 can be interpreted as showing that 3/9 of the patients with common variable hypogammaglobulinemia and one of the three patients with Bruton-type agammaglobulinemia were suppressed in SLMC, while all except one of the other patients were within normal limits. The one patient with immunodeficiency-thymoma syndrome had markedly enhanced spontaneous cytotoxicity and, in view of the similar results obtained with both controls, and the degree of enhancement observed, this is probably significant biologically as well as mathematically. Miscellaneous

Experiments

In order to further explore the extent of the similarity between normal and patient SLMC characteristics, a number of experiments were performed on selected

170

PROSS ET AL. TABLE 6 Tabular Summary of SLMC Activity by Lymphocytes from Immunodeficient Patients as a Proportion of Normal Control Lymphocyte Activity Control L/T ratio giving equivalent lysis”

Relative SLMC* (proportion)

SLMC(S/l) (%I”

AC

BC

vs A

vs B

Immunodeficiencythymoma syndrome ws

60

25.011

22.011

5.00

4.40

Bruton-type agammaglobulinemia BL RM MR

51 20 14

7.611 0.911 2.711

1.1/l 30.011

1.51 0.18 0.54

0.22 6.08

Common variable hypogammaglobulinemia TD CB MD cv ME EC MC RR JD

28 13 I 11 60 54 27 29 25

3.911 1.6/l 1.1/l 1.5/l 8.0/l 10.0/l I.511 2.211 2.211

4.711 2.0/l 1.411 1.711 5.011 17.011 9.9/l 7.311 -

0.78 0.32 0.22 0.30 1.61 2.00 1.50 0.44 0.45

0.94 0.39 0.21 0.33 1.00 3.42 1.99 1.45 -

Patient

a vs K562, calculated from linear regression lines based on cytotoxicity at three or more L/T ratios. b The expected range (95% confidence limits) based on a large group of normals is 0.38-2.80. A patient’s lymphocyte preparation which causes 25% SLMC at 5/l compared to a normal control preparation which requires only a 2.5/l LT ratio for equivalent lysis is defined as having a relative SLMC of 0.5, (8) (Materials and Methods). c A and B represent results obtained with normal control lymphocytes obtained and separated at Queen’s University and the Sloan-Kettering Cancer Center, respectively.

patients. Because the results were essentially negative, the data has not been shown. In one such experiment, lymphocytes from the CVH patients described in Table 5, group 2 were tested against K562 cells cultured for many months in human serum or in agamma calf serum, and, in each case, in the presence of either 5% agamma calf serum or immunoglobulin-free HSA in the assay. In each combination, the relative SLMC by the patients’ lymphocytes compared to each other and to the control was similar to that observed using FCS-cultured K562 in the standard assay, indicating the serum independence of the cytotoxic effect caused by patient lymphocytes, as has already been reported for normal donors (14). In another experiment, 50 ~1 of 1% of Ox-RBC coated with hyperimmune rabbit anti-Ox-RBC, uncoated Ox-RBC, sheep RBC coated with Cordis (Miami) 19s anti-SRBUmouse complement, or uncoated sheep RBC were added to the standard 15Oql assay mixtures, and left in the assay overnight. Of these reagents, only antibody-coated Ox-RBC inhibited SLMC, and the results were identical between the patient and the control. These results3 demonstrating that the effector cell is Fc receptor

