Autologous mechanisms generating natural killer activity within human peripheral lymphocytes depleted of NK target-binding cells

CELLULAR

IMMUNOLOGY

66, 342-351 (1982)

Autologous Mechanisms Generating Natural Killer Activity within Human Peripheral Lymphocytes Depleted of NK Target-Binding Cells’ RONALD

L. WHISLER, KATHY BAKENHASTER,

AND

E. MITCHEL

OPREMCAK

Division of Immunology, Department of Medicine, The Ohio State University, Columbus, Ohio 43210 Received September 17, 1981: accepted November 11, 1981 Human peripheral blood lymphocytes (PBL) were depleted and enriched for natural killer target-binding cells (NK-TBC) by sedimentation of MOLT 4 tumor conjugate suspensions over discontinuous gradients. NK-TBC-depleted PBL consistently demonstrated diminished NK cytolytic levels whereas the NK levels of PBL enriched for NK-TBC were at least sixto eight-fold greater. An equal ratio of NK-TBC-enriched and depleted PBL combined at the time of cytotoxicity assay demonstrated NK levels intermediate between those of TBC-enriched and depleted PBL. However, coculturing NK-TBC-enriched ahd depleted PBL for 18 hr resulted in levels equivalent to those of NK-TBC-enriched cells and greater than those predicted from either population cultured alone. The increased NK activity in 1%hr cocultures required protein synthesis by TBC-enriched cells but was not abrogated by anti-interferon antibodies. In other experiments both NK-TBC-depleted and -enriched populations demonstrated considerable NK activity after exposure to autologous non-T lymphocytes. Also, autologous monocytes were found to inhibit the generation of NK activity among TBC-depleted PBL exposed to autologous non-T lymphocytes. The results suggest that non-TBC PBL have the potential to develop functional NK activity and that differing autologous mechanisms might be reponsible for NK generation.

INTRODUCTION Peripheral blood lymphocytes (PBL)’ from normal humans demonstrate spontaneous cytotoxicity against virally infected cell lines, immature lymphoid cells, and in vitro tumor cell targets. The augmentation of natural killer (NK) activity after exposure to viruses, poly(I:C), BCG, and certain NK-sensitive targets is well documented to represent the stimulation of interferon (IF) production (l-3). Also, the boosting of NK activity by IF results from improved lytic efficiency of preexisting target-binding cells (TBC) and not the generation of NK activity by PBL residing within the non-TBC compartment (4). ’ Supported by Leukemia-Lymphoma Research Grant CA 15147. * Abbreviations used: PBL, peripheral blood lymphocytes; NK, natural killer; IF, interferon; TBC, target-binding cells; PBMC, peripheral blood mononuclear cells; SBSS, Seligmann’s balanced salt solution; HS, human serum; SRBC, sheep red blood cell; slg, surface immunoglobulin; MLR, mixed lymphocyte reaction; FBS, fetal bovine serum; ADCC, antibody-dependent cellular cytotoxicity; CPM, counts per minute; SR, spontaneous release; MR, maximal release. 342 0008-8749/82/020342-10$02.00/O Copyright Q 1982 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

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The current conceptual model of NK developmental pathways proposed by Herberman and co-workers indicates that PBL within the non-TBC compartment termed “pre-NK cells” should have the potential to express NK-functional activity (5). However, the cellular interactions or characteristics of mechanisms responsible for the autologous generation of NK activity within human non-TBC PBL have yet to be critically investigated. Our laboratories previously demonstrated that human PBL subpopulations enriched and depleted for NK-TBC could be purified by density gradient sedimentation of PBL suspensionscontaining tumor cell conjugates (6). Functionally, TBCdepleted cells displayed diminished NK levels while augmented NK activity was observed among TBC-enriched populations. Utilizing this approach, we have attempted to characterize the autologous mechanisms by which non-TBC populations might acquire functional NK activity. The results presented in this report indicate that two types of autologous cellular interactions can result in the generation of functional NK levels within non-TBC populations. The possible pertinence of these observations to the maintenance of basal NK levels is discussed. MATERIALS Isolation

