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Modulation of the expression of interleukin 2 receptors of human thymocytes by recombinant interleukin 2
ARCHIVES
Vol.
OF BIOCHEMISTRY
239, No. 2, June,
Modulation
AND
BIOPHYSICS
pp. 455-461,
1985
of the Expression of lnterleukin 2 Receptors Thymocytes by Recombinant lnterleukin 2’ GABRIELLE
Department
of Phamnacology, Received
H. REEM’ NQW
October
York
AND
NING-HSING
School
University
31, 1984, and in revised
of Medicine, form
of Human
YEH New
January
York,
NQW
York
10016
2, 1985
The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-y synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-y, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-y was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with antiTat remained low throughout the first week of culture and interferon-y synthesis was not observed. Nonetheless, interleukin Z-induced proliferation was inhibited by antiTat on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-y synthesis, and for the growth of thymocytes in vitro. 0 1985 Academic press, I~C.
The expression of the receptor for interleukin 2 [T-cell growth factor (IL 2)13 is important for the proliferation of human T cells and of human thymocytes in vitro. Human thymocytes proliferate spontaneously in vivo and isolated human thymocytes continue to proliferate spontaneously in vitro. This spontaneous proliferation is gradually lost and, unless thymocytes are induced to differentiate i This paper is dedicated to Dr. B. L. Horecker on the occasion of his 70th birthday. ’ To whom correspondence should be addressed. 3 Abbreviations used: IL 2, interleukin 2; Con A, concanavalin A; PMA, phorbol myristate acetate; HTLV, human T-cell leukemia virus; IFN, interferon. 455
and stimulated to synthesize IL 2 or are cultured with IL 2, they rapidly lose the capacity to proliferate (1, 2). Studies recently carried out in our laboratory have shown that human thymocytes can be induced to express receptors for IL 2 in vitro, and that highly purified IL 2 enhanced the expresion of IL 2 receptors by thymocytes induced with Con A or with phorbol myristate acetate (PMA) (3, 4). We and others have reported a similar enhancement by IL 2 of the expression of IL 2 receptors by human T cells (5, 6). Other investigators have shown that IL 2 receptors are expressed on human Tcell leukemia virus (HTLV)-infected T cell lines (7, 8). The receptors of these cell 0003-9861/85 Copyright All rights
$3.00
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
456
REEM
lines are not subject to modulation of expression by anti-Tat, in contrast to those of T cells induced with lectin (7, 9), and synthesis of IL 2 is not necessarily linked to the expression of IL 2 receptors (10). Some T-cell lines derived from human leukemic cells can be induced by PMA to display IL 2 receptors and, in this respect, thay may resemble thymocytes cultured with PMA and IL 2 (4, 10). The present study addresses the effect of purified recombinant IL 2 on thymocyte proliferation, on the expression of Tat antigen (IL 2 receptors), and on lymphokine synthesis by human thymocytes. It provides evidence that cloned IL 2 allows thymocytes to proliferate after they have lost their capacity to proliferate spontaneously. However, in contrast to lectinactivated thymocytes, cultures of thymocytes supplemented with IL 2 in the absence of lectin display a small percentage of thymocytes which express Tat antigen at a low density. Cloned IL 2 was shown to augment the expression of IL 2 receptors on Con A-activated thymocytes, and to partially reverse the inhibitory effect of the glucocorticoid dexamethasone on IL 2-dependent proliferation. MATERIALS
AND
METHODS
Reagents. Purified recombinant Interleukin 2 (lo7 units/mg IL 2) was a generous gift from Dr. John J. Farrar, Hoffman La-Roche (Nutley, N. J.), antiTat antibody was from Dr. Thomas A. Waldmann, Metabolism Branch, National Institutes of Health (Bethesda, Md.). Concanavalin A was purchased from Miles Laboratories (Elkhart, Ind.) and dexamethasone from Sigma (St. Louis, MO.). Fluorescein-conjugated rabbit anti-mouse IgG (IgG fraction) was obtained from Cappel Laboratory (Cochranville, Pa.). Isolation and culture of thymocytes. Sections of human thymus were obtained during cardiac surgery of infants and children. Thymocytes were immediately isolated, washed in medium, and cultured in RPM1 1640 medium (Associated Biomedic System, Buffalo, N. Y.) containing 5% heat-inactivated fetal calf serum (KC Biological Laboratories, Lenexa, KS), 2 mM glutamine, and antibiotics (penicillin, 100 lJ/ ml; streptomycin, 100 pgg/ml; amphotericin B, 0.25 pg/ml; Grand Island Biologicals, Grand Island, N. Y.). Thymocytes were cultured in flat-bottomed microtiter plates (200 ~1) at a density of 6 X lo6 cells/ ml at 3’7°C in a humidified atmosphere containing 5% co*.
