Cytotoxic studies in human newborns: Lessened allogeneic cell-induced (augmented) cytotoxicity but strong lymphokine-activated cytotoxicity of cord mononuclear cells

CELLULAR

IMMUNOLOGY

103,24 l-25 1 ( 1986)

Cytotoxic Studies in Human Newborns: Lessened Allogeneic CellInduced (Augmented) Cytotoxicity but Strong LymphokineActivated Cytotoxicity of Cord Mononuclear Cells’ TERRY W. CHIN, BONNIEJ. ANK, DANA MURAKAMI, MARTIN GILL, CELSA SPINA, SHEILA STROM, AND E. RICHARD

STIEHM*

Department of Pediatrics and The Centerfor Interdisciplinary Research in Immunology and Disease, University of California at Los Angeles School ofMedicine. Los Angeles, California 90024 Received December 2, 1985; acceptedJuly I, 1986 Nonspecific cytotoxic responses such as natural killer activity can be increased in vitro by incubating effector cells with soluble factors or allogeneic cells. We sought to determine if newborn cells, known to be deficient in most cytotoxic responses, including resting NK activity, could develop enhanced cytotoxic responses following incubation with allogeneic cells (augmented cytotoxicity) or with lymphokines (lymphokine-activated cytotoxicity). Cord whole mononuclear cells (WMC) incubated with irradiated Raji cells for 5 days develop lower levels of cytotoxicity toward K562 targets at both a 2O:l effector:target (ET) ratio (39 ? 2.7% vs 49 + 3.6%) and a 1O:l E:T ratio (29 + 2.6% vs 40 + 3.6%) than do adult cells. Lessenedspecific cytotoxicity of cord cells developed toward the sensitizing Raji cells was also observed at both E:T ratios. Attempts to enhance the induced cytotoxicity by incubation with interferon or isoprinosine were unsuccessful. In contrast, lymphokine (i.e., interleukin 2)-activated killer (LAK) cytotoxicity is not deficient in cord WMC. Indeed, the level of LAK cytotoxicity is equivalent to that observed with similarly treated adult cells despite a lower baseline level of cytotoxicity toward the target cells. In the presenceofpurified IL-2 for 5 days, cord WMC cytotoxicity against K562 cells increased from 12 + 2.6 to 71 + 4.2% and against Raji cells increased from 9.6 f 2.5 to 48 + 6.7%. Similarly treated adult cells increased their killing against K562 from 23 + 4.2 to 6 1 + 4.5% and against Raji from 12 + 3.0 to 36 + 5.3%. This substantial lymphokine-activated cytotoxicity of newborn cells suggeststhe possibility of therapeutic intervention with purified lymphokines in neonatal infections or neoplasms. 0 1986 Academic Press,Inc.

INTRODUCTION

The cytotoxic potential of human newborn cells is deficient when compared to adult or older infant cells. This may be a correlate of impaired resistance to certain intracellular bacteria, fungi, and viruses, diminished limitation of viral replication, and the occasional occurrence of maternal T-cell engraftment or malignancy in newborns. Diminished cytotoxic potential (when compared to adults) in newborns has been documented for antibody-dependent (ADCC) ( l-4), natural killer (NK) (4- 14), and ’ Supported by NIH Grants HD-09800, AI-07008, and AI-1 5332. 2 To whom reprint requests should be addressedat ERS, UCLA Department of Pediatrics, Los Angeles, Calif. 90024. 241 OOOS-8749186 $3.00 Copyright 0 1986 by Academic Press,Inc. All rights of reproduction in any form reserved.

