receptor reinduction were evaluated by restimulating cells with IL-2 in the presence of 30-150 nM actinomycin D. (Sigma) or 5-10 ,uM cycloheximide (Sigma).
Proc. Nati. Acad. Sci. USA
Vol. 82, pp. 4230-4234, June 1985 Immunology
Interleukin 2 (IL-2) augments transcription of the IL-2 receptor gene (in vitro transcription/lymphokine gene expression)
JOEL M. DEPPER, WARREN J. LEONARD, CYNTHIA DROGULA*, MARTIN KRONKE, THOMAS A. WALDMANN, AND WARNER C. GREENE Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205
Communicated by DeWitt Stetten, Jr., March 22, 1985
ABSTRACT We demonstrate that purified interleukin 2 (IL-2) can directly upregulate IL-2 receptor expression on
medium consisting of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (GIBCO)/2 mM L-glutamine/50 units of penicillin per ml/50 pug of streptomycin per ml, in the presence of E-PHA at 0.5 ,ug/ml (Burroughs Wellcome, Research Triangle Park, NC). After 3 days, cells were washed twice, and thereafter were maintained at 105_106 cells per ml in complete medium supplemented with 10% delectinated partially purified IL-2 (Cellular Products, Buffalo, NY). Cells were reactivated as described below after at least 7 days in culture and, where indicated, after a resting period of 12-72 hr. Cells were rested by thorough washing and reculturing in complete medium without added IL-2. Receptor Upregulation and Reagents. Two preparations of IL-2 were used in these experiments. Purified Jurkat-derived IL-2 (a generous gift of Richard Robb, E. I. DuPont de Nemours) was prepared as described (2). Purified recombinant IL-2 (lot LP214, a generous gift of Kirsten Koths, Cetus Corporation, Emeryville, CA) was 95% pure by NaDodSO4/ PAGE and reversed-phase HPLC (7). The specific bioactivity of the two preparations was essentially identical. The requirements for new RNA and protein synthesis in receptor reinduction were evaluated by restimulating cells with IL-2 in the presence of 30-150 nM actinomycin D (Sigma) or 5-10 ,uM cycloheximide (Sigma). Binding Assay. IL-2 receptor number was evaluated with monoclonal anti-Tac (8, 9), which recognizes both high- and low-affinity IL-2 binding sites (10). Purification, radiolabeling by reductive methylation, and binding with 3H-labeled antiTac were as described (3, 11). Briefly, cells were washed, and triplicate aliquots of 106 viable cells were incubated with 100,000 dpm of 3H-labeled anti-Tac (="19 ng). Cell-associated and free radiolabeled anti-Tac were separated by pelleting the cells through a 1 M sucrose cushion at 12,000 x g as described (3). Cell pellets were resuspended in a balanced salt solution and transferred to glass vials for liquid scintillation counting. Triplicate aliquots of the same cells were incubated with 3H-labeled anti-Tac in the presence of a 1000-fold excess of unlabeled antibody and were similarly counted. This value was subtracted from total counts bound to determine specific binding. Analysis of Specific RNA Levels by RNA Blotting. Aliquots of 108 7-day-old PHA blasts were cultured for 0-72 hr at 2 x 106 cells per ml in complete medium containing 3.8 nM Jurkat IL-2. At various times, cells were pelleted and flash-frozen in liquid nitrogen. Total RNA was isolated by the method of Chirgwin et al. (12) and RNA concentration and purity were determined by measurement of absorbance at 260 and 280 nm. Either 10 or 20 Ag of RNA was electrophoresed on
phytohemagglutinin-activated T lymphocytes maintained in culture until IL-2 receptor expression had markedly declined. The IL-2-induced increase in IL-2 receptor number is maximal within 12 hr, requires new RNA and protein synthesis, and is mediated by an interaction of ligand with the high-affinity receptors for IL-2. IL-2 stimulation results in increased accumulation of IL-2 receptor mRNA within 4 hr, while an increase in IL-2 receptor gene transcription is detected within 30 min in isolated nuclei. In addition, IL-2 incubation results in increased amounts of c-myc and transferrin receptor mRNA, but it does