AND
YEH
Detection of Tat antigen Tat antigen was determined by indirect fluorescence by flow cytofluorometry (Cytofluorograph, Ortho Pharmaceuticals, Raritan, NJ), or by immunoperoxidase staining as described (4). Incorporation of [*H]thymidine. Cells were pulsed with 1 &i PH]thymidine (sp act, 6.7 CVmmol; New England Nuclear, Boston, Mass.) for the last 12 h of incubation, unless otherwise stated, and harvested on Whatman glass fiber filters (GM/CF, Clifton, N. J.) with a cell harvester (Hoeffer Scientific Instruments, San Francisco, Calif.), precipitated with 10% ice-cold trichloroacetic acid, and washed with 100% ice-cold methanol. The filters were dried and counted in a Beckman L-250 spectrometer (Beckman Instruments Inc., Mountainside, N. J.). Determination of inte&ron-7. Interferon-gamma (IFN-7) was determined by a solid-phase radioimmunoassay with monoclonal antibodies purchased from Centocor (Malvern, Pa.) as described (4). Incorporation of [“C]leucine [i4C&eucine (237 mCi/ mmol; New England Nuclear, Boston, Mass.), 0.2 &i per well, was added 5 h before cells were harvested, and thymocytes were cultured in leucine-free medium for that period of time. Proteins were precipitated on glass fiber filters, washed, and counted as described above. RESULTS
Human thymocytes isolated and placed in culture without delay proliferate spontaneously at a rapid rate. Following 48 h of culture (Day 2) this spontaneous rate of proliferation decreased to approximately one-fourth of the initial rate (Table I). At the end of the third day of culture, the proliferative rate was further reduced to 5% of the initial rate, and by Day 7 thymocytes cultured in complete medium had virtually ceased to proliferate. Less than 2% of thymocytes so cultured bound anti-Tat, and in less than 1% was the fluorescence of the Tat binding cells bright. If purified recombinant IL 2 was added to the medium, the decrease in the spontaneous proliferative rate observed on Day 2 was similar to that of thymocytes cultured without IL 2, but subsequently the proliferative rate slowly increased and thymocytes continued to proliferate at a slow rate. This proliferative rate could be maintained for longer periods of time if cultures were supplemented with IL 2. These findings indicate that during the first 2 days of culture, thymocyte prolif-
EXPRESSION
OF
INTERLEUKIN
2 RECEPTORS TABLE
REQUIRES
INTERUXJK~N
2
457
I
MODULATIONOFTHEEXPRESSIONOFTACANTIGENPROLIFERATIONAND SYNTHESISBYHUMANTHYMOCYTES
IFN--r
Tat+ cellsb [‘HJTdR Additions” None IL2 Con Con Dex Dex Dex Dex
A A + IL2 + IL2 + Con A + Con A + IL2
incorporation
(cpm
X 103) on Days
0
1
2
3
4
7
Total
104.3 86.6 81.7 78.9 54.7 30.1 32.3 36.2
102.3 101.2 97.8 91.8 2.9 1.9 4.9 4.3
23.5 21.9 23.0 47.5 0.1 0.2 0.3 2.4
5.2 7.4 19.5 106.2 0.1 0.2 0.2 11.9
1.2 3.9 6.5 194.4 0.1 0.1 0.1 35.5
0.1 5.6 0.8 1.2 0.1 0.1 0.1 10.0
1.9 2.8 17.6 21.1 1.0 6.3 8.4
Note. The data recorded on this Table were obtained ’ IL 2,100 U/ml; Con A, 5 rg/mh Dex, dexamethasone, b Determined on Day 2 by cytofluorometry. ’ Determined on Day 2 and expressed as NIH units.
eration is independent of IL 2, whereas late proliferation of thymocytes is IL 2dependent and can be maintained by the addition of IL 2 to cultures devoid of lectin. The kinetics of proliferation of thymocytes cultured with Con A initially resembled those of thymocytes cultured with IL 2 alone but, unlike thymocytes cultured with IL 2, they lost their capacity to proliferate within 7 days unless IL 2 was added to the cultures (data not shown). The proliferative activity of thymocytes cultured with both Con A and IL 2 exceeded that of cultures supplemented with Con A alone and reached a peak on Day 4, but it fell to 1% of the maximal rate by Day 7. This decline in proliferative activity reflected the rapid consumption of IL 2 in cultures grown with Con A and IL 2, and it too could be prevented by addition of IL 2 to the cultures at the time of maximal proliferation (data not shown). Dexamethasone exerted an inhibitory effect on both early and late proliferation of uninduced thymocytes, and the addition of IL 2 to thymocytes cultured with dexamethasone did not reverse the inhibition of the early IL 2-independent and the late
IFN-y” (U/ml)
Bright 0.8 1.7 5.9 10.5 1.0 3.6 4.6
from a thymic specimen from a single 1O-6 M was added on Day 0.
0.1 0.1 0.7 4.7 0.0 0.1 0.3 1.1 donor.
IL 2-dependent proliferation (Table I). On the second day of culture the viability of uninduced thymocytes was approximately 80% and the difference in viability between thymocytes cultured with or without dexamethasone was 15%. By Day 6, only 20% of thymocytes cultured with or without dexamethasone were viable (data not shown). Similarly, dexamethasone inhibited both the early and the late proliferative activity of thymocytes cultured with Con A; however, the inhibitory effect on Con A-induced, IL 2-dependent proliferation could be partially overcome by IL 2 (Table I). Cultures could be rescued and continued to grow provided they were supplied with the appropriate factors. Despite its marked inhibitory effect on the proliferative activity, dexamethasone decreased the viability of these cultures by less than 10% during the early proliferative phase. Even in cultures supplemented with IL 2 it caused a 50% decrease in viability within 6 days (data not shown). These cultures, however, regained their proliferative activity in the presence of IL 2 (Table I). A striking difference was observed in the expression of the number of thymocytes expressing Tat antigen, depending
458
REEM
AND
on the conditions of culture. Only 2% of thymocytes cultured with IL 2 alone expressed Tat antigen, while 17.6% of thymocytes cultured with Con A expressed Tat antigen on the second day of culture, before thymocytes had become dependent on IL 2 for growth. Addition of IL 2 to Con A-stimulated cultures increased the number of Tat antigen-bearing cells, as well as the density of receptors on the cell surface. This was demonstrated by the increase in the number of intensely fluorescent cells (5.9 to 10.5%). Dexamethasone decreased the number of receptorbearing cells in cultures supplemented with Con A, and the addition of IL 2 to these cultures resulted in a very moderate increase in the number of thymocytes expressing Tat. IFN-y synthesis was not induced by IL 2, but IL 2 augmented Con A-induced IFN--r synthesis more than sixfold (Table I). Dexamethasone inhibited Con A-induced 1FN-y synthesis by 57%, after 2 days of culture, at a time when spontaneous thymocyte proliferation was inhibited by dexamethasone and this inhibition was reversed by IL 2. In order to investigate the effect of IL 2 and dexamethasone on protein synthesis of spontaneously proliferating thymocytes and of thymocytes dependent on IL 2, the TABLE EFFECT
OF INTERLEUKIN
2 AND DEXAMETHASONE
YEH
incorporation of [14C]leucine into total protein was measured. The rate of protein synthesis of thymocytes cultured in complete medium decreased with time (Table III). This decrease was reversed by IL 2; in fact, protein synthesis on Day 6 of cultures supplemented with IL 2 exceeded that of the initial rate of protein synthesis by thymocytes cultured either with or without IL 2. IL 2 also increased protein synthesis of Con A-activated thymocytes. The increase in the rate of protein synthesis became apparent on the third day of culture and it was sustained throughout the period of culture. In cultures supplemented with Con A and IL 2 it exceeded that of cultures supplemented with Con A alone. IL 2 reversed the inhibitory effect of dexamethasone on protein synthesis by Con A-activated thymocytes during the late phase of the culture period, when thymocytes had developed dependence on IL 2. The overall effect of IL 2 on protein synthesis resembled that on the proliferative activity and became apparent when thymocytes required IL 2 for growth. The effect of IL 2 on IFN synthesis presented on Table II was consistent with that recorded on Table I. The data presented on Table III demonstrate that thymocytes proliferated for II ON PROTEIN
[i4CjLeucine
Additions” None IL2 Con Con Dex Dex Dex Dex
A A + IL2 + IL2 + Con A + Con A + IL2
Day 1.8 1.9 3.2 3.1 0.2 0.2 0.8 0.8
-t * ++ k -+ * f
1 0.1 0.1 0.3 0.3 0.0 0.0 0.1 0.0
incorporation
Day 0.4 0.8 1.4 10.7 0.1 0.1 0.1 1.4
3 k f + + + f f +
0.0 0.1 0.0 1.0 0.0 0.0 0.0 0.1
AND INTERFERON-T (cpm
SYNTHESIS
X 10m3 + SD)
Day 0.1 2.9 0.7 24.4 0.1 0.1 0.1 9.6
6 + + -t 2 +* f f
0.0 0.3 0.1 1.1 0.0 0.0 0.0 0.7
Note. Conditions of induction of IFN-y synthesis recorded on Tables I and II were suboptimal. with 1 rig/ml PMA and 5 pg/ml Con A resulted in the synthesis of 21.1 U/ml of IFN--y by Day a IL 2, 1000 U/ml; Con A, 5 rg/ml; Dex, 1O-6 M. ‘Determined on Day 2 and expressed as NIH units.