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several forms of T-cell cytotoxicity including lectin-dependent, cell-mediated lympholysis and viral-induced T-cell cytotoxicity (CTL) ( 15- 18). The most consistently observed neonatal defect has been in natural killer cytotoxicity, usually against the K562 target. In one study cell-mediated lympholysis in the newborn has been reported to be normal (19). In this report we extend our observations on neonatal cytotoxicity by examining nonspecific allogeneic cell-induced (i.e., augmented) cytotoxicity and lymphokineactivated killer (LAK) cytotoxicity. Augmented cytotoxicity is the nonspecific cytotoxicity of mononuclear cells that develops in a mixed-leukocyte culture (MLC) in parallel with specific cytotoxicity toward the sensitizing cocultured cells (20). Lymphokine activated killer cytotoxicity is a newly recognized form of nonspecific cytotoxicity induced by incubation of mononuclear cells with interleukin 2 (IL-2) (2 1). Our studies demonstrate a lessenedcytotoxicity (compared to adult cells) induced by allogeneic cell activation of cord cells, both against the sensitizing cells (Raji) and against a nonspecific NK target cell, K562. By contrast, cord LAK cytotoxicity after long-term incubation with IL-2 is equivalent to the cytotoxicity observed by adult mononuclear cells. MATERIALS AND METHODS

Isolation of efictor cells. Cord blood samples (heparinized) from normal term newborns were collected routinely at delivery and studied within 6 hr of delivery. Peripheral blood samples were obtained from normal adult volunteers, with informed consent in accordance with the regulations of the UCLA Human Subjects’ Protection Committee. Mononuclear cells were obtained using density centrifugation over lymphocyte separation media. In some of the experiments, the monocytes were removed from the mononuclear cell preparation by treatment with carbonyl iron followed by removal with a magnet. Carbonyl iron (50 mg) was added to lo-20 X lo6 cells in a total volume of 10 ml of culture medium supplemented with 20% pooled human AB serum. The cell suspensions were incubated with continuous rotation at 37°C for 30 min. Cells ingesting the iron were removed by a magnet, and the remaining lymphocyte-enriched population was washed twice with phosphate-buffered saline. The cells were counted and the percentage of monocytes remaining was determined by Wright’s stain. After iron magnet treatment, the percentage of monocytes in the cell preparations was 7.5 + 1.5%. The cells were resuspended in RPM1 1640 supplemented with antibiotics, 10 mMHepes, 2 mML-glutamine, and 10%heat-inactivated pooled human AB serum. NK assay.The assayas described by Jondal et al. (22) employed ‘ICr-labeled K562 or Raji target cells at 25: 1 and 10:1 effector:target cell ratios. Each target (100 ~1)( lo4 cells) in RPM1 1640 with 10% inactivated AB serum and effector cells (2.5 or 1.O X 1O5cells) was mixed in triplicate wells in V-bottom-well microtiter plates, centrifuged at 200g for 5 min, and then incubated for 3 hr at 37°C in 5% CO*. After incubation, the microtiter plates were then centrifuged at 450g for 10 min and lOO~1 supernatants were removed from each well for counting. Percentage specific lysis was calculated by the following formula: Percentage specific lysis = [(CPM,,, - CpM,,)/(CPM,, - CPM,,,,)] X 100, where CPM,, was determined in medium containing 6% perchloric acid. The mean percentage specific lysis was calculated and given with standard error.