SLMC IN IMMUNODEFICIENCY

DISEASE

1’11

positive, were also as expected (unpublished observations) and are similar to those reported (25). DISCUSSION In this paper, we have made a number of observations with respect to spontaneous and antibody-dependent lymphocyte-mediated cytotoxicity in patients with immunodeficiency disease. (a) SLMC and ADCC function were intact (and possibly enhanced) in a patient with a severe defect in both T- and B-cell function (immunodeficiency-thymoma syndrome). (b) SLMC was still detectable in three patients with Burton-type agammaglobulinemia. One of these three had lower than expected SLMC, the other two were probably not different from normal. (c) The inability of B lymphocytes to produce immunoglobulin in vivo or in vitro did not consistently affect the ability of (presumably) other lymphocytes to mediate SLMC and ADCC. In addition to the three Bruton-type agammaglobulinemic patients, nine patients with common variable hypogammaglobulinemia were all capable of SLMC. SLMC by lymphocytes from three of the common variable hypogammaglobulinemia patients was lower than normal, although it was not established whether this was due to the immune dysfunction, or because of factors secondary to the basic defect (e.g., recurrent infections). (d) In every case removal of Fc receptor-bearing cells from the patients’ lymphocyte preparations severely depleted SLMC. In those patients in which it was done, SLMC and ADCC were either unchanged or enhanced by depletion of E rosette-forming T ceils. These effects of Fc receptor-positive cell depletion or T-cell depletion were similar on both SLMC and ADCC effector cells (“NK” and “K” cells), and were similar to the results obtained using lymphocytes from normal donors. (e) Comparative levels of SLMC were similar between patients and controls when their lymphocytes were tested against KS62 cultured for many months in agamma calf serum or in human serum, and when the assay was performed in agamma calf serum or in an immunoglobulin-free HSA solution. (f) The addition of Ox-RBC coated with hyperimmune anti-Ox-RBC caused inhibition of immunodeficient patients’ SLMC against K562, while no suppression was observed with the addition of 19S-EAC cells, Ox-RBC alone or sheep RBC alone. These results were similar to those observed using normal donor lymphocytes. Assuming that these observations are valid, certain conclusions can be made from them. SLMC is not mediated by the majority of T cells or B cells, or by cells of the same lineage as the antibody-producing B cells. It is also not dependent upon immunoglobulin synthesis in vitro, and is presumably not caused by the same mechanism as ADCC. Although the mechanism of killing may be different, the effector cells of SLMC and ADCC are indistinguishable, whether judged by cell separation experiments or by the effects of immunodeficiency disease on their function. Finally, the surface receptors and functional characteristics of the SLMC effector cells in patients with immunodeficiency disease are indistinguishable from those of normal donors. These “experiments of nature” thus confirm a number of observations which

172

PROSS ET AL.

have been made in vitro using cells from normal donors. It has been shown using nylon wool columns (10,27) or columns coated with F(ab’), fragments of rabbit IgG anti-human F(ab’), of immunoglobulin G (28) that the presence of B cells in the assay system is not necessary for SLMC. It has also been shown that Protein A will block ADCC but not SLMC (26, 29), implying that SLMC does not occur by effector cell recognition of IgG complexed with target-cell antigens. On the other band, Takasugi and co-workers have shown that SLMC may be mediated by cytophilic antibodies (30) bound to effector cells possibly bearing B-cell alloantigens (31). That immunoglobulins are on the SLMC effector cells is supported by the work of Eremin et al. (32) who have shown depletion of SLMC activity by depletion of cells rosetting with rabbit anti-human immunoglobulincoated SRBC, and by the work of Perlmann’s group (33) who found that Fab fragments of rabbit anti-human Ig inhibited SLMC when added to the assay, although, as stated above, removal of B cells on columns coated with anti-human IgG had no effect (28). The conclusion of these groups that cell-bound immunoglobulins are involved in SLMC is difficult to reconcile with our own data, and that of others (see below), on patients with B-cell defects, although there is no reason to believe that any one mechanism is exclusive of all others. Since none of the patients we have studied was completely devoid of serum immunoglobulin, it is possible that some cytophilic antibody may have become bound to the killer-cell surface in viva, and remained to be effective in vitro. At the opposite extreme, several groups have suggested that the SLMC effector cells are T cells, based on the observation that they weren’t B cells (34), or by experiments on rosette-depleted or enriched lymphocyte preparations (25,26). The data presented here, demonstrating enhanced SLMC in association with a severe T-cell defect, and repeated experiments by ourselves and others (10,23,24,34-36) showing unchanged or enhanced SLMC after depletion of E rosettes do not support a role for T cells as the principle effector cell population. The fact that the E-depletion data can be reconciled with the conclusion that a subpopulation of SLMC effector cells may be Fc receptor-bearing T cells has already been discussed (24). The immunodeficiency-thymoma patient did in fact have higher than normal Fc receptor-positive T cells (T, = 27%) and this may have contributed to some of this patient’s cytotoxic activity (Gupta-unpublished results). While conclusions drawn by different investigators as to the surface characteristics of NK and K cells have not always coincided, within each laboratory the two effector cell types have always been demonstrated to behave similarly with respect to the effects of various depletion or enrichment experiments, suggesting that the effector cells are the same. An exception to this generalization is the recent work of Koren ef al. (37). These investigators also studied immunodeficient patients, and found that in their population of X-linked agammaglobulinemic subjects, SLMC activity was intact but ADCC was markedly suppressed. This result suggests that, although the surface characteristics of the two cell types may be similar, at some point in their differentiation they develop along different pathways. This discrepancy with our own work on these patients may have resulted because of the assay system used to detect ADCC. Koren et al. used specific mouse antibody-coated TNP-conjugated tumor cells, whereas we have used rabbit antibody-coated tumor-cell targets. The two target-cell systems have been shown to behave similarly in other experiments (37), but differences are observed when rabbit and mouse antisera are compared (Koren-personal