AND METHODS

and Culture of Human Peripheral

Blood Lymphocytes

Peripheral blood mononuclear cells (PBMC) were obtained from healthy adult volunteers by centrifugation over Ficoll-Hypaque gradients (7). After rinsing twice in Seligmann’s balanced salt solution (SBSS), the cells were suspended in RPM1 1640 supplemented with 20% human serum from normal males (RPMI-20% HS). PBL were isolated by the passage of PBMC over nylon wool columns. Eighty percent of the cells were recovered in the effluent which consisted of less than 1% contaminating monocytes as assessedby nonspecific esterase staining (8). Non-T-cell-enriched subpopulations were obtained by centrifugation of sheep red blood cell (SRBC) rosetting suspensions over Ficoll-Hypaque gradients (9) or by selective elution from nylon wool columns. The nonrosetting cells consisted of 6070% surface immunoglobulin (sIg)-bearing cells, less than 2% monocytes, and no more than 3% SRBC rosetting cells. Approximately 30% of the cells lacked detectable surface markers. Monocyte-enriched populations consisting of 94% or greater esterase-positive cells were harvested with a rubber policeman after surface adherence to plastic petri dishes (9). The viabilities of the adherent cells were more than 97% as assessedby trypan blue exclusion. Cultures were performed in RPMI-20% HS supplemented with 200 nM L-glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin. In the recruitment and autologous mixed lymphocyte reaction (MLR) experiments, the final cell density of all cultures was 1.2 X 106/ml. The cultures were incubated at 37°C in 95% air and 5% CO? in No. 3033 culture tubes (Falcon Plastics, Oxnard, Calif). The viabilities of the cell populations recovered for 18-hr and 6-day cultures were assessedby trypan blue exclusion. The viabilities were routinely -90% or greater with no consistent differences among the various subpopulations. Isolation

of PBL Subpopulations

Enriched

and Depleted of TBC

PBL were fractionated into MOLT 4 TBC-enriched and -depleted populations by sedimentation over fetal bovine serum (FBS) gradients exactly as previously

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described in detail (6). The TBC-depleted populations localizing to the upper third of the gradients had less than 1% contaminating cells within tumor cell conjugates. More than 40% of the TBC-enriched population sedimenting to the bottom of the gradients were conjugate bound. After dissociation of the conjugates followed by density gradient sedimentation, only 2% residual MOLT 4 cells were present in the TBC-enriched and -depleted PBL suspensions. Control PBL were similarly velocity sedimented over the FBS gradients in the absence of MOLT 4 cells. Under these conditions, greater than 80% of the cells were localized to the upper third of the gradients and the NK activity of the PBL recovered from the various gradient fractions did not differ from unfractionated PBL. NK Cytotoxicity

Assay

The tumor cell lines MOLT 4 and Raji were maintained in RPMI-10% FBS supplemented with glutamine, penicillin, and streptomycin. The lines were passed twice weekly. For “Cr labeling, 10-l 5 X lo6 target cells were suspended in 200 ~1 SBSS and incubated at 37°C for 1 hr with 150 &i 51Cr (No. NEZ 030-S New England Nuclear, Boston, Mass.). The suspension was then diluted with SBSS containing 5% human albumin (ALB) and the nonviable tumor cells removed by Ficoll-Hypaque gradient centrifugation. The tumor cells recovered from the interface were >97% viable. Unbound Wr was removed by rinsing the cells four times with SBSS-5% ALB. “Cr-labeled antibody-coated Raji targets for the antibody-dependent cellular cytotoxicity (ADCC) assay were prepared as previously detailed (6). Cytotoxicity assayswere performed in round-bottom microtiter plates (ISMRC96TC, Linbro, New Haven, Conn.). Each well received 50 ~1 of the target cell suspension containing 0.5 X lo4 cells. Different dilutions of effector cells corresponding to effector-target cell ratios of 5: I-50: 1 were suspended in 200 ~1 so as to yield 2.5 X lo4 to 2.5 X lo5 effecters/well. The microtiter plates were centrifuged at 200 g for 1 min and then incubated at 37°C. After 4 hr, the plates were centrifuged 5 min at 200g. The counts per minute (cpm) “Cr present in 125 ~1 of supernatant were determined in a gamma counter. The percentage specific lysis was calculated from the standard formula cpm experimental - cpm SR x 100, % specific lysis = cpm MR - cpm SR where spontaneous release (SR) was defined as the cpm present in the supernatant of the target cells incubated alone and maximal release (MR) representing the cpm released into the supernatant after detergent lysis of targets ( 1% Triton X-100). In all experiments, SR was less than 10%. The data were expressed as percentage specific lysis per triplicate sample at the various log effector cell concentrations per milliliter. The variation among individual samples did not exceed 15%. Cycloheximide

