Synergistic effect of thymosin α1 and αβ-interferon on NK activity in tumor-bearing mice

Int. J. Immunopharmac., Vol. 11, No. 5, pp. 443-450, 1989. Printed in Great Britain.

0192-0561/89 $3.00 + .00 International Society for Imunopharmacology.

SYNERGISTIC EFFECT OF THYMOSIN a 1 AND a/J-INTERFERON ON NK ACTIVITY IN TUMOR-BEARING MICE CARTESIO FAVALLI,* ANTONIO MASTINO,* TERESA JEZZI,* SANDRO GRELLI,* ALLAN L. GOLDSTEINt:i: and ENRICO GARACI* *Department of Experimental Medicine and Biochemistry, II University of Rome Tor Vergata, 00173 Rome, Italy; *Department of Biochemistry, The George Washington University Medical Center, 2300 Eye Street, N.W., Washington, DC 20037, U.S.A. (Received 20 July 1988 and in final form 1 May 1989)

-We have investigated the possibility of thymosin a~ (TH) cooperating with a/J-interferon (IFN) in boosting natural killer (NK) activity in tumor-bearing, immunosuppressed mice in vivo. Treatment with a single injection of 30,000 IU of IFN 24 h before testing enhanced NK activity in tumor-bearing mice if the IFN was administered 9 days after tumor inoculation, when the animals have normal NK responsiveness. On the other hand, the same treatment led to lower or no improvement of NK responses if the treatement was given 13 or 17 days after tumor inoculation, at a time when tumor growth causes immunosuppression. However, combination treatment with TH (200/ag/kg) for 4 days, followed by IFN was found to restore normal NK cell activity. Selective depletion of antigen-positive cells showed that killer cells stimulated by combination treatment with TH and IFN seem to bear phenotypic characteristics of NK cells. These studies provide the first documentation of a novel combination approach to reconstitution of immunosuppressed tumor-bearing mice using TH and IFN. We hypothesize that TH restores NK boosting activity by IFN by effecting the differentiation/inductionof precursor populations of IFN-responsive cells.

Abstract

Thymosin al (TH), the first of the biologically active polypeptides isolated from thymosin fraction 5, has been found to enhance a number of T-cell responses and to increase thymus-dependent immunity in immunosuppressed humans and to protect immunosuppressed mice following microbial infections (Goldstein, Low, Zatz, Hall & Naylor, 1983; Sztein & Goldstein, 1986; Bistoni, Marconi, Frati, Bonmassar & Garaci, 1982; Ishitsuka, Umeda, Nakamura & Yagi, 1983). TH has been also found to increase the production of a number of different lymphokines and cytokines, including migration inhibition factor (MIF) (Thurman, Seals, Low & Goldstein, 1984), aft-interferon (IFN) (Huang, Kind, Jagoda & Goldstein, 1981) and interleukin-2 (IL-2) (Zatz, Oliver, Samuels, Skotnicki, Sztein & Goldstein, 1985). Furthermore, additional studies have shown that TH, as well as a number of lymphokines and cytokines, each exhibit some, but not complete, antitumor activity in experimental animal models (Umeda, Sakamoto, Nakamura, Ishitsuka & Yagui, 1983) and in humans (Cohen, Chretien, Ihde, Fossieck, Makuch, Bunn, Johnston,

Shackney, Matthews, Lipson, Kenady & Minna, 1979; Schulof, 1985). The possibility that thymic hormones and lymphokines/cytokines might have additive effects in enhancing immunity and slowing tumor growth was suggested by preclinical experiments using TH and IFN together in cyclophosphamide-suppressed C57bl/6 mice (Favalli, Jezzi, Mastino, RinaldiGaraci, Riccardi & Garaci, 1985). In this study, we found that neither TH or INF, when administered alone, were effective in restoring natural killer (NK) cell activity. However, when both were given in combination, there was a synergistic response and this treatment was effective in restoring NK activity. Over the last few years much attention has focused on the importance of NK cell activity in the control of neoplastic growth, and, in particular, of metastatic spread (Herberman, Nunn & Lavrin, 1975; Trinchieri & Perussia, 1984; Uchida, 1986; Hanna, 1986). IFN has proved to be an important agent in activation of NK activity in vitro as well as in vivo in a number of animal models (Herberman, Ortaldo, Mantovani, Hobbs, Kung & Pestka, 1982;