SLMC

IN IMMUNODEFICIENCY

DISEASE

173

communication). Other studies of ADCC in Bruton-type agammaglobulinemic patients have shown them to have intact ADCC when antibody-coated red blood cells were used (38), but when antibody-coated lymphocytes were the target this type of killing was low or absent (38,39). ADCC by lymphocytes from patients with common variable hypogammaglobulinemia was slightly but significantly reduced whichever target cell was used (38). SLMC was not assessed in these studies, although detectable and usually normal levels of both SLMC and ADCC were reported in a later study (37). Peter et al. (40), Rosenberg er al. (4 I), and Livnat (personal communication) have also studied SLMC in immunodeficient patients. Peter er al. (40) reported that in three patients with common variable hypogammaglobulinemia SLMC vs the melanoma line IGR3 was present but lower than that seen with normal donors. A similar result was oberved by Rosenberg et al. (41) on two patients with chronic mucocutaneous candidiasis and anergy. In studying a large number of patients with a variety of primary and secondary immunodeficiencies, Livnat (personal communication) has observed that SLMC against K562 was intact in the majority of these patients, and indistinguishable from that of normal donors. Two patients with severe combined immunodeficiency were also studied, and in these patients SLMC and ADCC were absent, as has been reported by Koren et al. (37). Unfortunately, this result does not help to definitively characterize the effector cells as far as their T- and B-cell lineage is concerned, although the result does suggest that a severe defect early in cell differentiation is required for the SLMC effector cell to be affected (37). Patients who have advanced malignant disease and leukemia have also been reported to have reduced SLMC (8,9,41,42) and ADCC (43) and this may account for some of the apparent decrease in disease-related cytotoxicity observed, for instance, in patients with melanoma (44, 45). Although the effect may be attributable to immunodeficiency secondary to malignancy, SLMC vs K562 in our patients did not correlate well with their ability or inability to mount a vigorous delayed hypersensitivity response (8), suggesting, as expected, that different cell types are involved in the two types of reaction. Speculation as to the in vivo relevance of SLMC has largely centered around its role as a type of surveillance mechanism against malignancy. This is, of course, difficult to prove, and the type of arguments which are used to support this theory are exactly analogous to those supporting a surveillance role for the immune response in general. Early epidemiological studies showing that there is an increased incidence of malignancy in patients with immunodeficiency diseases (46) were later disputed as evidence for immune surveillance by the argument that the malignancies involved were of lymphoreticular origin, and that the incidence of different types of cancer did not reflect the incidence seen in the population in general. It has also been pointed out that congenitally athymic mice do not have an increased incidence of spontaneous tumors, again suggesting that T cells are not of paramount importance in immune surveillance (47) [discussed in (3)]. On the other hand, however, SLMC or “natural killing” is still intact (or enhanced) in both immunodeficient patients and athymic mice (6, 48), suggesting that this is an alternative surveillance mechanism. It has also been shown by Kiessling er al. (49) that the ability of certain strains of mice to reject a tumor transplant is proportional to the genetically-determined in vitro NK activity of these mice, and similarly (50), using bone marrow reconstituted, thymectomized mice, the resistance of the

174

PROSS ET AL.