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Mixed leukocyte culture activation. Mononuclear cells (2 X 106) were incubated with or without Raji cells at a 10:1 responder:stimulator cell ratio in RPM1 1640 with 10% heat-inactivated human cord serum. The Raji cells were pretreated with 50 pg/ ml mitomycin C for 30 min at 37°C washed three times with large volumes of phosphate-buffered saline or Hanks’ buffered saline, and resuspended in culture medium. The leukocyte mixtures were incubated in a total volume of 2 ml in 16 X 125mm Corning round-bottom plastic culture tubes at 37°C with 5% CO2 for 5 days. After incubation, contents of tubes containing like cultures were pooled, counted, and used as effector cells in the NK assayas described above. In parallel with the MLC activation, aliquots of cells were also tested for proliferative activity. At the end of the 5-day incubation period, responding cells (2 X 104) were incubated in triplicate in round-bottom well microtiter plates with 2 PCi [3H]thymidine per well for 4 hr. The cultures were then harvested on a Multiple Automated Sample Harvester (M. A. Bioproducts) and the amount of radioactivity incorporated was determined by counting in a liquid scintillation counter. Lymphokine-activated killer (LAK) cytotoxicity assay.Mononuclear cells were incubated for 5-7 days at 37°C in 5% COZ in the presence of interleukin 2. The final cell concentration was 1 X 1O6cells/cc incubated in 16 X 125-mm Coming roundbottom plastic culture tubes. When supematants from MLA 144 cells were used as the source of IL-2, their final concentration was 25%. Dose-response curves established 50 U/cc to be the optimal dose for recombinant IL-2 from Amgen (Thousand Oaks, Calif.) and 100 U/cc for the preparation from Cetus Corporation (Emeryville, Calif.). At the end of the incubation period, the cells were centrifuged at 400g for 10 min and supernatants removed. The cells were resuspended in RPM1 1640 media with 10% AB serum and assayed as described above except that the effector cells added to each well were 4 X 105.The final effector:target cell (E:T) ratio was 40: 1. In these experiments, a baseline NK assayon Day 0 was also performed at an E:T ratio of 40: 1. Lymphokine and isoprinosine sources.Isoprinosine (inosine: N,N-Dimethylamino-2-propanol para-acetamidobenzoate, 1:3 molar ratio) was supplied by Newport Pharmaceuticals International, Inc. (Newport Beach, Calif.). It was diluted in RPM1 1640 medium and added in a loo-p1 volume on Day 0 for a final concentration of 250 pg/cc. Interferon-a was obtained from Hoffman-La Roche (Nutley, N.J.). It was also diluted in RPM1 1640 medium and added in loo-p1 aliquots on Day 3 of the 5day incubation. MLA 144 cells were obtained from Dr. Sidney Golub (UCLA) and grown in RPM1 1640 supplemented with 5% fetal calf serum. The conditioned media from this Gibbon leukemia cell line has been shown to contain interleukin 2 and no interferon (23). At the time of splitting, cells were centrifuged and supematants removed. They were stored at 4°C and used to activate mononuclear cells within 5 days at a final concentration of 25%. Purified, recombinant IL-2 was obtained from two commercial sources, Amgen and Cetus Corporation. They were diluted in RPM1 1640 with 10%AB serum. RESULTS

LessenedAugmented Cytotoxicityfollowing Mixed-Leukocyte Culture Cytotoxic activity of cord cells against K562 target cells following a 5&y incubation with Raji cells in a mixed-leukocyte culture was lessthan that of equivalent adult

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FIG. 1. Mixed-leukocyte culture (MLC) activation. Mononuclear cells (2 X 106)from adult (A) peripheral blood and cord (C) blood are incubated with mitomycin-treated Raji cells at a 10:1 responder:stimulator cell ratio for 5 days at 37°C. Cellular cytotoxicity was then assayedat two effector:target cell ratios as shown, 20: 1 and 10:1.Results are expressedas percentage specific lysis + standard error ofthe mean. (A) Decreased cord cytotoxicity toward KS62 cell targets representing augmented NK activity. (B) Decreased cord cellmediated lympholysis toward the sensitizing cell (Raji).