Treatment and Anti-IF

Serum

For inhibition of protein synthesis, TBC-enriched PBL were incubated with 50 pg/ml cycloheximide (Sigma, St. Louis, MO.) for 45 min at 37’C and rinsed three times in SBSS. This concentration of cycloheximide has previously been shown to

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inhibit protein synthesis >80% (5). The cells were then resuspended in RPMI-20% HS. As assessedby trypan blue exclusion, this treatment did not adversely affect cell viability. The rabbit anti-human leukocyte IF antibody (B-24, No. DAB 0006) was obtained from the Resources Branch, NIAID, NIH. A 1:lOO final dilution of this antibody neutralized 1000 U/ml of human leukocyte IF as determined by inhibition assay. RESULTS Characteristics

of NK and ADCC Acquisition

during Coculture

To determine if PBL enriched for TBC might influence the development of functional NK activity by non-TBC populations, human PBL were enriched and depleted for MOLT 4 TBC. The following populations or coculture combinations were incubated at 37°C for 18 hr and tested for NK activity: (a) TBC depleted, (b) TBC enriched, or (c) a 1:1 ratio of TBC depleted plus TBC enriched. As a control, TBC-enriched and -depleted PBL previously incubated alone for 18 hr were combined in a I:1 ratio at the time of cytotoxicity assay. The data presented in Table 1 demonstrate that the NK levels of the TBC-enriched:depleted PBL combined just prior to assay were intermediate between those observed for TBCenriched and -depleted PBL. In contrast, the PBL from the 1%hr cocultures which had contained a 1:1 ratio of TBC-enriched:depleted cells demonstrated NK levels TABLE NK Levels of 18-hr Cocultures Containing

1

TBC-Depleted

PBL and TBC-Enriched

PBL”

Log effector cell concentration Effector

population

5.0

5.4

5.1

6.0

Percentage specific lysis Expt 1 Unseparated PBL TBC enriched TBC depleted TBC enriched:depleted TBC enriched:depleted Expt 2 Unseparated PBL TBC enriched TBC depleted TBC enriched:depleted TBC enriched:depleted

( 18 hr) (0 hr)

10 18 2 16 8

15 29 3 27 13

19 41 5 37 18

36 83 12 74 41

(18 hr) (0 hr)

12 21 5 23 10

14 38 6 34 16

21 54 9 49 22

31 68 16 59 31

“PBL subpopulations enriched or depleted for MOLT 4 TBC were prepared as described under Materials and Methods. The indicated subpopulations were either cultured 18 hr alone and a 1:l ratio combined immediately prior to NK assay (0 hr) or a 1: 1 ratio of TBC enriched:depIeted was cocultured together for 18 hr. The NK activity was assayed with MOLT 4 target cells at the specified effector cell concentrations. Results are for two representative experiments of six.

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identical to the TBC-enriched PBL. Similar coculture experiments performed with undepleted PBL and varying ratios of TBC-depleted populations are shown in Fig. 1. Note that at a 1:10 and 1:5 ratio of undepleted:depleted PBL, the NK levels of the 18-hr cocultured cells did not differ from identical ratios combined at the time of assay. These levels were equivalent to the depleted PBL. However, the cells from cocultures containing a 1:2 or 1:1 ratio of undepleted:TBC-depleted PBL displayed NK levels significantly greater than identical ratios combined just prior to cytotoxicity assay (P < 0.001 and 0.01, respectively). To ascertain whether the acquisition of cytotoxic potential during the 18-hr cocultures was restricted to NK or might also include K cells mediating ADCC of tumor targets, the following experiments were performed. PBL were depleted of MOLT 4-binding cells. Coculture experiments identical to those described above were performed with nondepleted and TBC-depleted PBL. PBL depleted of MOLT 4 TBC displayed diminished cytotoxicity against both MOLT 4 and antibodysensitized Raji targets (Fig. 2). In addition, 18-hr cocultures of a 1:1 ratio of nondepleted:TBC-depleted cells demonstrated greater cytotoxicity levels against both targets compared to an identical ratio combined at the time of cytotoxicity assay. Similar results kere obtained in reciprocal experiments in which PBL depleted of TBC for Raji AB targets were assayed against either MOLT 4 or Raji Ab targets (data not shown).

