Cytotoxic T-lymphocyte and spontaneous killer cell activity against human T- and B-lymphoid cell lines

CELLULAR

IMMUNOLOGY

47, 46-56 (1979)

Cytotoxic T-Lymphocyte and Spontaneous against Human T- and B-Lymphoid RICHARD

A. MILLER,~

ABRAHAM

J. TREVES,~

Killer Cell Activity Cell Lines1

AND HENRY

S. KAPLAN

Cancer Biology Research Laboratory, Department of Radiology, Stanford University School of Medicine, Stqnford, California 94305 Received December 5, 1978 We have investigated cell-mediated immune responses to cultured human T- and B-cell lines. Two effector mechanisms were explored and found to have different capabilities for mediating cytotoxic reactions. Cytotoxic T lymphocytes were generated by stimulation with irradiated B-cell lines and demonstrated cross-reactive cytotoxicity against these lines but not against T-cell lines. Unseparated mononuclear cells showed spontaneous cytotoxicity for both T- and B-cell lines; however, T-cell lines appeared more susceptible. Cell separation procedures were employed to determine functional differences in effector cells. In contrast to cytotoxic T lymphocytes induced in vitro, spontaneous killer cells (SKC) were shown to be nylon wool adherent, non-T lymphocytes with receptors for IgG-coated sheep erythrocytes.

INTRODUCTION Mixed lymphocyte cultures (MLC) and cell-mediated lympholysis (CML) assays have been widely utilized as in vitro models of allograft and tumor rejection. MLC-CML requires a recognition phase which is thought to involve activation by lymphocyte-defined (LD) antigens and a subsequent effector phase which is mediated by thymus-derived (T) cells (1,2). These effector cells are directed against cell surface antigens coded for within the major histocompatibility complex (3). Natural or spontaneous cell-mediated cytotoxicity has recently been detected in rodents and man (4- 11). Although their identity is still uncertain, spontaneous killer cells (SKC) appear to have distinctive characteristics (7, 12- 17). It is likely that antigens or targets for SKC-mediated cytotoxicity are also different from those involved in T-cell-mediated cytotoxicity (17- 19). The biological significance of SKC activity is unknown but it has been postulated that it may act in vivo as a surveillance system against tumor development (20). In the evaluation of in vitro immune responses to cultured human cell lines, it is essential to determine the relative contribution of each of the various cytotoxic effector mechanisms. In the present study the in vitro immune response of human 1 Supported by Research Contract NOI-CP-43228 and by gifts to the Joseph Edward Luetje Memorial Fund for Lymphoma Research. 2 American Cancer Society Postdoctoral Fellow. 3 Present address: Sharett Institute of Oncology, Hebrew University-Hadassah Medical Center, Jerusalem, Israel. 46 0008-8749/79/110046-l 1$02.00/O Copyright All rights

0 1979 by Academic Press. Inc. of reproduction in any form reserved.

CYTOTOXICITY

AGAINST

T- AND

B-CELL

LINES

47

peripheral blood lymphocytes (PBL) to cultured human T- and B-lymphoid cell lines was investigated. Lymphoid cell lines were shown to have varying susceptibilities to cytotoxic effector cells. Two effector mechanisms were explored and found to have different capabilities for mediating cytotoxic reactions against Tand B-cell lines. MATERIALS