cells at two different effector:target cell ratios (Fig. IA). The mean adult cytotoxicity was 49 f 3.6% at a 2O:l E:T ratio and 40 f 3.6% at a 1O:l E:T ratio, values which were significantly greater than the comparative values of 39 f 2.7% at 20: 1 (P c 0.05) and 29 + 2.6% at 10:1 (P < 0.01) for cord cells. There was no significant correlation between the level of cytotoxicity observed against K562 on Day 0 (spontaneous NK activity) and after activation with Raji cells on Day 5 for either cord or adult cells (data not shown). Specific cytotoxicity against Raji cells is shown in Fig. 1B for adult and cord cells. There were significant differences between adult and cord cells at both 20: 1 and 10:1 E:T ratios. The levels of cytotoxicity of the adult cells were 34 f 5.0% at 20: 1 and 26 f 4.0% at 10:1 compared to lower values for cord cells, 14 f 2.5% at 20: 1 (P < 0.001) and 11 k 2.4% at 10:1 (P < 0.0 1). Therefore, both specific and nonspecific cytotoxic responsesof cord cells were lessthan adult cells. No Correction with Interferon or Isoprinosine We attempted to correct the deficient responsesof cord cells using interferon-a and isoprinosine, two agents reported to affect NK and T-cell cytotoxicity. No increase of specific cytotoxicity toward sensitizing cell (Raji) or nonspecific cytotoxicity toward K562 targets was observed (Table 1). Neither agent affected MLC-generated proliferation. The mean stimulation for cord cells (n = 6) was 11,100 f 2300 cpm with Raji cells, 13,400 f 2600 cpm with Raji cells plus interferon, and 11,400 f 3700 with Raji cells plus isoprinosine. For adult cells (n = 4) the stimulation was 7100 & 2000 cpm with Raji cells, 8300 + 2500 with Raji plus interferon, and 6400 k 2000 cpm with Raji cells plus isoprinosine. Resting or unstimulated proliferative activity was unaffected by either isoprinosine or interferon alone (2300 cpm f 1400 for cord; 2300 cpm + 400 for adult). Lymphokine-Activated (LAK) Killer Cell Activity in Cord Blood We next examined the effect of lymphokine preparations on the cytotoxic activity of cord and adult whole mononuclear cells (WMC). Initial experiments used a super-

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TABLE 1 Effect of Interferon and Isoprinosine on Adult and Cord Blood MLC-Activated Cytotoxicity against K562 and Raji Target Cells” Percentage specific lysis (mean k SE)b Activity K562 Target cells Cord Adult Raji target cells Cord Adult

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Control

Interferon

Isoprinosine

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39 + 7.3 (8) 30 zk6.6 (8) 50 -c 7.1 (6) 41 + 6.6 (6)

22 + 7.4 (5) 15 + 5.8 (5) 35 + 9.8 (4) 27 + 8.6 (4)

2o:l lo:1 20: 1 IO:1

14 + 2.5 ( 19)d 11 ?2.4(15)’ 34&5.0(12) 26+4.0(11)

14 f 4.5 (8) 9k3.1 (8)’ 27 + 3.9 (6) 19 f 3.4 (6)

9 f 5.3 (5) 6 AI4.0 (5) 12 f 2.8 (4) 10 f 2.3 (4)

a The activated cytotoxicity was generated after a 5-day incubation with Raji cells at an effector:stimulator ratio of 10:1.Values represent the mean results of percentagesof specific lysis asdefined under Materials and Methods after a 3-hr incubation using K562 or Raji cell targets. bThe number in parenthesesrepresents the number of individuals studied. ’ Compared to adult controls, P < 0.0 1. d Compared to adult controls, P < 0.00 1. ‘Compared to adult controls, P < 0.05.

natant from the MLA 144 cell line, which contains interleukin 2 (and probably other factors) (23). We noted markedly enhanced cord cell cytotoxicity against both K562 targets as well as the NK-resistant Raji cell targets when MLA supematants were added at a concentration of 25% and incubated for 5 days (Fig. 2). However, marked variability was also seen depending on the cell concentration of the MLA 144 cells and the age of the supematant. To minimize these variables as well as to better define the lymphokine responsible, purified recombinant interleukin 2 from two different sources was utilized. Dose dependent responseswere observed with an optimal concentration of 50 U/cc for the Amgen product and 100 U/cc for the Cetus preparation (data not shown). Both adult and cord WMC show an enhancement of cytotoxicity against both K562 and Raji cells after incubation with IL-2 for 5-7 days (Fig. 3). Cord cells show a 5.9-fold increasein cytotoxicity against the K562 targets (12 f 2.6 to 7 1 f 4.2%) compared to a 2.7-fold increase observed for adult WMC (23 f 4.2 to 6 1 * 4.5%) at an E:T ratio of 40: 1. Similarly, a greater enhancement was also observed in LAK activity toward Raji cell targets. Cord WMC increased their cytotoxic activity 5-fold (9.6 + 2.5 to 48 f 6.7%) compared to a 3-fold increase for adult WMC (12 f 3.0 to 36 15.3%).