recipient mice to tumor take was found to reflect the in vitro NK activity of the donor strain. Thus, in animals at any rate, a strong case can be made for the existence of NK cell function in vivo [reviewed in (51)]. In man, the in vivo relevance of SLMC is more difficult to assess. It has been shown that short-term human tumor cell lines are less susceptible than established lines to SLMC (12,52), but this cannot be used as evidence against the in vivo susceptibility of malignant cells to being killed by NK cells in vivo. The majority of mechanisms which have been postulated to allow tumor cells to “escape” immunological attack would also apply to SLMC, and it should be expected that only those tumor cells which are capable of avoiding immunological or spontaneous cytotoxicity would subsequently develop into clinically apparent tumors. If the recent data (53.) demonstrating the HLA dependence of immune T-cell cytotoxicity in humans is found to apply to anti-tumor cytotoxicity, the potential role of SLMC in surveillance will become more significant. The presence of HLA is not necessary on the target cells in this system, and HLA-loss variants (e.g., K562) are still susceptible to attack. This, in conjunction with the observation that “NK” destruction of tumor target cells occurs rapidly and without apparent presensitization of the effector cells, suggests that SLMC is fundamentally different from other postulated mechanisms of surveillance, and may be complementary to them. ACKNOWLEDGMENTS We would like to thank Mr. Lee Boudreau, Mrs. Iva Kosatka, Mrs. Mabel Chau, Mrs. Debbie Alward, Mr. Joseph Cavallo, and Ms. Judith Miller for excellent technical assistance, and Mrs. Nancy Wainman for typing the manuscript. This project was funded by a grant from the Ontario Cancer Treatment and Research Foundation, by National Institutes of Health Grants CA-19267, CA-17404, CA-08748, AI-l 1843, and NS-11457, and by the Fund for the Advanced Study of Cancer.

REFERENCES 1. Thomas, L.,Zn “Cellular and Humoral Aspects of the Hypersensitive state” (H. S. Lawrence, Ed.), pp. 529-530, Harper & Row (Hoeber), New York, 1959. 2. Bumet, F. M., Prog. Exp. Tumor Res. 13, 1, 1970. 3. Allison, A. C., Cancer Zmmunol. Zmmunother. 2, 151, 1977. 4. Pross, H. F., and Baines, M. G., Cancer Zmmunol. Zmmunother. 3, 75, 1977. 5. Kiessling, R., Petranyi, G., Klein, G., and Wigzell, H., Znt. J. Cancer 15, 933, 1975. 6. Sendo, F., Akoi, T., Boyse, E. A., and Buofo, C. K., J. Nat. Cancer Inst. 55, 603, 1975. 7. Haller, O., Hansen, M., Kiessling, R., and Wigzell, H., Nature (London) 270, 609, 1977. 8. Pross, H. F., and Baines, M. G., Znt. J. Cancer 18, 593, 1976. 9. Takasugi, M., Ramseyer, A., and Takasugi, J., Cancer Res. 37, 413, 1977. 10. Jondal, M., and Pross, H. F., Inst. J. Cancer 15, 596, 1975. 11. McCoy, J. L., Herberman, R. B., Rosenberg, E. B., Donnelly, F. C., Levine, P. H., and Alford, C., Nat. Cancer Inst. Monogr. 37, 59, 1973. 12. Takasugi, M., Mickey, M. R., and Terasaki, P. I., Cancer Res. 33, 2898, 1973. 13. Ortaldo, J. R., Oldham, R. K., Cannon, G. C., and Herberman, R. B., J. Nat. Cancer Inst. 59,77, 1977. 14. Pross, H. F., Luk, S., and Baines, M. G., Znt. J. Cancer 21, 291, 1978. 15. Gupta, S., and Good, R. A., Clin. Zmmunol. Zmmunopath. 8, 520, 1977. 16. Gupta, S., Good, R. A., and Siegal, F. P., Clin. Exp. Zmmunol. 26, 204, 1976. 17. Schwartz, S. A., Choi, Y. S., Shou, L., and Good, R. A., J. Clin. Invest. 59, 1176, 1977. 18. Coombs, R. R. A., Gumer, B. W., Wilson, A. B.. Holm, G., and Lindgren, B.,Znt. Arch. Allergy 39, 658, 1970. 19. Dickler, H. B., and Kunkel, H. G., J. Exp. Med. 138, 191, 1972. 20. Gupta, S., and Grieco, M. H., Znt. Arch. Allergy Appl. Zmmunol. 49, 734, 1975. 21. Lozzio, C. B., and Lozzio, B. B., J. Nat. Cancer Inst. 50, 535, 1973.

SLMC IN IMMUNODEFICIENCY 22. 23. 24. 25.