not augment levels of mRNA encoding the fi chain of the T-cell receptor for antigen. These results demonstrate that IL-2 can directly upregulate transcription and expression of its own receptor and, therefore, indicate that IL-2 may regulate IL-2-dependent immune responses, in part, by influencing the expression of IL-2 receptors. As T lymphocytes are activated in normal immune responses, or after mitogen or antigen stimulation in vitro, they are induced to secrete a 15,500-Da glycoprotein hormone, interleukin 2 (IL-2; also referred to as T-cell growth factor), and express specific membrane receptors for this lymphokine (1, 2). Resting peripheral blood lymphocytes (PBL) do not express receptors for IL-2. These receptors appear within 4-8 hr after phytohemagglutinin (PHA) stimulation, reach peak expression at 2-3 days, and then decline over a period of 7-12 days to levels 5-20% of that maximally expressed (3). Restimulation of cells that have lost the majority of IL-2 receptors with either lectin or appropriate antigen results in an increase in the number of IL-2 receptors expressed and in cellular proliferation (3-5). Thus, IL-2 receptor expression and amount of IL-2 produced may both contribute to regulation of T-cell immune responses. Using dexamethasone to inhibit IL-2 production during lectin activation of human lymphocytes, Reem and Yeh recently observed that IL-2 itself may be necessary for optimal IL-2 receptor expression (6). While evaluating the requirements for reexpression of IL-2 receptors on cultured lymphoblasts, we observed that purified IL-2 alone increased the number of IL-2 receptors expressed. In the present study, we characterize the effects of purified IL-2 on IL-2 receptor expression in such a restimulation system.
MATERIALS AND METHODS Cells. Human peripheral blood mononuclear cells were obtained by Ficoll-diatrizoate density gradient centrifugation of heparinized venous blood. Cells were cultured in complete
Abbreviations: IL-2, interleukin 2; PBL, peripheral blood lymphocytes; PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
acetate.
*Present address: Department of Biochemistry, George Washington University Medical School, Washington, DC 20037.
4230
Immunology: Depper et A formaldehyde gels, transferred to Gene-Screen Plus nylon membranes (New England Nuclear), baked at 80'C in vacuo for 2 hr, and hybridized with various DNA probes radiolabeled with [32P]dCTP by the random primer method of Feinberg et al. (13). The IL-2 receptor cDNA used has been described (14), the c-myc DNA was a generous gift of Kathleen Kelley (National Cancer Institute), transferrin receptor cDNA was the generous gift of Claudio Schneider (European Molecular Biology Laboratory) and Melvyn Greaves (Chester Beatty Laboratories, London), and the f3 chain of the murine T-cell antigen receptor cDNA was a generous gift of Mark Davis (Stanford University). Hybridizations were performed in a solution containing 5 x SSPE (1 x SSPE = 180 mM NaCl/10 mM NaH2PO4/1 mM EDTA), 1% NaDodSO4, 50% formamide, and 10% dextran SO4. Filters were washed twice in 2x SSPE/0. 1% NaDodSO4 at room temperature, followed by two washes in O.1x SSPE/0.1% NaDodSO4 at 60'C, and they were autoradiographed at -70'C with intensifying screens (DuPont). In Vitro Nuclear Transcription Assay. In vitro transcription with isolated nuclei was performed according to the method ofMcKnightandPalmiter(15)asdescribed(16). The [32P]UTPlabeled nascent RNA transcripts obtained were hybridized to an excess of specific denatured cDNA immobilized on nitrocellulose filters (16) for 72 hr at 42°C in 40% formamide/4x NaCl/Cit (lx NaCl/Cit = 0.15 M NaCl/0.015 M sodium citrate, pH 7)/5 mM EDTA/0.4% NaDodSO4 lx Denhardt's solution (0.02% bovine serum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone)/150 ,g of yeast tRNA per ml. Filters were washed twice in 2x NaClI/Cit/0.1% NaDodSO4 and twice in O.1x NaClI/Cit/0.1% NaDodSO4 at 50°C. Filters were exposed at -70°C to Kodak XAR film using intensifying screens.