Efector Cells without either Lymphokine or Cellular Activation In parallel cultures, cord as well as adult cells incubated for 5-7 days with neither IL2 nor irradiated Raji cells show a decline in their ability to lyse K562 and Raji cells (Fig. 4). The mean percentage of specific lysis was lessthan 5% for the two effector cells and both target cells.

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DISCUSSION The cytotoxicity of cord blood mononuclear cells induced by a 5-day mixed-leukocyte culture with mitomycin-treated Raji cells toward Raji and K562 cells is lessthan that generated by similarly treated adult cells. At least two distinct types of cytotoxic cell activities are generated in an MLC (25-27): (i) NK-like activity against the K562 target cells, and (ii) specific cytotoxicity toward the sensitizing (Raji) cell line. Unlike resting cytotoxicity, the NK-like activity is mediated by a Leu- 11-negative cell population. Treatment with antisera which inhibit NK cytotoxicity will not affect Raji cell cytolysis (25). Conversely, treatment with pan-T-cell monoclonal antibody eliminates the cytotoxicity generated against the sensitizing tumor cell line without diminishing cytotoxicity against K562 cells (26). Furthermore, the development ofcytotoxicity against Raji cells is not an NK function since human NK cells in the resting state do not kill Raji cells. Thus, this form of cytotoxicity is specific and mediated by T cells, i.e., comparable to cell-mediated lympholysis. Our observation of diminished cell-mediated lympholysis in newborn cells is in agreement with that of Granberg et al. ( 17, 18), who noted decreasedlysis of concanavalin A-activated peripheral mononuclear cells by cord blood cells. An intrinsic defect of the cytotoxic machinery was speculated. Andersson et al. (28) confirmed deficient neonatal cytotoxic generation with isolated effector T cells toward haptenconjugated, Epstein-Barr virus-transformed, and phytohemagglutinin (PHA)-activated lymphocytes. In contrast, Rayfield et al. (19) could not demonstrate a defect in newborn cytotoxicity utilizing two lymphoblastoid cells as both stimulatory and target cells. The lessenedaugmented cord cytotoxicity toward K562 targets reported here is not surprising in view of deficient cord spontaneous or resting NK cytotoxicity previously reported by numerous investigators (4-14). Three mechanisms for diminished cord NK activity have been identified. There appear to be decreasednumbers of mature NK cells identified morphologically as large granular lymphocytes and by monoclonal antibody HNK- 1 or Leu-7 analysis ( 13, 14). However, when other monoclonal antibodies with broader specificities have been used, cord blood mononuclear cells have normal proportions of B73.1 -positive (29) or Leu- 11-positive (30) cells. Therefore, although mature NK cell numbers are less in cord blood, immature or pre-NK cells appear to be similar. Second, there are indications that the cytotoxic activity of cord cells is qualitatively inferior to that of adult cells. Single cell assay studies have revealed significantly less lysis as well as binding by cord WMC when compared with adult cells (14). Finally, Nair et al. have recently found an association between decreasednatural cytotoxicity and the production of natural killer cytotoxic factor and interferon-y (32). Decreasedproduction of the latter by PHA-stimulated cord cells is well established (33) but whether this occurs in vivo is not known. Indeed, such a defect may be an immunoregulating abnormality (34) or may be attributed to defective monocyte-macrophage function (35).

PIG. 2. Activated killer cellular cytotoxicity of adult (A) and cord (C) cells by crude lymphokine preparation. Mononuclear cells (10’) were incubated in 7.5 cc of RPM1 1640 medium supplemented with 10% human AB cerum and 2.5 cc of supernatants from MLA 144cell line for 5 days at 37°C. Cellular cytotoxicity against chromium-labeled K562 (A) and Raji (B) cells was then examined at an effector:target cell ratio of 40: 1. Also shown is the spontaneous (baseline) natural killer activity on Day 0.