AND METHODS

Preparation ofmononuclear cells. Venous blood was collected from normal male and female donors into 100 x 16-mm Vacutainer tubes (Becton-Dickinson, Rutherford, N.J.), which contained 143 USP units of sodium heparin. Blood was diluted 2:l with phosphate-buffered saline (PBS) and the mononuclear cells separated by centrifugation over a cushion of Ficoll-Hypaque (21). The interface was removed and the cells were washed three times with PBS before use in MLC or cell separation procedures. Lymphoid cell lines. Lymphoid cell lines MOLT-4 and HSB-2 express T-lymphocyte characteristics (22, 23). Lymphoblastoid cell lines LB-2 and LB-10 were established from spleen and peripheral blood, respectively, and exhibit nonneoplastic B-lymphocyte characteristics (24). These cell lines were grown in RPM1 1640 medium (Grand Island Biological Company, Grand Island, N.Y.) with 2 mM glutamine, 1% sodium pyruvate, 1% vitamin solution, 10 mM Hepes buffer, 50 units/ml penicillin, and 50 pg/ml streptomycin (Grand Island Biological Company) supplemented with 15% heat-inactivated fetal calf serum (FCS, Microbiological Associates, Bethesda, Md.). DHL-4 is a neoplastic cell line established from a patient with a histopathologic diagnosis of diffuse histiocytic lymphoma; it has been demonstrated to bear monoclonal cytoplasmic immunoglobulin, consistent with a B-cell origin (25). This line was grown in the above medium containing 20% FCS and 10% heat-inactivated pooled human serum. All cell lines were passaged twice per week and usually used in MLC or cytotoxicity assays just prior to passage. Cell separation procedures. Unseparated mononuclear cells, in medium containing human serum, were incubated on nylon wool columns by the method of Julius et al. (26). After 1 hr, nonadherent cells were eluted from the column and suspended in medium. Recovery of nonadherent cells ranged from 60 to 80% of the total unseparated cells. Mononuclear cells were separated into T- and non-T-cell populations by their capacity to form spontaneous rosettes with sheep erythrocytes (E), as previously described (27). Sheep erythrocytes (Microbiological Media, Concord, Calif.) were washed three times with Hanks’ balanced salt solution (HBSS, Grand Island Biological Company) and suspended at 0.7% in HBSS. Mononuclear cells were washed three times in HBSS and suspended at 5 x lo6 cells/ml in HBSS. One milliliter each of mononuclear cells and E were mixed in 5-ml plastic tubes (Falcon No. 2058, Oxnard, Calif.). Two-tenths milliliter of human serum, adsorbed with E, was added. The mixture was incubated at 37°C for 5 min, spun at 2008 for 5 min, and incubated at 4°C for 1.5 hr. The cells were then gently resuspended and transferred to 40-ml glass conical centrifuge tubes. E-rosette-forming cells (T cells) were separated by centrifugation over Ficoll-Hypaque at 400g for 40 min at 4°C. Pelleted and interface cells were collected. E were lysed in the pelleted fraction by treatment with Tris buffer-NH&l solution for 3 min. Both E-rosette- and non-rosette-

48

MILLER,

TREVES,

AND

KAPLAN

forming cells were washed three times with PBS before suspension in medium. Non-rosette-forming cells usually contained less than 5% contaminating T cells. Total cell recovery usually ranged from 50 to 75%. Fc receptor-bearing mononuclear cells were separated by rosetting with antibody-coated sheep E (EA). Five percent washed E in HBSS was incubated with a subagglutinating titer of rabbit IgG anti-E antiserum (Cordis Laboratories, Miami, Fla.) at a 1:l ratio at 37°C for 30 min and then washed two times in GVBZ+ buffer (Cordis Laboratories, Miami, Fla.) before suspending at 0.7% in HBSS. Mononuclear cells suspended in HBSS at 5 x lo6 cells/ml were mixed 1: 1 with EA for 30 min at 37°C. EA-rosetting cells were separated by centrifugation over Ficoll-Hypaque as above. Tris buffer-NH&l solution was used to lyse remaining E in both EA-rosetting and non-EA-rosetting fractions. Both fractions were washed three times in PBS before suspension in medium. Total cell recovery ranged from 50 to 75%. Preparation of MLC. Responder lymphocytes were suspended at 10 x lo6 cells/ml in FCS-free medium containing 10% pooled human serum. Stimulator cells, in the same medium, were irradiated with 3000 rad (J. L. Shepherd Mark I irradiator, model 25, containing 200 Ci cesium- 137 source) and 1.O ml added to 1.O ml of responder cells in 35-mm wells of Costar tissue culture plates (Costar, Cambridge, Mass.) for a responder-stimulator ratio of 20: 1. Control wells without stimulator cells had 1.O ml of medium added to responder cells. On Day 3,0.5 ml of medium was added to all cultures. Cultures were incubated at 37°C in a humidified atmosphere of 5% CO* in air for 5 days. Preparation of target cells. Target cells were washed once and labeled with [3H]leucine (New England Nuclear, Boston, Mass.; specific activity 60 Ci/mmol) as described (28). Briefly, cells were suspended in 2.0 ml of leucine-free Eagle’s minimum essential medium plus 15% dialyzed FCS with 50 @/ml [3H]leucine for 2 hr, washed, and cultured in medium with 15% FCS overnight before use in the CML assay. This has been found to be a convenient method of target cell labeling for studies involving human lymphoma cell lines (28). CML and SKC assay. Effector and target cells were collected, washed, and suspended in medium with 15% FCS. Target cells (1 X 104/0. 1 ml) and various concentrations of effector cells were added to triplicate wells of V-bottom microtiter plates (Cooke Microtitre System II, Cooke Engineering Co., Alexandria, Va.) to give a final volume of 0.2 ml. For determination of SKC activity fresh effector cells were added to wells as above. Plates were incubated at 37°C for 6 hr and then centrifuged at 200g for 5 min. One-tenth milliliter supernatant from each well was removed and counted in 2.5 ml Aquasol solution (New England Nuclear) in a Searle Isocap 300 scintillation counter. Spontaneous target cell release was determined from the supernatant of target cells cultured without effector cells and was less than 30% of maximum release. Maximum target cell release was determined by treatment with distilled water for 1 hr. Percentage cytotoxicity was calculated as described previously (29): (mean cpm in experimental) - (mean cpm of spontaneous (mean cpm of maximum target cell release) - (mean cpm of spontaneous