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CONCENTRATION

FIG. 1. Effects of coculturing differing ratios of PBL with TBC-depleted PBL. Nondepleted PBL populations (A) were incubated 18 hr at 37°C and the NK levels were assayed with MOLT 4 target cells (see Materials and Methods). The levels were compared to TBC-depleted cells (a), 18-hr cocultures of TBC enrichedzdepleted (o), and nondepleted:depleted cells combined immediately prior to assay (0). The ratios of nondepleteddepleted cells cocultured or immediately combined were: (a) 1: 10, (b) 15, (c) 1:2, and (d) 1:l.

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8 65

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EFFECTOR

FIG. 2. NK and ADCC activity of cells from cocultures containing NK TBC-depleted and non-depleted PBL. PBL were depleted of NK-TBC and the ability of nondepleted PBL to recruit either NK (A) or ADCC (B) activity was determined as described in Table 1 and under Materials and Methods. PBL in 1:l ratio of nondepleted:depleted were either cocultured for 18 hr (0) or immediately combined at the time of cytotoxicity assay (0). The cytotoxic levels were compared to those of nondepleted PBL (A) or depleted PBL (0).

Dependency of Coculture

NK Augmentation

on TBC Protein Synthesis

Basal NK effector levels are not reduced by cycloheximide inhibition of protein synthesis (5). To analyze the effects of cycloheximide on the enhancement of NK activity in 1%hr cocultures of TBC-enriched:depleted PBL, the following experiments were performed. An aliquot of PBL enriched for TBC was pretreated with cycloheximide as described under Materials and Methods. The following PBL populations were then cultured for 18 hr and NK levels determined: (a) TBC-enriched, (b) cycloheximide-treated TBC-enriched, (c) TBC-depleted, (d) cycloheximidetreated TBC-enriched cocultured with TBC-depleted, and (e) TBC-enriched cocultured with TBC-depleted. Note (Table 2) that the NK levels of untreated and TABLE Effects of Treating

2

NK-Enriched PBL with Cycloheximide with TBC-Depleted PBL”

Prior to Coculturing

Log effector cell concentration PBL effector population

5.0

5.4

5.1

6.0

Percentage specific lysis Unseparated PBL TBC enriched TBCcy enriched TBC depleted TBC enriched:depleted TBCcy enriched:depleted

9 17 15 2 16 4

14 29 2-l 3 23 6

21 41 36 5 31 15

21 52 49 I 49 21

n TBC-enriched PBL were treated with cycloheximide (Cy) as described under Materials and Methods. The specified populations or 1:l cocultures were incubated 18 hr and the NK activity was assayed with MOLT 4 targets. Results are for one of three experiments.

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OPREMCAK

cycloheximide-treated TBC-enriched PBL were identical at all effector cell concentrations, being sevenfold greater than the TBC-depleted PBL. The NK levels of cells from cocultures containing TBC-enriched and TBC-depleted PBL were equivalent to those of TBC-enriched PBL. In contrast, the PBL from cocultures containing cycloheximide-pretreated TBC-enriched and TBC-depleted PBL demonstrated a two- to fourfold reduction of NK levels compared to cycloheximidetreated TBC-enriched PBL. Failure of Human Leukocyte IF Antibodies to Abrogate Coculture Augmentation Since the coculture augmentation could represent the effects of endogenous IF, we next examined whether the presence of anti-IF antibodies might block the enhancement. A concentration of anti-human leukocyte IF antibodies capable of neutralizing 1000 U/ml IF was added to cocultures of depleted and enriched PBL. The cytotoxicity percentages of 18-hr control cocultures at the four effector cell concentrations were 12, 30, 50, and 5 1, compared to 12,27,42, and 55, respectively, for the anti-IF cocultures. These values were not significantly different (P > 0.5). Acquisition of NK Activity after Exposure to Autologous Non-T Cells The next series of experiments investigated whether PBL depleted of TBC might be capable of developing functional NK activity after exposure to autologous nonT cells. The following lymphocyte subpopulations or cell combinations were incubated for 6 days in RPMI-20% HS and assayed for NK activity against MOLT 4 cells: (a) nondepleted PBL, (b) TBC-depleted PBL, (c) TBC-depleted non-T cells, (d) nondepleted PBL plus TBC-depleted non-T cells, and (e) TBC-depleted PBL plus TBC-depleted non-T cells. As shown in Fig. 3, the TBC-depleted PBL and non-T cells incubated separately for 6 days failed to develop NK activity, being

60

4.5

5.0 Log Effector

5.5

6.0

Concentration

FIG. 3. Generation of NK activity within TBC-depleted populations after coculturing with autologous non-T cells. The following PBL cell populations were incubated at a final density of 1.2 X lo6 ml in RPM1 1640 supplemented with 20% HS: nondepleted PBL (A), TBC-depleted PBL (m), or TBC-depleted non-T cells (0). &cultures were performed consisting of a 1: 1 ratio of TBC-depleted non-T cells with nondepleted PBL (0) or with TBC-depleted PBL (0). After 6 days, the cells were removed and tested for NK activity. Results are representative of three separate experiments.