RESULTS Purified IL-2 Upregulates Expression of IL-2 Receptors on Long-Term Cultured PHA Lymphoblasts. Fig. 1 summarizes the results of 12 experiments in which cells activated with PHA and then maintained in IL-2-containing medium for 7-15 days were incubated with purified Jurkat-derived IL-2, 15-75 ng/ml (1-5 nM) for 12-24 hr. As previously reported (3, 4), after an initial lectin-induced increase in IL-2 receptor number, these cultured cells lose 80-95% of their receptors for IL-2. In each case, exposure to purified IL-2 augmented IL-2 receptor number 2- to 5-fold, as measured by 3H-labeled anti-Tac binding. Similar increases were seen whether or not the cells were exhaustively washed and precultured in the absence of IL-2 for 24-72 hr. The IL-2-induced increase in receptor number was also observed with purified recombinant IL-2 and was independent of the number of receptors present on the cells prior to IL-2 stimulation. These data suggest that purified IL-2 alone is capable of upregulating the number of IL-2 receptors displayed on T cells previously activated by lectin and maintained in IL-2-containing conditioned media. Time Course of IL-2-Mediated IL-2 Receptor Upregulation Compared with Upregulation Induced by PHA and Phorbol 12-Myristate 13-Acetate (PMA). To evaluate the kinetics of IL-2-induced IL-2 receptor upregulation, 10-day-old PHA blasts were rested in complete medium for 48 hr and then stimulated with purified Jurkat IL-2 (7.5 ng/ml, 0.5 nM), E-PHA (0.5 ,ug/ml), or PMA (50 ng/ml) (Fig. 2). IL-2-induced increases in IL-2 receptor number were maximal at 12 hr, after which time the number of receptors declined. PMA and PHA stimulation also resulted in increased IL-2 receptor number, which was maximal after 24 hr of incubation, as reported (3). Cells maintained in the absence of IL-2 expressed progressively fewer receptors with time in culture.
Proc. Natl. Acad. Sci. USA 82 (1985)
4231
m50 0
x
23.5 10 J -o
I I
0. 0
Medium
IL-2
FIG. 1. Purified IL-2 increases IL-2 receptor expression on PHA-activated lymphocytes. PBL activated with PHA were maintained in culture for 7-15 days until IL-2 receptor number had declined. Cells were resuspended in IL-2-free medium for 0-72 hr prior to incubation with purified Jurkat IL-2 (1-5 nM) for 12-24 hr. IL-2 receptor number was determined by incubation with 3H-labeled anti-Tac as described. Mean receptors per cell: unstimulated, 6910 + 1470; IL-2 incubation, 17,200 ± 3900.
IL-2-Mediated Upregulation of IL-2 Receptors Requires New RNA and Protein Synthesis. To investigate the possibility that the IL-2 effect was the result of the mobilization of cryptic preformed receptors to the cell surface, cells were incubated with IL-2 in the presence of actinomycin D or cycloheximide (Table 1). In three experiments, IL-2 alone resulted in a 2.5-fold increase in receptor expression, while no significant increase in receptor number occurred in cells stimulated with IL-2 in the presence of actinomycin D or cycloheximide. Thus, de novo RNA and protein synthesis
° 20 x
10o 5-
4.
0.
0.5
-2t4
0 4 8 12
24
48
Time after stimulant added, hr FIG. 2. Time course of IL-2-mediated upregulation of IL-2 receptor expression. PHA Iymphoblasts maintained in culture for 10 days were incubated in IL-2-free medium for 48 hr prior to restimulation with 0.5 ,ug of PHA (A), 50 ng of PMA per ml (e), or S nM IL-2 (o. At the indicated times, IL-2 receptor number was determined on triplicate aliquots of 106 viable cells with 3H-labeled anti-Tac. Binding to cells maintained in IL-2-free medium is also shown (S). Similar results were obtained in three additional experiments.