target cell release) x loo target cell release)

CYTOTOXICITY

AGAINST

T- AND B-CELL

Results were expressed as percentage cytotoxicity mean of triplicate samples.

49

LINES

? the standard error (SE) of the

RESULTS MLC-CML

with T- and B-Cell Lines

Nylon wool nonadherent responder lymphocytes were cultured with irradiated cell lines DHL-4, LB-2, MOLT-4, and in medium alone. After 5 days, responder lymphocytes were tested in a CML assay against each of these cell lines as targets. Nylon wool separation of lymphocytes was performed because previous experiments had revealed that this procedure reduced background cytotoxicity (see below). As shown in Table I, both DHL-4 and LB-2 were potent stimulators of cytotoxic effector cells from four normal allogeneic donors. These effector cells exhibited cross-reactivity for both of these lines but cytotoxicity was usually greatest for identical stimulator-target cell combinations. In contrast, MOLT-4 did not stimulate a CML reaction in any of the donors. In addition, it was not susceptible to killing by effector cells generated by stimulation with B-cell lines. Control lymphocyte cultures in medium alone exhibit levels of cytotoxicity comparable to those cultured with MOLT-4. In Table 2 a similar experiment was performed using different T- and B-cell lines, HSB-2 and LB-IO. Identical results were obtained. Both DHL-4 and LB-10 induced specific cytotoxic effector cells which showed partial cross-reactivity on these B-cell targets but not on the T-cell TABLE MLC-CML

1

against Lymphoid Lines Using Nylon Wool Nonadherent Responder Cells Percentage cytotoxicity against target cells GE” MLC”

DHL-4

LB-2

MOLT-4

Donor 1

DHL-4 LB-2 MOLT-4 Medium

45 k 2 38 + 1 020 Ok0

26 c 1 56 k 1 221 020

Sk1 821 6+1 051

Donor 2

DHL-4 LB-2 MOLT-4 Medium

40 f 1 43 + 1 020 -1 k-0

15 i- 2 43 + 2 2kO 020

4k2 121 -52 1 -5 ‘-c 1

Donor 3

DHL-4 LB-2 MOLT-4 Medium

60 k 2 32 k 1 Ok1 2t1

31 + 3 57 27 2 2+0 5*0

9?2 022 I*3 722

Donor 4

DHL4 LB-2 MOLT-4 Medium

43 + 2 48 + 3 121 3-r-2

22 L 1 61 f 1 O? 1 221

950 721 121 620

” Effector-target ratio of 50: 1. b Four allogeneic donors stimulated by cell lines in an MLC and tested in a CML assay against target cells.