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6- to 1%fold less than nondepleted PBL. In contrast, the cells from cocultures of the TBC-depleted PBL and non-T cells demonstrated 4.7- to 7.0-fold greater NK levels than either subpopulation cultured alone. The NK levels of nondepleted PBL cocultured with a 1:1 ratio of TBC-depleted non-T cells were twofold greater than expected, being slightly more than the nondepleted PBL cultured alone. Less than 10% specific lysis of autologous lymphocytes was observed for all cells recovered from the various cultures (data not shown). Monocyte Inhibition

of NK Activity

Stimulated

by Non-T Lymphocytes

To examine if the development of NK activity in the cocultures of non-TBC PBL and autologous non-T lymphocytes might be modulated by monocytes, cocultures were prepared containing 25, 35, or 50% autologous monocytes. After 6 days incubation, the cells were removed and depleted to less than 5% residual monocytes. The NK levels of these PBL were compared to cells from cocultures of non-TBC PBL plus non-T lymphocytes not supplemented with additional monocytes. As shown in Fig. 4, maximal NK activity was observed for the PBL recovered from the cocultures without additional autologous monocytes. In comparison, the NK activity of PBL from cocultures supplemented with 25-50s autologous monocytes was reduced 1.5- to 5.0-fold. DISCUSSION These data demonstrate that human PBL which reside within the non-TBC compartment and manifest diminished NK levels can develop functional NK activity by autologous cellular interactions. Two types of mechanisms were found to result in the acquisition of NK function. 60-roY!?

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60-

; 50E ; 40:: 30: oz zoif IO-

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;o LOG EFFECTOR

6.0 CELL CONCENTRATION

FIG. 4. Effects of monocytes on the autoactivation of NK. Autologous PBL and non-T cells were depleted of NK TBC and cocultured in RPM1 1640 with 20% HS at 1.2 X 106/ml. After 6 days the NK levels of these cocultures (0) were compared to identical cocultures containing 25 (0) 35 (A), or 50% (A) autologous monocytes. Prior to cytotoxicity assay, monocytes were removed to less than 5% by multiple surface adherence. The cytotoxicity levels of the depleted cells or the monocytes cultured alone did not exceed 8% at any of the effector cell concentrations.

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One mechanism which resembled a recruitment process was apparent from the studies examining the effects of coculturing non-TBC and TBC-enriched cells for 18 hr. In these experiments, the NK activity of the cocultured cells was greater than that predicted from either subpopulation alone, being equivalent to the TBCenriched PBL. The expected intermediate levels of NK were observed for the TBCenriched and non-TBC populations combined just prior to assay. Besides time dependency, the acquisition of NK activity was also determined by the cell ratios cocultured. Thus, a 1:1 or 1:2 ratio of TBC nondepleted:depleted populations displayed greater NK levels than identical ratios combined immediately prior to assay. In contrast, no coculture augmentation was observed at a 15 or 1:lO ratio of nondepleted:depleted cells. Similar results were obtained using TBC-enriched PBL (R. L. Whisler, unpublished observations). Another characteristic of the augmented NK levels in cocultures was the requirement for protein synthesis by the TBC-enriched PBL. Similar to previous studies (5), cycloheximide treatment did not diminish existing NK levels. However, the inhibition of protein synthesis among TBC-enriched PBL substantially abrogated coculture development of augmented NK. Experiments in progress are attempting to ascertain if coculture enhancement is similarly inhibited by cycloheximide pretreatment of TBC-depleted PBL. Several lines of evidence argued against the NK elevation in 18-hr cocultures being secondary to endogenous IF synthesis. First, tumor cell contact with TBC was not an absolute requirement for the coculture enhancement of either NK or ADCC. Thus, the stimulation of endogenous IF production by tumor cells did not explain the coculture augmentation. Second, concentrations of anti-interferon antibodies capable of neutralizing endogenous IF produced by cultured human PBL (2, lo), failed to block the enhanced NK levels in cocultures. Third, the augmentative effects of IF on NK activity are documented to be secondary to greater lytic efficiency of TBC and not the development of non-TBC into NK effecters (4). Although admittedly indirect, the latter suggeststhat the augmentation of coculture NK activity equivalent to TBC-enriched cells does not represent the action of IF. Indeed, others have suggested that basal NK levels may be relatively independent of endogenous IF synthesis (10). It should be emphasized that these experiments do not absolutely exclude the possibility that at least some of the enhanced NK activity in the 18 hr cocultures might represent the further activation of the enriched populations through interactions with TBC-depleted PBL. Human PBL depleted of NK effecters have been reported to acquire NK-like activity following in vitro incubation with FBS or after allosensitization in the MLR ( 1l- 14). Tomonari ( 15) showed that coculturing human PBL with autologous non-T cells generated NK-like activity, but the PBL populations were not depleted of NK TBC. Therefore it was impossible from these experiments to assesswhether the autologous enhancement represented improved lytic efficiency of preexisting NK effecters (e.g., TBC populations) and/or the de nova generation of NK-like activity by non-TBC. In the present report, there was substantial autologous enhancement of NK among TBC-depleted PBL after exposure to non-T lymphocytes. However, considerable augmentation of NK activity also occurred among PBL not depleted of TBC, since one would expect an intermediate value upon coculture at a one-to-one ratio. It should be mentioned that 6-day incubation was performed since in our laboratories this incubation interval closely corresponds to brisk au-