4232
Immunology: Depper et al.
appear to be required for IL-2 to upregulate expression of its own receptor. IL-2 Augments IL-2 Receptor mRNA Expression and IL-2 Receptor Gene Transcription. To more directly study the mechanism of IL-2-mediated receptor upregulation, we isolated total RNA from cells at various times after stimulation with IL-2. After size fractionation on formaldehyde gels and transfer to nylon membranes, the blots were probed with a radiolabeled IL-2 receptor cDNA probe (Fig. 3). As evaluated by densitometry, IL-2 receptor mRNA levels were increased E-nfold within 12 hr after IL-2 incubation, and they remained elevated for 72 hr. In additional experiments, IL-2 receptor mRNA levels declined at 48 and 72 hr. We next evaluated whether this upregulation was associated with the induction of other mRNAs known to be associated with lymphocyte activation. We hybridized the same blot with probes for c-myc and the transferrin receptor (Fig. 4). Specific mRNA for both c-myc and the transferrin receptor were similarly increased after IL-2 incubation. Temporally, the enhancement of transferrin receptor mRNA occurred after the increases in IL-2 receptor and c-myc mRNA levels. In contrast to these activation antigens, mRNA for the 1 chain of the T-cell-antigen receptor was not increased as a result of IL-2 stimulation (Fig. 4). Increased IL-2 receptor mRNA levels could reflect changes in mRNA synthesis, stability, processing, or transport. We performed nuclear run-off assays to investigate whether IL-2 augmented transcription of the IL-2 receptor gene. An increase in IL-2 receptor-specific nuclear mRNA transcripts was detectable 30 min after IL-2 incubation, peaked at 2-6 hr, and then declined (Fig. 5.) Thus, augmented IL-2 receptor mRNA levels were the result, at least in part, of IL-2-induced transcriptional activation of the IL-2 receptor gene. We cannot exclude additional IL-2 effects on post-transcriptional events involving IL-2 receptor mRNA. IL-2 Induction of IL-2 Receptor Formation Is Mediated Through an Interaction of IL-2 with the High-Affinity Receptor for IL-2. Robb et al. have recently demonstrated the presence of two affinity classes of receptors for IL-2 (10). In addition to the initially described high affinity IL-2 receptors (Kd = 5 x 10-12 M), a second and proportionally larger class of specific receptors is present (Kd = 10-9 M). To evaluate which class of receptors mediated IL-2-induced receptor upregulation, we incubated cells with various concentrations of either Jurkat or recombinant IL-2 and measured 3Hlabeled anti-Tac binding and [3H]thymidine incorporation after 12 and 36 hr of incubation, respectively (Fig. 6). Maximal DNA synthesis occurred when cells were incubated with 0.2 nM IL-2, with 50% of maximal thymidine incorporation at 10-70 pM IL-2, consistent with previous observations that IL-2 interaction with the high-affinity IL-2 receptor is responsible for the proliferative response mediated by Table 1. IL-2 upregulation of IL-2 receptors requires new RNA and protein synthesis IL-2 receptors per cell (mean ± SD) 1 with Cells incubated Exp. 2 Exp. 2609 ± 561 1791 ± 189 Medium alone 6130 ± 136 4951 ± 157 Purified IL-2 1789 ± 4 1716 ± 540 With actinomycin D 3006 ± 196 2154 ± 336 With cycloheximide Eight-day-old PHA blasts rested out of IL-2 for 48 hr (Exp. 1) or 14-day-old PHA blasts rested for 10 hr (Exp. 2) were incubated with purified Jurkat IL-2 (5 nM) in the presence or absence of actinomycin D (200 ng/ml) or cycloheximide (10 uM), and 3HI-labeled anti-Tac binding was measured 12 hr later. Similar results were obtained in three additional experiments.