*Author to whom correspondence should be addressed. 443

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Gidlund, Orn, Wigzell, Senik & Gresser, 1978; Brunda & Rosenbaum, 1984). These findings have led to clinical trials in cancer patients to study the effects of IFN administration on tumor progression and NK activity. Unfortunately, despite the well documented effects of IFN on NK activity in vivo in normal animals and humans, the trials with IFN in immunosuppressed cancer patients have been disappointing to date. Furthermore, IFN administration has not always increased NK responses (Bash, Woody & Neefe, 1982; Maluish, Ortaldo & Herberman, 1982). An increased NK activity has also been observed in animals treated with a number of thymic hormones (Bardos & Bach, 1982; Kaiserlian, Bardos& Bach, 1983; Bistoni, Baccarini, Puccetti, Marconi & Garaci, 1984). Given our observations that there is synergistic activity between TH and IFN in cyclophosphamide-pretreated immunosuppressed animals, and the demonstration that TH is able to accelerate the recovery rate of NK activity in bone marrow reconstituted chimeras, a possible explanation of these results could be that the unresponsiveness is the result of a partial or total lack of mature cells of the NK lineage, able to respond to the IFN boosting activity. Moreover, it was recently shown that the combination of thymosin fraction 5 or TH and recombinant a-IFN, significantly increased NK activity of both peripheral blood lymphocytes and large granular lymphocytes, when compared to either agent alone, in vitro (Serrate, Schulof, Leondaridis, Goldstein & Sztein, 1987). In the experiments reported here, we have studied separately and in combination, the effect of TH and murine IFN on NK activity in B-16 melanomabearing mice. The treatment was started at different days after tumor cell inoculation to document the correlation between tumor-induced immunosuppression and effects on NK responses. Given the inability of IFN to consistently stimulate NK activity in tumor-bearing mice and humans, and the suggestion from our previous studies that this anergy may be due, in part, to a TH-dependent NK cell precursor, we have utilized TH, a purified thymic hormone in an attempt to increase the number of IFN-responsive NK cells.

E X P E R I M E N T A L PROCEDURES

Animals Four-week-old male C57BL/6NCrlBR mice, purchased from Charles River Italia (Calco, Como, Italy), were used.

Drugs Thymosin al (TH) kindly provided as a lyophilized synthetic preparation by Alpha 1 Biomedicals, Inc. (Washington, D.C.), was dissolved in sterile 1.4070 NaHCO3 at a concentration of 1 mg/ml and stored at - 2 0 ° C . Mice were injected with 200 gg/kg of TH. IFN (a mixture of a and/3), kindly suppfied by Dr F. Berardelli (Istituto Superiore di SanitY, Rome, Italy), was prepared from suspension cultures of sarcoma mouse 243 cells inoculated with Newcastle disease virus; 30,000 international units in 0.1 ml of PBS were administered. Treatment schedules Mice were divided into different experimental groups and received the following treatments: (a) control diluent at the same time as treated animals; (b) four daily i.p. injections of TH starting 4 days before testing the NK activity; (c) a single i.p. injection of IFN 24 h before testing the NK activity; (d) the same treatment as group (b), followed by the treatment of group (c) 3 h after the last pre-treatment injection. Treatments started at various days (i.e. 6, 10, 14) beginning after tumor inoculation. Dosages and treatment schedules were selected on the basis of our previous studies (Favalli et al., 1985). Tumors

B-16 malignant melanoma (obtained originally from Dr Zupi, Istituto Regina Elena, Rome, Italy), was maintained in vivo by serial subcutaneous transplantations in C57B1/6NCrlBr male mice. In these experiments the tumors, freshly excised from tumor-bearing animals, were minced in Hanks' balanced salt solution (HBSS) (Flow Laboratories, lrvine, Ayrshire, U.K.) and strained through a fine stainless steel mesh. Cells were then collected by centrifugation, washed twice, counted and resuspended to the desired concentration. Viability, as determined by trypan blue exclusion, ranged between 10 and 20070. Mice of different experiments were inoculated with 2 × 105 viable B-16 melanoma cells in 0.1 ml of HBSS subcutaneously in the right flank.