IFN-y (U/ml)
*
0.6 0.7 3.1 8.0 0.6 0.7 1.1 2.7 Induction 2.
EXPRESSION
OF TABLE
EFFECT
OF ANTI-TAC
PROLIFERATION ANTIGEN
1” 0 30 100 1ooo
2 RECEPTORS
III
ON INTERLEUKIN
Z-INDUCED
AND THE EXPRESSION OF TAC BY HUMAN THYMOCYTES
[‘HvdR (cpm Additions (U/ml IL2)
INTERLEUKIN
incorporation X lo-’ f SD) +anti-Tat
-anti-Tat
Tat+
cells
Total
Bright
1.9 2.6 2.8 1.7
0.8 1.7 1.7 1.7
Exp
2’ 0 100 1000
1.0 3.8 4.8 11.1
f 0.1 + 0.4 CL 0.3 k 0.7
1.1 + 0.2 1.5 + 0.1 7.9 + 0.4
0.1 + 0 3.0 f 0.6 15.8 f 2.7
0.1 * 0.0 0.5 A 0.1
Exp
-
a Tat+ cells were determined by flow cytofluorometry on Day 2. [8H]TdR incorporation was determined on Day 4. Anti-Tat ascites (diluted lo-‘) was added on Day 0. ” Anti-Tat ascites (diluted lo-‘) was added on Day 0. Thymocytes were harvested on Day 5 and Tat+ cells were determined by staining with immunoperoxidase. ‘Faintly stained.
at least 4 days when cultured with IL 2 and that the increase in the proliferative activity of thymocytes induced by IL 2 was dose-dependent and could be inhibited by anti-Tat. Although thymocytes proliferated, the number of Tat+ thymocytes was 3% on Day 2 (Exp. 1) and did not increase within 5 days (Exp. 2). Nonetheless anti-Tat was effective in inhibiting IL 2-induced proliferation. In similar experiments carried out on thymocytes obtained from different donors, the number of Tat+ cells remained low for the first 7 days and the intensity of the fluorescence of these Tat+ cells was low. Following 15 days, 10% of thymocytes cultured expressed Tat antigen and they became brightly fluorescent (data not shown). DICUSSION
The data reported in this study provide evidence that purified recombinant IL 2 supports the growth and proliferative activity of thymocytes in culture. This growth is slow, but steady, and initially thymocytes grown in complete medium supplemented with IL 2 only displayed a
REQUIRES
INTERLEUKIN
2
459
very low number of receptor-bearing cells. The number of receptor-bearing cells increased slowly and almost imperceptibly in the first week of culture; following the second week of culture no more than 10% of all cells displayed IL 2 receptors. AntiTat added at the start of cultures inhibited thymocyte proliferation. The degree of inhibition was dependent upon the relationship between the concentrations of IL 2 and anti-Tat. If the concentration of IL 2 was high (1000 U/ml) and that of antiTat was low (diluted 10W4), the proliferative rate was inhibited by only 29% whereas in cultures containing 100 U/ml IL 2 the proliferative rate was inhibited by 68% within 4 days. A lo-fold increase in concentration of anti-Tat (10m3) inhibited the proliferative activity almost completely, even in cultures containing 1000 U/ml IL 2. This relationship strongly suggests that the proliferation of thymocytes cultured in IL 2 is dependent upon IL 2 receptor-bearing cells. The very low number of thymocytes which displayed significant numbers of receptors during the first week of culture is puzzling and is at present under investigation in our laboratory. It would suggest that a very small percentage of thymocytes in culture responded to IL 2, and that the growth of these cells ultimately resulted in an increase in Tacf thymocytes. Alternatively, IL 2 could indeed induce IL 2 receptors in thymocytes which do not display these receptors initially. The initial lag phase in the expression of receptors raises the question whether the number of binding sites for anti-Tat may be below the threshold of detection. During the initial period of culture, therefore, the number of receptors recognized by anti-Tat appears low relative to the proliferative rate. Other investigators have reported that IL 2 augmented the activity of human natural killer cells (NK) in vitro. This effect of IL 2 could not be inhibited by anti-Tat, but was susceptible to inhibition by antibodies to IFN--y (11). These findings imply that IL 2 can induce IFN-7 synthesis by large granular lymphocytes and that this induction is not mediated by
460
REEM
IL 2 receptors which are recognized by anti-Tat. The present study demonstrates that purified recombinant IL 2, like highly purified natural IL 2, augments the expression of IL 2 receptors by thymocytes activated with Con A and that it amplifies the synthesis of IFN-7 before thymocytes respond to the proliferative effect of IL 2 (4). Mature T cells, both human and murine, can be induced with IL 2 to synthesize IFN-y, even if cells are not activated by lectins, and IL 2 amplifies IFN synthesis by lectin-activated cells (12-15). The population of human thymocytes is quite hetereogenous (16), and it is conceivable that subpopulations of thymocytes maintained in long-term culture would acquire more of the characteristics of mature T lymphocytes. We have previously reported the inhibitory effect of dexamethasone on