50

MILLER,

TREVES,

AND

TABLE MLC-CML

Against

Lymphoid

Lines

Using

Percentage MLC

2 Nylon

ratio

Nonadherent against

Responder

target

cells

28 57 -2 -2

Cells

*SE”

LB-10

38 t 1 42 k 1 Ok0 Ok0

a Effector-target

Wool

cytotoxicity

DHL-4

DHL-4 LB-10 HSB-2 Medium

KAPLAN

HSB-2

2 A A k

0 3 0 0

822 5+0 4kO 0+2

of 50: 1.

line. HSB-2 did not induce cytotoxic activity against any of the cell lines, as was the case with MOLT-4. Unseparated PBL from two donors were also used as responder cells. When such effector cells were tested in a CML assay, differences in reactivity were noted (Table 3). As with the above experiments, DHL-4 and LB-2 demonstrated both the ability to generate cytotoxic effector cells and cross-reactivity when used as target cells. Again, it was found that these effector cells were not cytotoxic to MOLT-4. Unlike the previous experiments, however, responder cells cultured in FCS-free medium alone or with irradiated MOLT-4 showed substantial levels of cytotoxicity for targets DHL4, LB-2, and MOLT-4. Since this activity was found with lymphocytes cultured in medium alone it appeared that unseparated PBL contained effector cells with spontaneous cytotoxic activity against these cell lines. Spontaneous

Killer

Cell Activity

Fresh peripheral blood mononuclear cells were tested for spontaneous killer cell activity against B-cell lines DHL-4 and LB- 10, and against T-cell lines MOLT-4 and TABLE MLC-CML

Against

Lymphoid Percentage

MLC”

Lines

3 Using

cytotoxicity

Unseparated against

DHL-4

Responder target

cells

Cells &SE”

LB-2

MOLT-4

Donor

5

DHL-4 LB-2 MOLT-4 Medium

59 40 18 13

+ + ? ?

6 1 3 2

22 33 14 24

k k k +

1 4 3 2

3-co 2kO 33 + 1 58 k 1

Donor

6

DHL-4 LB-2 MOLT-4 Medium

69 60 22 35

k -c k k

1 1 2 7

20 59 30 38

r + 2 k

3 6 5 4

8+4 0+2 64 ” 13 53 k 4

a Effector-target b Two allogeneic target cells.

ratio of 50: 1. donors stimulated

by cell lines

in an MLC

and tested

in a CML

assay

against

CYTOTOXICITY

AGAINST

T- AND

B-CELL

51

LINES

HSB-2 at various effector-target cell ratios. In Fig. 1, it can be seen that all four lines are susceptible to SKC activity. In Table 4, the average SKC activity against these four cell lines by effector cells from four different donors shows a greater susceptibility of T-cell lines to spontaneous killing. It is possible that the reactivity seen with effector cells cultured with MOLT-4 or in medium alone represents spontaneous killer cell activity and that this activity is removed by nylon wool separation. In Fig. 2 SKC activity from two donors was tested on DHL-4, LB-IO, MOLT-4, and HSB-2. When effector cells were separated by passage over nylon wool columns SKC activity was abrogated or markedly decreased. This may explain why the nylon wool nonadherent cells cultured in medium alone or with MOLT-4 in Tables 1 and 2 showed no cytotoxicity whereas effector cells not separated by nylon wool (Table 3) did demonstrate cytotoxicity toward the test cell lines. The data suggest that B-cell lines DHL-4, LB-2, and LB-10 stimulate specific cytotoxic effector cells which are unaffected by nylon wool separation in contrast to cells cultured in medium alone or with MOLT-4. MLC-CML

and SKC Activity

with Lymphocyte Subpopulations

To further differentiate SKC activity from that generated in MLC, cell fractionation procedures were employed to determine functional differences in effector cells. Fresh mononuclear cells and separated T- and non-T-cell populations were tested for SKC activity against DHL-4 and MOLT-4. It can be seen in Table 5 that the major SKC activity resides in the non-T-cell population, confirming observations made by others (12, 14-17). Concurrently with the testing of SKC activity, various lymphocyte subpopulations from the same donor were cultured in an MLC for 5 days and tested in a CML assay for cytotoxicity. Table 6 shows that

25:l EFFECTOR-TARGET

FIG. I. Spontaneous mononuclear cells from

5O:l

1OO:l RATIO

killer cell activity against T- and B-lymphoid cell lines. Fresh a single donor were tested in a SKC assay at various effector-target

unseparated cell ratios.