Preparation o f effector cells Spleen cells were obtained from normal or tumorbearing mice by gentle teasing of individual spleens in RPMI 1640 (Flow Lab.). The resultant cell suspension was filtered through a Nytex mesh, washed once with RPMI 1640 and the pellet was then re-suspended in the assay culture medium; it

Synergy of TH and IFN in Boosting NK Cells

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Table 1. Treatment schedule

Group Control TH IFN TH + IFN

ist day

2nd day

c.d. TH c.d. TH

c.d. TH c.d. TH

Treatment 3rd day c.d. TH c.d. TH

4th day

5th day

c.d. TH IFN TH,IFN

NK assay NK assay NK assay NK assay

c.d., control diluent; TH, thymosin a~ 200/~g/kg i.p.; IFN, murine a/J-interferon 30,000 I.U.i.p. consisted of RPMI 1640 supplemented with 10°70 fetal bovine serum (Flow Lab.), 200 mM L-glutamine (Flow Lab.), 26 mM HEPES (Flow Lab.) and 50/ag/ml gentamycin (GRS, Shering Co., Kenilworth, N J).

Target cells YAC-1, a Moloney virus-induced mouse T-cell lymphoma of A/SN origin was used as target cell in the chromium release assay. A cell suspension of 5 x 106 Yac-1 cells in 0.9 ml of culture medium was labeled with 100/aCi of sodium 51chromate (New England Nuclear, Boston, MA) for 60 min at 37°C in a CO2 incubator. After labeling, the cells were washed three times in RPMI 1640 and re-suspended in the complete culture medium at 1 × 105 cells/ml.

N K assay The NK activity of effector cells collected from mice was individually measured by a 4 h 5tchromium release assay as described by Herberman et al. (1975). Briefly, effector cells were adjusted to varying concentrations and added to 1 × 104 5~chromium labeled Yac-1 cells in U-shaped 96-well microtiter plates (Flow Lab.) in a total volume of 0.2 ml. The plates were then incubated for 4 h at 37°C in a CO2 incubator. After the incubation period, the plates were centrifuged at 350 × g for 10 min. Successively, 0.1 ml samples of supernatant were collected and the radioactivity was measured using a Beckman Biogamma Counting System (Beckman Instruments, Irvine, KY). All assays were performed in quadruplicate and three effector/target (E/T) cell ratios were employed. The baseline 51chromium release was that of labelled Yac-1 cells incubated alone in 0.2 ml of culture medium, and in no case did it exceed 10% of the total counts incorporated by target ceils. Experimental results were expressed as cytotoxicity obtained at three different (100:1, 50:1, 25:1) effector/target cell ratios. Specific cytotoxicity (SC) was calculated as follows:

SC=

test counts/min- baseline counts/min Total counts/rain incorporated- baseline" counts/min

Cell depletion The phenotypes of spleen cytolytic cells obtained from normal and tumor-bearing mice, treated or not with single or combination therapies with TH and IFN, were examined by treating fresh spleen cells with appropriate predetermined dilutions of the antibodies described below, plus low-toxic rabbit complement (Cedarlane Laboratories Ltd, Ontario, Canada; final dilution 1:8), before testing NK activity. Cells obtained from four mice for each experimental group were pooled, adjusted to 107/ml and incubated with antibodies for 30 min at 4°C. After washing twice, the cells were incubated for 1 h with complement. The cells were then washed twice and the cytotoxicity against YAC-1 target cells was determined as described above. Cell numbers were not readjusted after antibody and complement treatment, to prevent selective enrichment of surviving cells. Experimental groups are described in "treatment schedules", with treatment starting on day 14. A group of normal untreated mice was also examined. The following antibodies were used: monoclonal antibodies rat anti-mouse Thy 1.2 clone 30H12 and Lyt-2 clone 53-6.7 (Becton, Dickinson, Mountain View, CA), monoclonal antibody rat antimouse macrophage (M+) clone M1/70.15 (Sera-lab Ltd, Crawley Down, W. Sussex, U.K.) and rabbit antiserum anti asialo GM1 (Wako Chemicals GmbH, Neuss, W. Germany).

Statistical analysis Data obtained from animals of different experimental groups were analyzed for differences by the Student's two-way t-test. RESULTS

Effect o f thymosin a~ and aO-interferon on N K activity in B-16 melanoma-bearing mice 10 days after tumor inoculation

C. FAVALLIet al.

446

Table 2. Effect of thymosin aj and a// interferon on NK activity in B-16 melanoma-bearing mice* Mean percentage cytotoxicity + S.E.M. against YAC-I Group t

E: T ratio 50:1

100:1

Cells/spleen Mean + S.D. (× 106)

25:1

Exp. 1 First day Control* of treatment: B-16 day6after B-16+TH tumor B-16+IFN inoculation B-16+TH+IFN