DISEASE

175

Dunn, T. B., and Potter, M., .I. Nat. Cancer Inst. 18, 587, 1957. Pross, H. F., and Jondal, M., Clin. Exp. Immunol. 21, 226, 1975. Pross, H. F., Baines, M. G., and Jondal, M., Inr. J. Cancer 20, 353, 1977. West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., and Herberman, R. B.,J. Immunol. 118, 355, 1977. 26. Kay, H. D., Bonnard, G. D., West, W. H., and Herberman, R. B.,J. Immunol. 118, 2058, 1977. 27. Bakacs, T., Gergely, P., Comain, S., and Klein, E., Int. J. Cancer 19, 441, 1977. 28. Pape, G. R., Troye, M., and Perlmann, P., J. Immunol. 118, 1925, 1977. 29. Vessela, R. L., Gormus, B. J., Lange, P. H., and Kaplan, M. E., In?. J. Cunrrr 21, 594, 1978. 30. Akira, D., and Takasugi, M., Znt. J. Cancer 19, 747, 1977. 31. Koide, Y., Takasugi, M., and Billing, R., Eur. J. Zmmunol. 7, 732, 1977. 32. Eremin, O., Coombs, R. R. A., Plumb, D., and Ashby, J., Int. J. Cancer 21, 42, 1978. 33. Troye, M., Perlmann, P., Pape, G. R., Spiegelberg, H. L., Naslund, I., and Gidlof, A., J. Immunol. 119, 1061, 1977. 34. Hersey, P., Edwards, A., Edwards, J., Adams, A. E., Milton, G. W., and Nelson, D. S., Int. .I. Cancer 16, 173, 1975. 35. Kiuchi, M., and Takasugi, M., J. Nat. Cancer Inst. 56, 575, 1976. 36. Peter, H. H., Pavie-Fischer, J., Fridman, W. H., Aubert, C., Cesarini, J., Roubin, R., and Kourilsky, F. M., J. Immunol. 115, 539, 1975. 37. Koren, H. S., Amos, D. B., and Buckley, R. H., J. Immunol. 120, 796, 1978. 38. Sanal, S. O., and Buckley, R. H., J. C/in. Invest. 61, 1, 1978. 39. Rachelefsky, G. S., McConnachie, P. R., Ammann, A. J., Terasaki, P. I., and Stiehm, E. R., C/in. Exp. Immunol. 19, 1, 1975. 40. Peter, H. H., Korn-Nitschmann, I., Krapf, F., Siewertsen, H., C., Schmidt, P., Leibold, W., [n “Recent Trends in Immunodiagnosis and Immunotherapy-Relevance for Surgery?‘. (H. D. Flad, and C. Herfahrt, Eds.), Springer-Verlag, New York, in press. 41. Rosenberg, E. B., McCoy, J. L., Green, S. S., Donnelly, F. C., Siwarski, D. F., Levine, P. H., and Herberman, R. B., J. Nat. Cancer Inst. 52, 345, 1974. 42. Behelak, Y., Banejee, D., and Richter, M,, Cancer 38, 2274, 1976. 43. Ting, A., and Terasaki, P. I., Cancer Res. 34, 2694, 1974. 44. Peter, H. H., Kalden, J. R., Seeland, P., Diehl, V., and Eckert, G., C/in. ~xp. Immunol, 20, 193, 1975. 45. Hersey, P., Edwards, A., Milton, G. W., and McCarthy, W. H., Br. J. Cancer 37, 505, 1978. 46. Good, R. A., Advan. Biosc. 12, 123, 1974. 47. Rygaard, J., and Povlsen, C. O., Transplant. Rev. 28, 43, 1976. 48. Kiessling, R., Klein, E., Pross, H., and Wigzell, H., Eur. J. Immune/. 5, 117, 1975. 49. Kiessling, R., Petranyi, G., Klein, G., and Wigzell, H., hr. J. Cancer 17, 275, 1976. 50. Haher, O., Hansson, M., Kiessling, R., and Wigzell, H., Nature (London) 270, 609, 1977. 51. Herberman, R. B., and Holden, H. T., In “Advances in Cancer Research” (G. Klein and S, Weinhouse, Eds.) Vol. 27, p. 305, Academic Press, New York, 1978. 52. DeVries, J. E., Cornain, S., and Rumke, P., Znr. J. Cuncer 14, 427, 1974. 53. Tursz, T., Fridman, W. H., Senik, A., Tsapis, A., and Fellous, M., Nature (London) 269,806,1977.