52

MILLER,

Average

Target

SKC

TREVES,

Activity

t standard

KAPLAN

TABLE 4 against T- and B-Cell

Lines”

Percentage cytotoxicity +SD

cell

DHL-4 LB-10 MOLT-4 HSB-2 n Mean

AND

5.0 11.5 16.7 26.5 deviation

of SKC

activity

Range

-r- 3.6 k 4.6 t 14.8 + 21.6

of four

donors.

1-9 6-17 6-38 lo-58 Effector-target

ratio

of 5O:l.

both unseparated cells and T cells stimulated with DHL-4 mediate specific cytotoxic reactions against DHL-4. Minimal cytotoxicity is seen against MOLT-4. With lymphocytes cultured in medium alone, most of the cytotoxic activity is recovered in the non-T-cell fraction. These data show that specific cytotoxic cells induced by stimulation with B-cell lines are T cells. SKC activity, however, is mediated by non-T cells. Furthermore, this activity can persist in culture for up to 5 days. Several other investigators have shown that SKC have Fc receptors (7, 12- 15, 17). We tested this in our system by separating fresh mononuclear cells into Fc receptor-positive and Fc receptor-negative populations by mass EA-rosette formation. As seen in Table 7, both unseparated and EA-rosette-forming cells exhibited SKC activity against DHL4 and HSB-2. Non-EA-rosette-forming cells showed minimal or no cytotoxicity. The unseparated cells demonstrated slightly greater cytotoxicity than the Fc receptor-enriched population. This may be related to the Tris buffer-NH&l treatment of the EA-rosette-forming population, which has been shown to decrease SKC activity (30). DISCUSSION Several investigators have shown that lymphoblastoid cell lines are potent stimulators in MLC (23, 3 1, 32). Such stimulation induces the formation of T

FIG. 2. The effect of nylon wool separation on spontaneous killer cell activity. Unseparated (closed) and nylon wool nonadherent (open) mononuclear cells from two donors (circle, triangle) were tested in a SKC assay against four cell lines at an effector-target cell ratio of 1OO:l.

CYTOTOXICITY

AGAINST

T- AND

TABLE SKC

Activity

B-CELL

53

LlNES

5

of Mononuclear

Cell

Cytotoxic

Subpopulations”

activity

against

target

cells

DHL-4 Cell population Unseparated T cells Non-T cells Spontaneous target cell release Maximum target cell release

cpm + SEb

MOLT-4 Percentage

cpm

k SE0

Percentage

6,347 3,688 5,945

+ 839 it 218 + 271

19

8,945

L 424

13

-2 16

8,079 9,829

2 k

8 19

3,978

t

154

-

6,876

f

k 645

-

22,219

16,665

’ Fresh mononuclear cells separated by mass E-rosette formation and tested for cytotoxicity at effector-target ratio of 100: 1. b Counts per minute + standard error of triplicate samples.

20 9

316

-

+ 511

-

into T- and non-T-cell

populations

lymphocytes capable of mediating cytotoxic reactions against a broad range of autologous and allogeneic lymphoblastoid cell lines (33-35). Lymphoid cell lines with T-cell characteristics, however, have been reported not to stimulate in MLC or to generate cytotoxic lymphocytes (23,3 1,36). Our data are in agreement with these observations. In MLC, the B-cell lines DHL4, LB-2, and LB-10 were capable of generating cytotoxic lymphocytes. These effector cells had limited specificity, since partial cross-reactivity was noted on B-cell lines but not on the T-cell lines TABLE MLC-CML

of Mononuclear

6 Cell

Subpopulations”

Cytotoxicity

against

target

DHL-4 MLC Unseparated DHL-4 T cells DHL-4 Non-T cells DHL-4 Unseparated Medium T cells Medium Non-T cells Medium Spontaneous target cell release Maximum target cell release

cells MOLT-4

cpm

k SEb

Percentage

cpm

2 SE*

8,063

_f 234

8,142 4,238 3,539

2 212 + 135 t 81

2,996 3,441

i +

151 28

2.604

+

74

-

12,092

k

1,598

-

58 58

5,991 6,348

-t 256 +- 102

10

17 10

6,491 4,743

" 313 % 161

13 2

4 9

4,770 5,904

* 130 + 167

2 9

4,418

k 107

(1 Mononuclear cells separated by mass E-rosette formation used in MLC-CML at effector-target ratio of 50: 1. * Counts per minute -t standard error of triplicate samples.