24.44 28.63 31.79 56.43 53.94

+ + + + +

1.51 1.48 2.44 3.258 2.108

16.52 16.53 19.50 40.60 37.20

+ + + + +

1.08 0.95 1.68 2.79 ~ 1.87~

10.83 10.54 11.80 22.32 25.63

+ + + + +

1.10 0.82 1.14 2.88 ~ 1.68~

92 86 89 86 82

+ + + + +

19 16 17 21 22

Exp. 2 First day Control of treatment: B-16 day 10 after B-16+TH tumor B-16+IFN inoculation B-16+TH+IFN

21.06 18.73 20.64 24.19 29.08

+ + + + +

1.13 1.60 0.87 1.321 1.33~+t

14.80 15.00 14.98 22.36 23.93

+ + + + +

1.52 2.42 1.34 1.20I** 2.731~t

7.73 12.71 9.08 14.44 18.77

+ + + + +

2.00 1.25 2.75 0.76 0.84 TM

99 97 94 103 87

+ + + + +

10 11 11 13 16

Exp. 3 First day Control of treatment: B-16 day 14 after B-16+TH tumor B-16+IFN inoculation B-16+TH+IFN

26.76 14.67 15.88 22.04 33.47

+ + + + +

1.55HH 1.62 2.40 3.21 2.12 ~tt***

19.38 10.98 9.67 14.82 22.19

+ + + + +

1.2211 1.64 1.88 2.74 1.48~**

11.44 6.19 2.85 7.00 14.45

+ + + + +

1.451 1.39 1.19 1.76 2.211tt**

109 111 134 118 129

+ + + + +

38 13 181 43 15

*B-16 melanoma (2 × 105 viable cells) was inoculated s.c. in the flank 10 (exp. 1), 14 (exp. 2) or 18 (exp. 3) days before testing. Exp. 3 was repeated three times and similar results were obtained. tEach group consisted of five mice individually tested. C57BI/6NCrlBR B-16 melanoma-bearing mice, were randomized, divided into four groups and treated, respectively, with control diluent (B-16; thymosin a~ 200/ag/kg (B-16 + TH), IFN 30,000 I.U. (B-16+ IFN) or thymosin a~ 200 Mg/kg plus IFN 30,000 I.U. (B-16+TH+ IFN), according to treatment schedule shown in Table 1, starting 6 (exp. 1), 10 (exp. 2.) or 14 (exp. 3) days after tumor inoculation. Spleen cell NK activity was tested 24 h after last injection. *Normal untreated control mice. Statistical analysis by Student's two-way t-test among values obtained at corresponding E: Tratio: ~P<0.001 against tumorbearing control and TH treated group; HP<0.005 against tumor-bearing control and P<0.01 against TH-treated group; ~P<0.05 against tumor-bearing control; **P<0.005 against TH-treated group; tiP<0.05 against IFN-treated group; **P<0.05 against TH-treated group; ~sP<0.01 against IFN-treated group; m~P<0.001 against tumor-bearing control; 11P<0.005 against tumor-bearing control; ***P<0.05 against normal untreated group.

In order to estimate the effects o f T H and IFN alone a n d / o r in c o m b i n a t i o n on the cytolytic activity o f NK cells during t u m o r progression, groups c o m p o s e d o f 20 mice each were inoculated with 2 × 105 B-16 m e l a n o m a cells. Beginning at either 6, 10 or 14 days after t u m o r i n o c u l a t i o n , mice were r a n d o m i z e d , divided into four experimental groups (five mice each), a n d injected with control diluent a n d / o r single or c o m b i n e d t r e a t m e n t , as illustrated in Table 1 and as described in the Experimental P r o c e d u r e s . Ten, 14 and 17 days after t u m o r inoculation (i.e. 24 h after the last administration), NK activity was tested. A n experimental g r o u p consisting o f five age and sex-matched controls was also tested. As s h o w n in Table 2, 10 days after t u m o r i m p l a n t a t i o n (see experiment 1), t u m o r bearing mice showed a n o r m a l N K response as c o m p a r e d to n o n - t u m o r - b e a r i n g control animals. In

this case the animals treated with IFN showed a significant increase in N K activity at all ratios tested, while t r e a t m e n t with T H was without significant effects. Small, but not significant, differences were observed in mice treated with T H associated with IFN compared to those treated with IFN alone. Thus, when NK activity was not suppressed in tumor-bearing animals, administration o f T H or IFN alone or in combination were not additive. We have previously reported a similar finding in terms o f modulation o f NK activity in n o r m a l mice (Favalli et al., 1985).