IFN--y synthesis, and we and others have found that dexamethasone inhibited the synthesis of IL 2 (17-20) and the expression of Tat antigen (4, 5). Dexamethasone also inhibits the synthesis of IL 2 and IFN-y mRNA (21). The inhibitory effect of dexamethasone on human thymocytes, however, cannot be ascribed exclusively to its effect on IL 2. Our data in this study indicate that dexamethasone inhibits the early IL 2-independent phase of proliferation, and depresses total protein synthesis during that time. This effect was not reversed by IL 2, and therefore is independent of IL 2. The inhibitory effect on the early, IL 2-independent proliferation of thymocytes is comparable to that observed on the activation of B cells by hydrocortisone. Hydrocortisone has been reported to inhibit the activation of tonsillar B cells, but had no effect on the proliferative response of preactivated B cells (22). Other investigators have shown that dexamethasone inhibited the proliferation of lectin-activated peripheral blood lymphocytes by arresting the cell cycle in G1, and that this effect on the lectininduced proliferative response could be reversed by IL 2 (23). This inhibitory effect on the lectin-induced proliferative response of mature T lymphocytes, which,
AND YEH
unlike thymocytes, do not proliferate spontaneously, but require activation by lectins or antigens and are dependent upon IL 2 in order to proliferate, resembles that on thymocytes which have become dependent on IL 2 for growth in vitro. Our data clearly demonstrate that IL 2 can reverse the antiproliferative effect of dexamethasone on thymocytes which have lost their ability to proliferate spontaneously. In conclusion, the findings reported in this study provide evidence that IL 2 is required for the optimal expression of IL 2 receptors by human thymocytes. In the absence of IL 2, Tat antigen is not expressed and thymocytes cease to grow in vitro. The observation that thymocytes can be induced by IL 2 to proliferate and grow in vitro without prior activation by lectin is of interest, and the mechanism by which IL 2 exerts this effect requires further investigation. ACKNOWLEDGMENTS This work was supported by NIH Grant RO 1 CA 33653-OlAl. We thank Dr. T. A. Waldmann for his gift of anti-Tat and Dr. J. J. Farrar (Hoffman-La Roche) for recombinant IL 2; Karen L. Davis for her help in some of the experiments, Dr. R. Basch for the use of the cytofluorograph; Betty L. Goon for her able technical assistance; and Julia Cohen for her assistance in preparing this manuscript. REFERENCES 1. REEM, 2. 3.
G. H., COOK, L. A., AND VILCEK, J. (1983) science (Washington, D. C) 221, 63. REEM, G. H., COOK, L. A., AND PALLADINO, M. A. (1984) J. Biol Resp. Mod 3, 195. YEH, N.-H., DIPRE, M., AND REEM, G. H. (1984)
Thymes 6.255. 4. REEM, G. H., AND YEH, N.-H. (1985) J. Immunol 134, 953. 5. REEM, G. H., AND YEH, N.-H. (1984) Science (Washington, D. C) 225,429. K., ANDREEFF, M., PLATZER, E., HOLLOWAY, K., RUBIN, B. Y., MOORE, M. A. S., AND MERTEILSMANN, R. (1984) J. Exp. Med 160,
6. WELTE,
1390. 7. TSUDO, M., UCHIYAMA, 8.
T., UCHINO, H., AND YODOI, J. (1983) Blood 61. 1014. DEPPER, J. M., LEONARD, W. J., KRONKE, M., WALDMANN, T. A., AND GREENE, W. C. (1984) J. Immunol 133, 1691.
EXPRESSION
OF
INTERLEUKIN
2 RECEPTORS
9. TSUDO, M., UCHIYAMA, T., TAKATSUKI, K., UCHINO, H., AND YODOI, J. (1982) J. Immunol 129, 592, 10. GREENE, W. C., ROBB, R. J., DEPER, J. M., LEONARD, W. J., DRONILA, C., SVETLIK, P. B., WONGSTAAL, F., GALLO, R. C., AND WALDMANN, T. A. (1984) J. Immund 133, 1042. 11. ORTALDO, J. R., MASON, A. T., GERARD, J. P., HENDERSON, L. E., FARRAR, W., HOPKINS III, R. F., HERBERMAN, R. B., AND RABIN, H. (1984) J. Immunol. 133, 779. 12. FARRAR, W. L., JOHNSON, H. M., AND FARRAR, J. J. (1981) J. Immunol. 126, 1120. 13. YAMAMOTO, J. K., FARRAR, W. L., AND JOHNSON, H. M. (1982) Cell Immunol. 66, 333. 14. KASAHARA, T., HOOKS, J. J., DOUGHERTY, S. F., AND OPPENHEIM, J. J. (1983) J. Immunol 130, 1784. 15. PEARLSTEIN, K. T., PALLADINO, M. A., WELTE, K., AND VILCEK, J. (1983) Cell Immurwl 80, 1.
REQUIRES
INTERLEUKIN
2
461
16. GELIN, C., BOUMSELL, L., DAUSSET, J., AND BERNARD, A. (1984) Proc. Nat1 Aud Sci. USA 81, 4912. 1’7. LARSSON, E.-L. (1980) J. Immunol 124, 2828. 18. GILLIS, S., CRABTREE, G. R., AND SMITH, K. A. (1979) J. Immund 123,1624. 19. CRABTREE, G. R., GILLIS, S., SMITH, K. A., AND MUNCK, A. (1979) Arth Rheum, 22,1246. 20. SMITH, K. A., CRABTREE, G. R., GILLIS, S., AND MUNCK, A. (1980) in Hormones and Cancer (Iacobelli, S., ed.), pp. 125-134, Raven Press, New York. 21. ARYA, S. K., WONG-STAAL, F., AND GALLO, R. C. (1984) J. Immunol. 133,273. 22. BOWEN, D. L., AND FAUCI, A. S. (1984) J. Immurwl. 133,1885. 23. BETTENS, F., KRISTENSEN, F., WALKER, C., SCHWULERA, U., BONNARD, G. D., ANDDEWECK, A. L. (1984) J. Immunol. 132, 261.