20,423

Percentage

12

-

” 1,488

into T- and non-T-cell

populations

and

54

MILLER,

TREVES,

AND

TABLE SKC

Activity

KAPLAN

7

of EA and Non-EA-Rosette-Forming Cytotoxicity

against

Cells” target

cells

DHL-4 Cell population Unseparated EA Non-EA Spontaneous target cell release Maximum target cell release

cpm

-r- SE*

HSB-2 Percentage

cpm

k SE”

Percentage

5,051 4,616 3,994

? 329 +- 191 -t 264

18 12 3

3,935 3,608 2,560

” 130 k 64 k 126

14 10 -3

3,811

+

38

-

2,798

k

26

-

? 723

-

10,983

+ 513

-

10,720

” Fresh mononuclear cells separated by mass EA-rosette formation effectortarget ratio of 50: 1. * Counts per minute ? standard error of triplicate samples.

and tested

for cytotoxicity

at

MOLT-4 and HSB-2. This pattern of reactivity was found for both unseparated and nylon wool nonadherent responder cells and is mediated by T lymphocytes. T-Cell lines were incapable of inducing cytotoxic effector cells in MLC. Other workers have reported lymphoblastoid cell line-induced cytotoxicity against T-cell lines but it is possible that SKC activity was responsible since no cell separation procedures were employed (36,37). It has also recently been shown that SKC cytotoxicity can be induced in culture (38). When nylon wool nonadherent responder cell populations cultured with medium alone or with T-cell lines were used as effecters, no cytotoxicity was observed on any target cells (Table 1 and 2). Unseparated responder cells, however, were cytotoxic to both T- and B-cell targets (Table 3). Further characterization of these effector cells showed them to be distinctly different from cytotoxic T lymphocytes. They were nylon wool adherent non-T cells which possessed Fc receptors as indicated by their ability to form rosettes with IgG-coated sheep erythrocytes. These characteristics are similar to those of spontaneous or natural killer cells described by others (7, 12- 17). This effector mechanism had the ability to manifest broad-range cytotoxic reactions against both T- and B-cell lines in contrast to cytotoxic T lymphocytes induced by B-cell lines which were not cytotoxic to T-cell lines. In contrast, T-cell lines seemed more susceptible to SKC activity than B-cell lines, as has been described by others (39, 40). In Table 3, unseparated responder cells stimulated with B-cell lines demonstrated effective cytotoxicity against DHL-4 and LB-2 but none against MOLT-4. Since unstimulated responders (cultured in medium alone or with MOLT-4) were cytotoxic for MOLT-4, one might expect that background SKC activity would result in some cytotoxicity against T-cell lines in responders stimulated by B-cell lines. That this was not the case may indicate a preferential selection of cytotoxic T-lymphocyte effecters rather than SKC. It is possible that this selective process would be advantageous as cytotoxic T-lymphocyte responses against B-cell lines appear to be greater than spontaneous killer cell responses (Tables 4, 5, and 6).