Effects o f thymosin ch and ap-interferon on N K activity in B-16 melanoma-bearing mice 14 and 18 days after tumor inoculation The same above described experimental design was utilized in exp. 2 and exp. 3 in which treatments began 10 or 14 days after t u m o r inoculation. In these

Synergy of TH and IFN in Boosting NK Cells

447

Table 3. Phenotypic characterization by selective depletion with antibodies plus complement of cytotoxic cell from B-16 bearing mice stimulated by TH and IFN* % cytotoxicity ~ Treatment None Complement Anti-Thy 1.2 Anti-Lyt-2 Anti-aGM1 Anti-Mo

Normal*

B-16

23.2 24.4 18.6 21.9 1.3 22.8

9.0 8.9 8.1 7.5 0.6 7.5

B-16(TH) 10.4 10.5 10.4 10.5 3.5 14.0

B-16(IFN)

B- 16(TH + IFN)

20.8 20.5 19.8 21.4 4.5 20.7

29.9 27.6 24.4 26.3 3.8 23.9

*C57B1/6NCrlBR B-16 melanoma-bearing mice (2 x 105 viable cells s.c. in the flank), were randomized, divided into four groups (four animals each) and treated respectively with control diluent (B-16), thymosin a~ 200/ag/kg (B-16 + TH), IFN 30,000 I.U. (B-16 + IFN) or thymosin aj 200/ag/kg plus IFN 30,000 I.U. (B-16+TH + IFN), according to treatment schedule shown in Table 1, starting 14 days after tumor inoculation. Spleen cells from mice of the same experimental group were pooled, selective depletion with antibodies and complement was performed and cytotoxic activity against YAC-1 cells was tested 24 h after last injection. +Effector: target ratio of 100:1. *Normal untreated control mice. cases, as shown in Table 2, we obtained results different from those described above. In fact, 14 days after inoculation with B-16 melanoma cells (exp. 2) TH alone did not significantly improve mean % cytotoxicities against YAC-1 cells. IFN treatment alone only partially improved depressed NK activity. When IFN was given in association with TH, significant increases in NK activity were noted above those seen with IFN alone. This synergistic effect phenomenon was more evident with tumor progression as immunosuppression overtly came out. In fact, as shown in Table 2 (exp. 3), 18 days after tumor inoculation a dramatic suppression in NK response was observed at all E / T tested ratios. At this time, treatments with IFN or TH alone were unable to restore the suppressed NK-cytolytic activity, while the combined treatment with TH and IFN was highly effective in restoring normal activity. In fact, there appeared to be an overshoot effect with NK-cell activity observed being higher than normal non-tumor-bearing animals.

Phenotypical characterization o f cytolytic cells stimulated by combination therapy with TH and IFN In order to characterize the cytolytic effector spleen cells stimulated in vivo in B-16 melanomabearing mice by combination therapy with TH and IFN, a depletion experiment was performed. The phenotypes of cytotoxic spleen cells collected from tumor-bearing mice treated or not with TH and IFN in single or combination therapy, were determined by elimination of cells bearing various cell surface antigens with specific antibodies and complement,

before testing the NK activity. Results, summarized in Table 3, showed no evident phenotypic difference among effector cells obtained from different experimental groups and exerting various cytotoxic activities against YAC-1 target cells. In particular, phenotypical characterization of cytolytic cells collected from B-16-bearing mice in vivo treated with combination therapy with TH and IFN, showed that they seem to bear the surface markers of NK cells.

DISCUSSION IFN, a major modulator of NK activity in normal mice, is not as effective when injected into tumorbearing animals. This anergy could be related to observations previously reported by our laboratory in mice immunosuppressed by cyclophosphamide treatment. In these studies, we found that IFN was only effective in increasing NK cells in normal mice and following chemotherapy had little effect unless given in combination with TH. Moreover, these findings in mice are in agreement with reports in humans that prolonged treatment with IFN alone in cancer patients in an advanced stage was not able to significantly stimulate suppressed NK activity (Golub, D ' A m o r e & Rainey, 1982; Spina, Fahey, Durkos-Smith, Dorey & Sarna, 1983). Similarly, TH alone only slightly modified NK activity in tumorbearing animals or cyclophosphamide-treated animals. In this report, we have shown that in tumorbearing, immunosuppressed animals, combined treatment with TH and IFN can substantially

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C. FAVALLI et al.