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tologous MLR proliferative responses.Further studies are needed to determine the kinetics of NK generation after autoactivation. The autologous generation of NK activity within non-TBC PBL was impaired by monocytes. The mechanism(s) of monocyte inhibition has not been defined but soluble mediators such as prostaglandins or hydrogen peroxide have been described to impair lymphocyte function (16, 17). Alternatively, the presence of monocytes could be competitively inhibiting cellular interactions between pre-NK cells and non-T lymphocytes. Although many questions remain unanswered, these studies demonstrate that two types of interactions among autologous cells are capable of generating de nova NK activity. Hopefully, future studies of these autologous interactions might provide further information of the mechanisms responsible for maintaining basal NK levels. REFERENCES 1. 2. 3. 4. 5. 6.

Djeu, J. Y., Heinbaugh, J. A., Holden, H. T., and Herberman, R. B., Z. Zmmurwl. 122, 175, 1979. Trinchieri, G., and Santoli, D., J. Exp. Med. 147, 1314, 1978. Santoli, D., and Koprowski, H., Zrnmunol. Rev. 44, 125, 1979. Silva, A., Bonavida, B., and Targan, S., J. Zmmunol. 125, 479, 1980. Ortaldo, J. R., Phillips, W., Wasserman, K., and Herberman, R. B., .Z.Zmmurwl. 125, 1839, 1980. Opremcak, E. M., Bakenhaster, K., and Whisler, R. L., Cell. Zmmunol. 58, 415, 1981. 7. Boyum, A., &and. J. Clin. Lab. Invest. 21, 1, 1968. 8. Li, C. Y., Lam, K. W., and Yam, L. T., J. Histochem. Cytochem. 21, 1, 1973. 9. Bobak, D., and Whisler, R., J. Zmmunol. 125, 2764, 1980. 10. Copeland, C. S., Koren, H. S., and Jensen, P. J., Cell. Zmmunol. 62, 220, 1981. 11. Golub, S. H., Golightly, M. G., and Zielske, J. V., Cancer 24, 273, 1979. 12. Ortlado, J. R., Bonnard, G. D., Kind, P. D., and Herberman, R. B., J. Zmmunol. 122, 1489, 1979. 13. Seeley, J. K., and Golub, S. H., J. Zmmunol. 120, 1415, 1978. 14. Seeley, J. K., Masucci, G., Poros, A., Klein, E., and Golub, S. H., J. Zmmunol. 123, 1303, 1979. 15. Tomonari, K., J. Zmmunol. 124, 1111, 1980. 16. Metzger, A., Hoffeld, J. T., Oppenheim, J. J., J. Zmmunol. 124, 983, 1980. 17. Goodwin, J. S., and Webb, D. R., Clin. Zmmun. Zmmunopathol. 15, 106, 1980. 18. Goodwin, J. S., Messner, R. P., Bankhurst, A. D., Peake, G. T., Saiki, J., and Williams, R. C., N. Engl. J. Med. 297, 963, 1977.

19. Schecter, G. P., and Soehnlen, F., Blood 52, 261, 1978.