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AGAINST

T- AND

B-CELL

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55

Alternatively, culture conditions which exist during a vigorous MLC may not support SKC activity. In cultures with medium alone or with T-cell lines there is little or no stimulation and SKC activity may manifest itself. It is also possible that suppressor cells are induced in B-cell-stimulated MLCs which inhibit SKCmediated cytotoxicity. The nature of the antigens involved in both stimulation and the effector phase in our system is unclear. Since B-cell lines both stimulated cytotoxic T lymphocytes and were susceptible targets it is possible that B-cell- or I-region-associated differentiation antigens are involved. It has been demonstrated that Iregion-associated antigens can be the target for cell-mediated cytotoxic reactions (41). This would explain why T-cell lines were not susceptible targets for effector cells stimulated by B-cell lines. The nature of target antigens for SKC activity is also uncertain but the data presented here suggest that they are different from those antigens involved in T-lymphocyte-mediated cytotoxicity. We have demonstrated two different effector mechanisms involved in T- and B-cell line cytotoxic reactions. In the study of in vitro responses to lymphoid cell lines it will be important to distinguish each of the effector mechanisms involved. One can postulate that SKC activity represents a first-line defense against neoplastic T- or B-cell lines. Cytotoxic T lymphocytes are induced by B cells and later manifest an enhanced specific reactivity against these cell lines. Further elucidation of cellular immune responses to T- and B-cell lines may have value in understanding the mechanisms of in viva surveillance of T- and B-cell leukemias and lymphomas. REFERENCES 1. Bach, F. H., Grillot-Courvahn, C., Kuperman, 0. J., Sallinger, H. W., Hayes, C., Sondel, P. M., Alter, B. J., and Bach, M. L., Immunol. Rev. 35, 76, 1977. 2. Bach, F. H., Segall, M., Zier, K. S., Sondel, P. M., Alter, B. J., and Bach, M. L., Science 180,403, 1973. 3. Eijsvoogel, V. P., DuBois, M. J., Meinesz, A., Bierhorst-Eijlander, A., Zeylemaker, W. P., and Schellekens, P., Transplant. Proc. 5, 1675, 1973. 4. Greenberg, A. M., and Playfair, J. H., Clin. Exp. Immunol. 16, 99, 1974. 5. Kiessling, R., Klein, E., and Wigzell, H., Eur. J. Immunol. 5, 112, 1975. 6. Herberman, R. B., Nunn, M. E., and Larvin, D. H., Int. J. Cancer 16, 216, 1975. 7. Herberman, R. B., Nunn, M. E., Holden, H. T., and Larvin, D. H., Int. 1. Cancer 16,230, 1975. 8. Zarling, J. M., Nowinski, R. C., and Bach, F. H., Proc. Nat. Acad. Sci. USA 72, 2780, 1975. 9. Oehler, J. R., Lindsay, L. R., Nunn, M., and Herberman, R. B., Int. J. Cancer 21, 204, 1978. IO. Takasugi, M., Mickey, M. R., and Terasaki, P. I., Cancer Res. 33, 2898, 1973. 11. Rosenberg, E. B., McCoy, J. L., Green, S. S., Connelly, F. C., Siwarski, D. F., Levine, P. H., and Herberman, R. B., J. Nat. Cancer Inst. 52, 345, 1974. 12. Herberman, R. B., Bartram, S., Haskil, J. S., Nunn, M., Holden, H., and West, W., J. Immunol. 119, 322, 1977. 13. West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., and Herberman, R. B.,J. Immunol. 118, 355, 1977. 14. Pross, H. F., and Jondal, M., Clin. Exp. Immunol. 21, 226, 1975. 15. Bakacs, T., Gergely, P., Cornain, S., and Klein, E., Int. J. Cancer 19, 441, 1977. 16. Haller, O., Kiessling, R. A., Om, K., Kirre, K., Nilsson, K., and Wigzell, H., Int. J. Cancer 20,93, 1977. 17. Kiesshng, R., Petranyi, G., Karre, K., Jondal, M., Tracey, D., and Wigzell, H., J. Exp. Med. 143, 772, 1976. 18. Takasugi, M., Akira, D., Takasugi, J., and Mickey, M., J. Nat. Cancer Inst. 59, 69, 1977.

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19. Ortaldo, J. R., Oldham, R. K., Cannon, G. C., and Herberman, R. B., J. Nat. Cancer

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Pross, H. F., and Baines, M. G., Cancer Immunol. Immunother. 3, Boyum, A., Clin. Lab. Invest. (Suppl.) 21, 77, 1968. Minowada, J., Ohnuma, T., and Moore, G. E., J. Nat. Cancer Inst. Royston, I., Graze, P. R., and Pitts, R. B., J. Nat. Cancer Inst. 53, Epstein, A. L., Henle, W., Henle, G., Hewetson, J. F., and Kaplan, USA

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