Vol.
OF BIOCHEMISTRY
239, No. 2, June,
Modulation
AND
BIOPHYSICS
pp. 455-461,
1985
of the Expression of lnterleukin 2 Receptors Thymocytes by Recombinant lnterleukin 2’ GABRIELLE
Department
of Phamnacology, Received
H. REEM’ NQW
October
York
AND
NING-HSING
School
University
31, 1984, and in revised
of Medicine, form
of Human
YEH New
January
York,
NQW
York
10016
2, 1985
The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-y synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-y, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-y was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with antiTat remained low throughout the first week of culture and interferon-y synthesis was not observed. Nonetheless, interleukin Z-induced proliferation was inhibited by antiTat on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-y synthesis, and for the growth of thymocytes in vitro. 0 1985 Academic press, I~C.
The expression of the receptor for interleukin 2 [T-cell growth factor (IL 2)13 is important for the proliferation of human T cells and of human thymocytes in vitro. Human thymocytes proliferate spontaneously in vivo and isolated human thymocytes continue to proliferate spontaneously in vitro. This spontaneous proliferation is gradually lost and, unless thymocytes are induced to differentiate i This paper is dedicated to Dr. B. L. Horecker on the occasion of his 70th birthday. ’ To whom correspondence should be addressed. 3 Abbreviations used: IL 2, interleukin 2; Con A, concanavalin A; PMA, phorbol myristate acetate; HTLV, human T-cell leukemia virus; IFN, interferon. 455
and stimulated to synthesize IL 2 or are cultured with IL 2, they rapidly lose the capacity to proliferate (1, 2). Studies recently carried out in our laboratory have shown that human thymocytes can be induced to express receptors for IL 2 in vitro, and that highly purified IL 2 enhanced the expresion of IL 2 receptors by thymocytes induced with Con A or with phorbol myristate acetate (PMA) (3, 4). We and others have reported a similar enhancement by IL 2 of the expression of IL 2 receptors by human T cells (5, 6). Other investigators have shown that IL 2 receptors are expressed on human Tcell leukemia virus (HTLV)-infected T cell lines (7, 8). The receptors of these cell 0003-9861/85 Copyright All rights
$3.00
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
456
REEM
lines are not subject to modulation of expression by anti-Tat, in contrast to those of T cells induced with lectin (7, 9), and synthesis of IL 2 is not necessarily linked to the expression of IL 2 receptors (10). Some T-cell lines derived from human leukemic cells can be induced by PMA to display IL 2 receptors and, in this respect, thay may resemble thymocytes cultured with PMA and IL 2 (4, 10). The present study addresses the effect of purified recombinant IL 2 on thymocyte proliferation, on the expression of Tat antigen (IL 2 receptors), and on lymphokine synthesis by human thymocytes. It provides evidence that cloned IL 2 allows thymocytes to proliferate after they have lost their capacity to proliferate spontaneously. However, in contrast to lectinactivated thymocytes, cultures of thymocytes supplemented with IL 2 in the absence of lectin display a small percentage of thymocytes which express Tat antigen at a low density. Cloned IL 2 was shown to augment the expression of IL 2 receptors on Con A-activated thymocytes, and to partially reverse the inhibitory effect of the glucocorticoid dexamethasone on IL 2-dependent proliferation. MATERIALS
AND
METHODS
Reagents. Purified recombinant Interleukin 2 (lo7 units/mg IL 2) was a generous gift from Dr. John J. Farrar, Hoffman La-Roche (Nutley, N. J.), antiTat antibody was from Dr. Thomas A. Waldmann, Metabolism Branch, National Institutes of Health (Bethesda, Md.). Concanavalin A was purchased from Miles Laboratories (Elkhart, Ind.) and dexamethasone from Sigma (St. Louis, MO.). Fluorescein-conjugated rabbit anti-mouse IgG (IgG fraction) was obtained from Cappel Laboratory (Cochranville, Pa.). Isolation and culture of thymocytes. Sections of human thymus were obtained during cardiac surgery of infants and children. Thymocytes were immediately isolated, washed in medium, and cultured in RPM1 1640 medium (Associated Biomedic System, Buffalo, N. Y.) containing 5% heat-inactivated fetal calf serum (KC Biological Laboratories, Lenexa, KS), 2 mM glutamine, and antibiotics (penicillin, 100 lJ/ ml; streptomycin, 100 pgg/ml; amphotericin B, 0.25 pg/ml; Grand Island Biologicals, Grand Island, N. Y.). Thymocytes were cultured in flat-bottomed microtiter plates (200 ~1) at a density of 6 X lo6 cells/ ml at 3’7°C in a humidified atmosphere containing 5% co*.