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Natural killer activity in cord and adult cells can be augmented with interferon and inducers of interferon (7, 9, 10, 3 1, 32). However, since the magnitude of enhancement is the same for adult and cord cells, the enhanced lytic activity of cord cells remains lessthan that observed for adult cells. Long-term stimulation with allogeneic cells in an MLC can also enhance NK activity (25-27). However, as shown in this paper, decreasedcytotoxic activity is still present when compared to similarly treated adult cells. During a 5-day MLC, numerous cellular events are taking place, including activation, differentiation, and proliferation of various lymphocyte subpopulations. Elaboration of various lymphokines such as interferon occur (36). Our attempts to enhance the cytotoxic potential of cord cells (and adult cells) by incubating with interferon or isoprinosine as well as allogeneic cells were unsuccessful. Isoprinosine is an immunopotentiating drug which has been shown to augment NK activity in adults (37). Previous attempts to activate various forms of T or NK cellular cytotoxicity by various lectins also show deficient responsesby cord WMC ( 15). Production of interleukin 2 also occurs in a MLC (IL-2) (38,39). The 5- to 7-day incubation with IL-2 used for this paper resulted in a population of cytotoxic cells called lymphokine-activated killer cells, which are able to lyse NKFIG. 3. Activated killer cellular cytotoxicity of adult and cord cells by purified, recombinant interleukin 2. Mononuclear cells (106)were incubated in 50-100 U/cc of interleukin 2 for 5-7 days at 37°C. Cellular cytotoxicity against chromium-labeled K562 (A) and Raji (B) target cells was examined at an effector: target cell ratio of 40: 1. Also shown is the spontaneous NK activity on Day 0. Results are expressed as percentage specific lysis + standard error of the mean.

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resistant targets (2 1). These cells have properties which are unique and different from both NK and cytotoxic T lymphocytes (40). HLA-restriction does not exist despite the presence of T-cell surface markers OKT3 and OKT8. LAK cells are characterized by their ability to kill freshly isolated tumor cells and cell lines not readily lysed by freshly isolated NK cells. LAK precursor cells do not have T-cell markers (OKT3, OKT9) or NK cell surface antigens (Leu- 11) (40). So far, no cell surface markers are unique for LAK precursors. As demonstrated in this paper, although cord cells are capable of producing IL-2, they are able to be stimulated by exogenous IL-2 to achieve cytotoxic levels comparable with similarly treated adult cells. This effect was observed with three different sources of IL-2. This finding is somewhat surprising in view of deficient cytotoxicity observed after an MLC reaction for 5 days. Although we did not measure IL-2 levels in MLC supernatants, Hausser et al. have found levels in cords which should be sufficient to induce LAK activity (39). No defects have been described with IL-2 production by cord cells (38). One possible explanation may reside in the heterogeneous population of lymphocytes generated in an MLC. D’Amore and Golub (41) have described suppression of NK cell activity by cells bearing Fc receptors and HNK1 surface markers in long-term MLC. NK suppressor cells have been found more frequently in cord blood than in normal adults (42). In the murine model, suppression mediated by placental cells can be demonstrated toward natural killing as well as antibody-dependent cellular cytotoxicity and cytotoxic T lymphocytes (43). This suppression could not be overcome by the addition of IL-2 to the MLC (44). There may be a population of nonspecific cytotoxic cells which lyse a variety of target cells. IL-2 has been shown to act on several types of cytotoxic cells, including cytotoxic T-cell clones, null cells mediating antibody-dependent cellular cytotoxicity, and extravascular NK cells (45). Specific antiviral cytotoxic T lymphocytes can be regenerated with IL-2 (46, 47). A larger population of immature, nonspecific cytotoxic cells may be present in cord blood. Subsequent enhancement and proliferation by IL-2 of such cells may explain their high level of cytotoxicity. In addition, suppressive influences present in MLC may have been avoided by direct activation by IL2. Mononuclear cell populations from cord blood have been noted to have greater spontaneous proliferative ability than adult peripheral blood (33). This may reflect greater cell population or cellular density with the IL-2 receptor. These observations of the presence of lymphokine-activated killer cells in cord blood may be of clinical significance in view of the availability of purified recombinant IL-2 (48). Since the human neonate is relatively immunoincompetent (compared to adults), IL-2 and/or LAK therapy may be of benefit in the treatment of neonatal viral infections or neoplastic diseases.Clinical trials have demonstrated that the adoptive transfer of LAK cells is well tolerated and safe in human (49). Dosage and route of administration are now being studied (50). This lymphokine administration is a potent new form of therapeutic intervention. REFERENCES 1. McConnachie, P. R., Rachelefsky, G., Stiehm, E. R., and Terasaki, P., Pediatrics 52,795, 1973. 2. Campbell, A. C., Walter, C., Wood, J., Aynsley-Green, A., and Yu, V., Clin. Exp. Immunol. 18,469, 1974. 3. Shore, S. L., Milgrom, H., Wood, P. A., and Nahmias, A. J., Pediatrics 59,22, 1977. 4. Hallberg, A., and Malmstrom, P., Acta Puediatr. Sand. 71,43, 1982. 5. Timonen, T., and Saksela, E., Cell. Immunol. 33,340, 1977.