improve NK responses. We have found that in an early phase after tumor implantation, when animals are not NK-suppressed or even stimulated, i.e. when treatment began 6 days after tumor inoculation, IFN tends to keep its boosting capacity, but loses its effectiveness as tumor growth progresses. On the other hand, the stimulating activity of this cytokine is restored if tumor-bearing immunodepressed animals are pre-treated with the TH. A possible explanation for such data could be the partial or total lack of NK target cells for the IFN stimulation in tumor-suppressed mice. In fact, the results of experiments on bone marrow reconstituted chimeras previously published by our laboratory suggest that TH affects in v i v o the differentiation process of immature cells in the bone marrow into cytolytic effectors. Interestingly, a similar hyporesponsiveness state to IFN-induced NK activity boosting, was evidenced by other authors either during various experimental tumor growth or in different model of NK suppression in mice (Lala, Santer, Libenson & Parhar, 1985; Parhar & Lala, 1985; Saito, Welker, Fukui, Herberman & Chirigos, 1985). The existence of a non-lyric, IFN-unresponsive step during NK cells maturation has recently been demonstrated (Hackett, Tutt, Lipscomb, Bennet, Koo & Kumar, 1986; Kalland, 1986). Therefore, it seems likely that the activity of TH can be connected to the stimulation of the maturation of the NK progenitor cells on the low-reactive cell population (pre-NK). These pre-NK cells can be modified by the IFN boosting activity (Riccardi, Vose & Herberman, 1983). Our hypothesis is in agreement with recently reported results on TH in vitro on the regulation of NK activity through induction of IL-2 production and IL-2 receptor expression and ),-interferon production in mitogen-stimulated human peripheral blood lymphocytes or large granular lymphocytes (Serrate et al., 1987). However, we cannot rule out the hypothesis that TH can directly affect the maturation of T-helper lymphocytes that could, in turn, regulate NK activity, as has been suggested (Flexman, Holt, Mayrhofer, Lathan & Shellam, 1985). Alternatively, TH could negatively modulate suppressor cells in case they should be involved in NK activity depression during tumor growth. Transition from an early NK-stimulating phase to a late NK-depressing phase during tumor progression, could be in fact attributable to an imbalance between enhancing effects caused by endogenous cytokines production and suppressing activity exerted by cells and/or

factors, induced by tumors, as proposed (Parhar & Lala, 1985). In exp 1 and 2, NK activity was presumably tested just near this transition phase. This could perhaps explain either the different effectiveness of combination therapy we observed in exp. 3 and some of the differences obtained when comparing results of different E / T ratios, between normal and tumor-bearing mice in these experiments. Finally, if the enhancement of NK activity after combined TH and IFN administration is due to a synergistic effect between TH-induced endogenous cytokines and exogenous IFN, it will be possible to utilize this approval to develop a more effective strategy for immunotherapeutic approaches in cancer patients and in other patients with NK deficiencies. Interestingly, the induction of cytotoxic cells resembling NK cells after in v i v o combined treatment with IL-2 and IFN in mice, has recently also been observed (Brunda, Bellantoni & Sulich, 1987). Whatever exact mechanism could be implicated, direct or indirect, mediated or not by regulatory cells, the result of in v i v o combination therapy with TH and IFN in NK-depressed tumorbearing mice is a highly significant enhancement of cytotoxic activity against YAC-1 target by effector cells with a phenotype resembling that of NK cells, as demonstrated by depletion experiment. In summary, TH in v i v o acts synergistically with IFN in immunosuppressed tumor-bearing mice. The TH restored NK boosting activity by IFN, increases as tumor progression and immunosuppression increases and may be due to an effect on the differentiation/induction of precursor populations of NK cells. The utilization in combination of TH and IFN and/or other cytokines to restore NK activity in tumor-bearing mice provides a novel approach to immunotherapy, potentially applicable to immunoreconstitution of immunosuppressed cancer patients and patients with low NK activity. Studies are currently in progress to further define the nature of these thymic-dependent precursor cells and other co-factors necessary to maximize the killing of tumor cells in immunosuppressed mice. Acknowledgements - - We thank Giuseppe Febbraro for his

excellenttechnicalassistanceand Traci Black for her editorial and secretarial assistancein preparing the manuscript. This work was supported by grants 86-00406.44 from the Italian National Research Council, Special Project "Oncology"; and 86-01588.52, Special Project "Infectious Disease", and by the National Institutes of Health (CA24974).

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