AND
YEH
Detection of Tat antigen Tat antigen was determined by indirect fluorescence by flow cytofluorometry (Cytofluorograph, Ortho Pharmaceuticals, Raritan, NJ), or by immunoperoxidase staining as described (4). Incorporation of [*H]thymidine. Cells were pulsed with 1 &i PH]thymidine (sp act, 6.7 CVmmol; New England Nuclear, Boston, Mass.) for the last 12 h of incubation, unless otherwise stated, and harvested on Whatman glass fiber filters (GM/CF, Clifton, N. J.) with a cell harvester (Hoeffer Scientific Instruments, San Francisco, Calif.), precipitated with 10% ice-cold trichloroacetic acid, and washed with 100% ice-cold methanol. The filters were dried and counted in a Beckman L-250 spectrometer (Beckman Instruments Inc., Mountainside, N. J.). Determination of inte&ron-7. Interferon-gamma (IFN-7) was determined by a solid-phase radioimmunoassay with monoclonal antibodies purchased from Centocor (Malvern, Pa.) as described (4). Incorporation of [“C]leucine [i4C&eucine (237 mCi/ mmol; New England Nuclear, Boston, Mass.), 0.2 &i per well, was added 5 h before cells were harvested, and thymocytes were cultured in leucine-free medium for that period of time. Proteins were precipitated on glass fiber filters, washed, and counted as described above. RESULTS
Human thymocytes isolated and placed in culture without delay proliferate spontaneously at a rapid rate. Following 48 h of culture (Day 2) this spontaneous rate of proliferation decreased to approximately one-fourth of the initial rate (Table I). At the end of the third day of culture, the proliferative rate was further reduced to 5% of the initial rate, and by Day 7 thymocytes cultured in complete medium had virtually ceased to proliferate. Less than 2% of thymocytes so cultured bound anti-Tat, and in less than 1% was the fluorescence of the Tat binding cells bright. If purified recombinant IL 2 was added to the medium, the decrease in the spontaneous proliferative rate observed on Day 2 was similar to that of thymocytes cultured without IL 2, but subsequently the proliferative rate slowly increased and thymocytes continued to proliferate at a slow rate. This proliferative rate could be maintained for longer periods of time if cultures were supplemented with IL 2. These findings indicate that during the first 2 days of culture, thymocyte prolif-
EXPRESSION
OF
INTERLEUKIN
2 RECEPTORS TABLE
REQUIRES
INTERUXJK~N
2
457
I
MODULATIONOFTHEEXPRESSIONOFTACANTIGENPROLIFERATIONAND SYNTHESISBYHUMANTHYMOCYTES
IFN--r
Tat+ cellsb [‘HJTdR Additions” None IL2 Con Con Dex Dex Dex Dex
A A + IL2 + IL2 + Con A + Con A + IL2
incorporation
(cpm
X 103) on Days
0
1
2
3
4
7
Total
104.3 86.6 81.7 78.9 54.7 30.1 32.3 36.2
102.3 101.2 97.8 91.8 2.9 1.9 4.9 4.3
23.5 21.9 23.0 47.5 0.1 0.2 0.3 2.4
5.2 7.4 19.5 106.2 0.1 0.2 0.2 11.9
1.2 3.9 6.5 194.4 0.1 0.1 0.1 35.5
0.1 5.6 0.8 1.2 0.1 0.1 0.1 10.0
1.9 2.8 17.6 21.1 1.0 6.3 8.4
Note. The data recorded on this Table were obtained ’ IL 2,100 U/ml; Con A, 5 rg/mh Dex, dexamethasone, b Determined on Day 2 by cytofluorometry. ’ Determined on Day 2 and expressed as NIH units.
eration is independent of IL 2, whereas late proliferation of thymocytes is IL 2dependent and can be maintained by the addition of IL 2 to cultures devoid of lectin. The kinetics of proliferation of thymocytes cultured with Con A initially resembled those of thymocytes cultured with IL 2 alone but, unlike thymocytes cultured with IL 2, they lost their capacity to proliferate within 7 days unless IL 2 was added to the cultures (data not shown). The proliferative activity of thymocytes cultured with both Con A and IL 2 exceeded that of cultures supplemented with Con A alone and reached a peak on Day 4, but it fell to 1% of the maximal rate by Day 7. This decline in proliferative activity reflected the rapid consumption of IL 2 in cultures grown with Con A and IL 2, and it too could be prevented by addition of IL 2 to the cultures at the time of maximal proliferation (data not shown). Dexamethasone exerted an inhibitory effect on both early and late proliferation of uninduced thymocytes, and the addition of IL 2 to thymocytes cultured with dexamethasone did not reverse the inhibition of the early IL 2-independent and the late
IFN-y” (U/ml)
Bright 0.8 1.7 5.9 10.5 1.0 3.6 4.6
from a thymic specimen from a single 1O-6 M was added on Day 0.
0.1 0.1 0.7 4.7 0.0 0.1 0.3 1.1 donor.
IL 2-dependent proliferation (Table I). On the second day of culture the viability of uninduced thymocytes was approximately 80% and the difference in viability between thymocytes cultured with or without dexamethasone was 15%. By Day 6, only 20% of thymocytes cultured with or without dexamethasone were viable (data not shown). Similarly, dexamethasone inhibited both the early and the late proliferative activity of thymocytes cultured with Con A; however, the inhibitory effect on Con A-induced, IL 2-dependent proliferation could be partially overcome by IL 2 (Table I). Cultures could be rescued and continued to grow provided they were supplied with the appropriate factors. Despite its marked inhibitory effect on the proliferative activity, dexamethasone decreased the viability of these cultures by less than 10% during the early proliferative phase. Even in cultures supplemented with IL 2 it caused a 50% decrease in viability within 6 days (data not shown). These cultures, however, regained their proliferative activity in the presence of IL 2 (Table I). A striking difference was observed in the expression of the number of thymocytes expressing Tat antigen, depending
458
REEM
AND
on the conditions of culture. Only 2% of thymocytes cultured with IL 2 alone expressed Tat antigen, while 17.6% of thymocytes cultured with Con A expressed Tat antigen on the second day of culture, before thymocytes had become dependent on IL 2 for growth. Addition of IL 2 to Con A-stimulated cultures increased the number of Tat antigen-bearing cells, as well as the density of receptors on the cell surface. This was demonstrated by the increase in the number of intensely fluorescent cells (5.9 to 10.5%). Dexamethasone decreased the number of receptorbearing cells in cultures supplemented with Con A, and the addition of IL 2 to these cultures resulted in a very moderate increase in the number of thymocytes expressing Tat. IFN-y synthesis was not induced by IL 2, but IL 2 augmented Con A-induced IFN--r synthesis more than sixfold (Table I). Dexamethasone inhibited Con A-induced 1FN-y synthesis by 57%, after 2 days of culture, at a time when spontaneous thymocyte proliferation was inhibited by dexamethasone and this inhibition was reversed by IL 2. In order to investigate the effect of IL 2 and dexamethasone on protein synthesis of spontaneously proliferating thymocytes and of thymocytes dependent on IL 2, the TABLE EFFECT
OF INTERLEUKIN
2 AND DEXAMETHASONE
YEH
incorporation of [14C]leucine into total protein was measured. The rate of protein synthesis of thymocytes cultured in complete medium decreased with time (Table III). This decrease was reversed by IL 2; in fact, protein synthesis on Day 6 of cultures supplemented with IL 2 exceeded that of the initial rate of protein synthesis by thymocytes cultured either with or without IL 2. IL 2 also increased protein synthesis of Con A-activated thymocytes. The increase in the rate of protein synthesis became apparent on the third day of culture and it was sustained throughout the period of culture. In cultures supplemented with Con A and IL 2 it exceeded that of cultures supplemented with Con A alone. IL 2 reversed the inhibitory effect of dexamethasone on protein synthesis by Con A-activated thymocytes during the late phase of the culture period, when thymocytes had developed dependence on IL 2. The overall effect of IL 2 on protein synthesis resembled that on the proliferative activity and became apparent when thymocytes required IL 2 for growth. The effect of IL 2 on IFN synthesis presented on Table II was consistent with that recorded on Table I. The data presented on Table III demonstrate that thymocytes proliferated for II ON PROTEIN
[i4CjLeucine
Additions” None IL2 Con Con Dex Dex Dex Dex
A A + IL2 + IL2 + Con A + Con A + IL2
Day 1.8 1.9 3.2 3.1 0.2 0.2 0.8 0.8
-t * ++ k -+ * f
1 0.1 0.1 0.3 0.3 0.0 0.0 0.1 0.0
incorporation
Day 0.4 0.8 1.4 10.7 0.1 0.1 0.1 1.4
3 k f + + + f f +
0.0 0.1 0.0 1.0 0.0 0.0 0.0 0.1
AND INTERFERON-T (cpm
SYNTHESIS
X 10m3 + SD)
Day 0.1 2.9 0.7 24.4 0.1 0.1 0.1 9.6
6 + + -t 2 +* f f
0.0 0.3 0.1 1.1 0.0 0.0 0.0 0.7
Note. Conditions of induction of IFN-y synthesis recorded on Tables I and II were suboptimal. with 1 rig/ml PMA and 5 pg/ml Con A resulted in the synthesis of 21.1 U/ml of IFN--y by Day a IL 2, 1000 U/ml; Con A, 5 rg/ml; Dex, 1O-6 M. ‘Determined on Day 2 and expressed as NIH units.