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6. Levin, C. L., Massey, R. J., and Deinhardi, F., J. Natl. Cancer Inst. 60, 1283, 1978. 7. Sato, T., Fuse, A., and Kuwata, T., Cell. Zmmunol. 45,458, 1979. 8. Toivanen, P., Uksila, J., Leino, A., Lassila, O., Hirvonen, T., and Ruuskanen, O., Zmmunol. Rev. 57, 89, 1981. 9. Kohl, S., Frazier, J. J., Greenberg, S. B., Pickering, L. K., and Loo, L. S., J. Pediatr. 98,379, 1981. 10. Antonelli, P., Steward, W., II, and Dupont, B., Clin. Zmmunol. Zmmunopathol. 19, 161, 1981. 11. Kaplan, J., Shope, T. C., Bollinger, R. O., and Smith, J., J. Clin. Zmmunol. 2,350, 1982. 12. Uksila, J., LassiIa, O., and Hirvonen, T., C’lin. Exp. Zmmunol. 48,649, 1982. 13. Abo, T., Cooper, M. D., and Balch, C. M., J. Exp. Med. 155,32 1, 1982. 14. Bayley, J. E., and Schacter, B. Z., J. Zmmunol. 134,3042, 1985. 15. L&ens, R. G., Gard, S. E., &ode&erg-Warner, M., and Stiehm, E. R., Cell. Zmmunol. 74,40, 1982. 16. Tsoukas, C. D., Fox, R. I., Slovin, S. F., Carson, D. A., Pellegrino, M., Fong, S., Pasquali, J.-L., Ferrone, S., Kung, P., and Vaughan, J. H., J. Zmmunol. 126, 1742, 1981. 17. Granberg, C., Manninen, K., and Toivanen, P., Clin. Zmmunol. Zmmunopathol. 6,256, 1976. 18. Granberg, C., and Hirvonen, T., Cell. Zmmunol. 51, 13, 1980. 19. Rayfield, L. S., Brent, L., and Rodeck, C. H., Clin. Exp. Zmmunol. 42,56 1, 1980. 20. Goto, M., and Zvaifler, N. J., J. Exp. Med. 157, 1309, 1983. 21. Grimm, E. A., Mazumder, A., Zhang, H., and Rosenberg, S. A., J. Exp. Med. 155,1823,1982. 22. Jondal, M., Spina, C., and Targan, S., Nature (London) 272,62, 1978. 23. Rabin, H., Hopkins, R. F., Ruscetti, F. W., Neubauer, R. H., Brown, R. L., and Kawakaui, T. G., J. Zmmunol. 127,1852,1981. 24. Chang, T. W., McKinney, S., Liu, V., Kung, P. C., Vilcek, J., and Le, J., Proc. Natl. Acad. Sci. USA 81,5219,1984. 25. Seeley, J. K., Masussi, G., Poros, A., Klein, E., and Golub, S. H., J. Zmmunol. 123, 1303, 1979. 26. Strassman, G., Bach, F. H., and Zarling, J. M., J. Zmmunol. 130, 1556, 1983. 27. Phillips, J. H., Le, A. M., and Lanier, L. L., J. Exp. Med. 159,993, 1984. 28. Anderson, U., Bird, A. G., B&ton, S., and Palacios, R., Zmmunol. Rev. 57,5, 1981. 29. Perussia, B., Starr, S., Abraham, S., Fanning, V., and Trinchieri, G., J. Zmmunol. 130,2133, 1983. 30. Abo, T., Miller, C. A., and Balch, C. M., Eur. J. Zmmunol. 14,6 16, 1984. 31. Ueno, Y., Miyawaki, T., Seki, H., Matsuda, A., Taga, K., Sato, H., and Taniguchi, N., J. Zmmnol. 135,180, 1985. 32. Nair, M. P. N., Schwartz, S. A., and Menon, M., Cell. Zmmunol. 94, 159, 1985. 33. Bryson, Y. J., Winter, H. S., Gard, S. E., Fischer, T. J., and Stiehm, E. R., Cell. Zmmunol. 55, 191, 1980. 34. Wakasugi, N., and Virelizier, J.-L., J. Zmmunol. 134, 167, 1985. 35. Taylor, S., and Bryson, Y. J., J Zmmunol. 134, 1493, 1985. 36. Trinchieri, G., and Perussia, B., Lab. Invest. 50,489, 1984. 37. Tsang, K. Y., Fudenberg, H. H., Galbraith, G. M. P., Donnelly, R. P., Bishop, L. R., and Koopman, W. R., J. Clin. Invest. 75, 1538, 1985. 38. Ilonen, J., and Karttunen, R., Zmmunobiology 167,399, 1984. 39. Hauser, G. J., Zakuth, V., Rosenberg, H., Bing, T., and Spirer, Z., J. Clin. Lab. Zmmunol. 16, 37, 1985. 40. Grimm, D. A., and Rosenberg, S. A., In “The Human Lymphokine-Activated Killer Cell Phenomenon in Lymphokines” (E. Pick, ed.), Vol. 9, p. 279. Academic Press,Orlando, 1984. 4 1. D’Amore, P. J., and Golub, S. H., J. Zmmunol. 134,272, 1985. 42. Tarkkanen, J., Saksela, E., and Paavolainen, M., Clin. Zmmunol. Zmmunopathol. 28,29, 1983. 43. Kolb, J.-P., Chaouat, G., and Chassoux, D., J Zmmunol. 132,2305, 1984. 44. Clark, D. A., Chaput, A., Walker, C., and Rosenthal, K. L., J. Zmmunol. 134, 1659, 1985. 45. Kimber, I., Bakacs, T., Roberts, K., and Moore, M., J. Clin. Lab. Zmmunol. 15,77, 1984. 46. Merluzzi, V. J., Welte, K., Mertelsmann, R. H., Souza, L., Boone, T., and Last-Barney, K., J. Virol. 51,20, 1984. 47. Walker, C. M., Paetkau, V., Rawls, W. E., and Rosenthal, K. L., J. Zmmunol. 135, 1401, 1985. 48. Rosenberg, S. A., Grimm, E. A., McGrogan, M., Doyle, M., Kawasaki, E., Koths, K., and Mark, D. F., Science223, 1412, 1983. 49. Rosenberg, S. A., J. Biol. ResponseMod$3,501, 1984. 50. Cheever, M. A., Thompson, J. A., Kern, D. E., and Greenberg, P. D., J. Zmmnol. 134,3895, 1985.