IFN-y (U/ml)
*
0.6 0.7 3.1 8.0 0.6 0.7 1.1 2.7 Induction 2.
EXPRESSION
OF TABLE
EFFECT
OF ANTI-TAC
PROLIFERATION ANTIGEN
1” 0 30 100 1ooo
2 RECEPTORS
III
ON INTERLEUKIN
Z-INDUCED
AND THE EXPRESSION OF TAC BY HUMAN THYMOCYTES
[‘HvdR (cpm Additions (U/ml IL2)
INTERLEUKIN
incorporation X lo-’ f SD) +anti-Tat
-anti-Tat
Tat+
cells
Total
Bright
1.9 2.6 2.8 1.7
0.8 1.7 1.7 1.7
Exp
2’ 0 100 1000
1.0 3.8 4.8 11.1
f 0.1 + 0.4 CL 0.3 k 0.7
1.1 + 0.2 1.5 + 0.1 7.9 + 0.4
0.1 + 0 3.0 f 0.6 15.8 f 2.7
0.1 * 0.0 0.5 A 0.1
Exp
-
a Tat+ cells were determined by flow cytofluorometry on Day 2. [8H]TdR incorporation was determined on Day 4. Anti-Tat ascites (diluted lo-‘) was added on Day 0. ” Anti-Tat ascites (diluted lo-‘) was added on Day 0. Thymocytes were harvested on Day 5 and Tat+ cells were determined by staining with immunoperoxidase. ‘Faintly stained.
at least 4 days when cultured with IL 2 and that the increase in the proliferative activity of thymocytes induced by IL 2 was dose-dependent and could be inhibited by anti-Tat. Although thymocytes proliferated, the number of Tat+ thymocytes was 3% on Day 2 (Exp. 1) and did not increase within 5 days (Exp. 2). Nonetheless anti-Tat was effective in inhibiting IL 2-induced proliferation. In similar experiments carried out on thymocytes obtained from different donors, the number of Tat+ cells remained low for the first 7 days and the intensity of the fluorescence of these Tat+ cells was low. Following 15 days, 10% of thymocytes cultured expressed Tat antigen and they became brightly fluorescent (data not shown). DICUSSION
The data reported in this study provide evidence that purified recombinant IL 2 supports the growth and proliferative activity of thymocytes in culture. This growth is slow, but steady, and initially thymocytes grown in complete medium supplemented with IL 2 only displayed a
REQUIRES
INTERLEUKIN
2
459
very low number of receptor-bearing cells. The number of receptor-bearing cells increased slowly and almost imperceptibly in the first week of culture; following the second week of culture no more than 10% of all cells displayed IL 2 receptors. AntiTat added at the start of cultures inhibited thymocyte proliferation. The degree of inhibition was dependent upon the relationship between the concentrations of IL 2 and anti-Tat. If the concentration of IL 2 was high (1000 U/ml) and that of antiTat was low (diluted 10W4), the proliferative rate was inhibited by only 29% whereas in cultures containing 100 U/ml IL 2 the proliferative rate was inhibited by 68% within 4 days. A lo-fold increase in concentration of anti-Tat (10m3) inhibited the proliferative activity almost completely, even in cultures containing 1000 U/ml IL 2. This relationship strongly suggests that the proliferation of thymocytes cultured in IL 2 is dependent upon IL 2 receptor-bearing cells. The very low number of thymocytes which displayed significant numbers of receptors during the first week of culture is puzzling and is at present under investigation in our laboratory. It would suggest that a very small percentage of thymocytes in culture responded to IL 2, and that the growth of these cells ultimately resulted in an increase in Tacf thymocytes. Alternatively, IL 2 could indeed induce IL 2 receptors in thymocytes which do not display these receptors initially. The initial lag phase in the expression of receptors raises the question whether the number of binding sites for anti-Tat may be below the threshold of detection. During the initial period of culture, therefore, the number of receptors recognized by anti-Tat appears low relative to the proliferative rate. Other investigators have reported that IL 2 augmented the activity of human natural killer cells (NK) in vitro. This effect of IL 2 could not be inhibited by anti-Tat, but was susceptible to inhibition by antibodies to IFN--y (11). These findings imply that IL 2 can induce IFN-7 synthesis by large granular lymphocytes and that this induction is not mediated by
460
REEM
IL 2 receptors which are recognized by anti-Tat. The present study demonstrates that purified recombinant IL 2, like highly purified natural IL 2, augments the expression of IL 2 receptors by thymocytes activated with Con A and that it amplifies the synthesis of IFN-7 before thymocytes respond to the proliferative effect of IL 2 (4). Mature T cells, both human and murine, can be induced with IL 2 to synthesize IFN-y, even if cells are not activated by lectins, and IL 2 amplifies IFN synthesis by lectin-activated cells (12-15). The population of human thymocytes is quite hetereogenous (16), and it is conceivable that subpopulations of thymocytes maintained in long-term culture would acquire more of the characteristics of mature T lymphocytes. We have previously reported the inhibitory effect of dexamethasone on IFN--y synthesis, and we and others have found that dexamethasone inhibited the synthesis of IL 2 (17-20) and the expression of Tat antigen (4, 5). Dexamethasone also inhibits the synthesis of IL 2 and IFN-y mRNA (21). The inhibitory effect of dexamethasone on human thymocytes, however, cannot be ascribed exclusively to its effect on IL 2. Our data in this study indicate that dexamethasone inhibits the early IL 2-independent phase of proliferation, and depresses total protein synthesis during that time. This effect was not reversed by IL 2, and therefore is independent of IL 2. The inhibitory effect on the early, IL 2-independent proliferation of thymocytes is comparable to that observed on the activation of B cells by hydrocortisone. Hydrocortisone has been reported to inhibit the activation of tonsillar B cells, but had no effect on the proliferative response of preactivated B cells (22). Other investigators have shown that dexamethasone inhibited the proliferation of lectin-activated peripheral blood lymphocytes by arresting the cell cycle in G1, and that this effect on the lectininduced proliferative response could be reversed by IL 2 (23). This inhibitory effect on the lectin-induced proliferative response of mature T lymphocytes, which,
AND YEH
unlike thymocytes, do not proliferate spontaneously, but require activation by lectins or antigens and are dependent upon IL 2 in order to proliferate, resembles that on thymocytes which have become dependent on IL 2 for growth in vitro. Our data clearly demonstrate that IL 2 can reverse the antiproliferative effect of dexamethasone on thymocytes which have lost their ability to proliferate spontaneously. In conclusion, the findings reported in this study provide evidence that IL 2 is required for the optimal expression of IL 2 receptors by human thymocytes. In the absence of IL 2, Tat antigen is not expressed and thymocytes cease to grow in vitro. The observation that thymocytes can be induced by IL 2 to proliferate and grow in vitro without prior activation by lectin is of interest, and the mechanism by which IL 2 exerts this effect requires further investigation. ACKNOWLEDGMENTS This work was supported by NIH Grant RO 1 CA 33653-OlAl. We thank Dr. T. A. Waldmann for his gift of anti-Tat and Dr. J. J. Farrar (Hoffman-La Roche) for recombinant IL 2; Karen L. Davis for her help in some of the experiments, Dr. R. Basch for the use of the cytofluorograph; Betty L. Goon for her able technical assistance; and Julia Cohen for her assistance in preparing this manuscript. REFERENCES 1. REEM, 2. 3.
G. H., COOK, L. A., AND VILCEK, J. (1983) science (Washington, D. C) 221, 63. REEM, G. H., COOK, L. A., AND PALLADINO, M. A. (1984) J. Biol Resp. Mod 3, 195. YEH, N.-H., DIPRE, M., AND REEM, G. H. (1984)
Thymes 6.255. 4. REEM, G. H., AND YEH, N.-H. (1985) J. Immunol 134, 953. 5. REEM, G. H., AND YEH, N.-H. (1984) Science (Washington, D. C) 225,429. K., ANDREEFF, M., PLATZER, E., HOLLOWAY, K., RUBIN, B. Y., MOORE, M. A. S., AND MERTEILSMANN, R. (1984) J. Exp. Med 160,
6. WELTE,
1390. 7. TSUDO, M., UCHIYAMA, 8.
T., UCHINO, H., AND YODOI, J. (1983) Blood 61. 1014. DEPPER, J. M., LEONARD, W. J., KRONKE, M., WALDMANN, T. A., AND GREENE, W. C. (1984) J. Immunol 133, 1691.
EXPRESSION
OF
INTERLEUKIN
2 RECEPTORS
9. TSUDO, M., UCHIYAMA, T., TAKATSUKI, K., UCHINO, H., AND YODOI, J. (1982) J. Immunol 129, 592, 10. GREENE, W. C., ROBB, R. J., DEPER, J. M., LEONARD, W. J., DRONILA, C., SVETLIK, P. B., WONGSTAAL, F., GALLO, R. C., AND WALDMANN, T. A. (1984) J. Immund 133, 1042. 11. ORTALDO, J. R., MASON, A. T., GERARD, J. P., HENDERSON, L. E., FARRAR, W., HOPKINS III, R. F., HERBERMAN, R. B., AND RABIN, H. (1984) J. Immunol. 133, 779. 12. FARRAR, W. L., JOHNSON, H. M., AND FARRAR, J. J. (1981) J. Immunol. 126, 1120. 13. YAMAMOTO, J. K., FARRAR, W. L., AND JOHNSON, H. M. (1982) Cell Immunol. 66, 333. 14. KASAHARA, T., HOOKS, J. J., DOUGHERTY, S. F., AND OPPENHEIM, J. J. (1983) J. Immunol 130, 1784. 15. PEARLSTEIN, K. T., PALLADINO, M. A., WELTE, K., AND VILCEK, J. (1983) Cell Immurwl 80, 1.
REQUIRES
INTERLEUKIN
2
461
16. GELIN, C., BOUMSELL, L., DAUSSET, J., AND BERNARD, A. (1984) Proc. Nat1 Aud Sci. USA 81, 4912. 1’7. LARSSON, E.-L. (1980) J. Immunol 124, 2828. 18. GILLIS, S., CRABTREE, G. R., AND SMITH, K. A. (1979) J. Immund 123,1624. 19. CRABTREE, G. R., GILLIS, S., SMITH, K. A., AND MUNCK, A. (1979) Arth Rheum, 22,1246. 20. SMITH, K. A., CRABTREE, G. R., GILLIS, S., AND MUNCK, A. (1980) in Hormones and Cancer (Iacobelli, S., ed.), pp. 125-134, Raven Press, New York. 21. ARYA, S. K., WONG-STAAL, F., AND GALLO, R. C. (1984) J. Immunol. 133,273. 22. BOWEN, D. L., AND FAUCI, A. S. (1984) J. Immurwl. 133,1885. 23. BETTENS, F., KRISTENSEN, F., WALKER, C., SCHWULERA, U., BONNARD, G. D., ANDDEWECK, A. L. (1984) J. Immunol. 132, 261.