Proc. Natl. Acad. Sci. USA 82 (1985) 28S -
18s-
0
24
12
48
72
FIG. 3. Time course of IL-2-mediated upregulation of IL-2 receptor mRNA. Total RNA (20 jig) from 7-day-old PHA blasts restimulated with 3.8 nM IL-2 for the indicated times was electrophoresed on formaldehyde gels, transferred to a nylon membrane, and hybridized with 32P-labeled IL-2 receptor cDNA. Both the 3500-base and 1600-base messages specific for the IL-2 receptor were increased U7-fold based on densitometric scanning. Slightly less RNA was loaded for the 24-hr sample as assessed by ethidium staining intensity of 28S and 18S ribosomal RNA. Numbers below lanes indicate hours of IL-2 exposure.
IL-2. Maximal increases in IL-2 receptor number were seen at -0.2 nM IL-2, with >50% of the maximal increase seen at 2-50 pM IL-2 (Fig. 6). Addition of quantities of IL-2 sufficient to saturate the low-affinity binding sites did not result in greater proliferation or IL-2 receptor upregulation. These results suggest that IL-2 receptor upregulation induced by IL-2 occurs via an interaction with the high-affinity class of IL-2 receptors. Two Signals May Be Required for IL-2-Induced Upregulation of Its Receptor. Purified IL-2 does not induce or upregulate IL-2 receptor expression on freshly isolated PBL. These cells require mitogen or antigen as a primary or "competence" signal (17), which, together with IL-2, results in optimal IL-2 receptor expression. The successful upregulation of IL-2 receptors by IL-2 alone in mitogen-activated
c-myc
Transferrin receptor
-chain of T-cell antigen receptor 0
12
24
48
72
FIG. 4. IL-2 upregulates c-myc and transferrin receptor mRNAs. The same blot shown in Fig. 3 was washed and reprobed with cDNA encoding c-myc and the 13 chain of the T-cell receptor for antigen. A separate blot, representing 10 ug per lane of the same RNAs, was probed with a transferrin receptor cDNA. c-myc expression is augmented to a similar degree and with similar kinetics to the IL-2 receptor. Increase in transferrin receptor expression is delayed and more transient. Expression of the /8 chain of the T-cell receptor for antigen is not augmented by IL-2 incubation. Numbers below lanes indicate hours after IL-2 activation of day-12 PHA lymphoblasts.
Immunology: Depper et al.
Proc. Natl. Acad. Sci. USA 82 (1985)
10.50
4233
28S -
;o
0.
-
2S
.Y.. .. C
10
18S-
41
1
0
0.5 2 6 12 Time after IL-2 stimulation, hr
0
24
FIG. 5. Kinetics of IL-2-induced transcriptional activation of the IL-2 receptor gene (a). An increase in transcription of the IL-2 receptor gene is detected within 30 mmn of IL-2 exposure, peaks between 2 and 6 hr, and then declines. HLA gene (a) transcription is constitutive and not significantly affected by IL-2 incubation. 0
lymphoblasts maintained in culture suggests that these cells are poised in an activated or competent state. This is evidenced by the expression of IL-2 receptors on a majority of cells (3), albeit _ at a level 4-fold increase in receptor number (data not shown). Thus, a submitogenic concentration of PHA is one such competence factor that can restore IL-2 responsiveness to cells that have become refractory to IL-2-induced upregulation. A synergistic interplay of IL-2 and small concentrations of PHA is also seen in cells that do remain responsive to IL-2 as a single signal (Fig. 7). While IL-2 and PHA each increased IL-2 receptor mRNA, the combination of the two signals resulted in a more than additive accumulation of IL-2 receptor mRNA. We would conclude that a second signal in addition to IL-2 appears to be required for optimal IL-2 receptor reexpression. We next investigated whether exposure of day-3 PHA lymphoblasts to large quantities of IL-2 could block the progressive decline in receptor number occurring from day 3 to day 10. Despite daily pulsing of such cells with saturating quantities (1 nM) of IL-2, IL-2 receptor number declined at a rate similar to that present in cells cultured under normal conditions.
DISCUSSION IL-2-dependent immune responses appear to be regulated by the amount of IL-2 secreted and by the number of cell-surface IL-2 receptors expressed. Reem and Yeh (6), using dexamethasone to block IL-2 secretion, and Welte et al. (18), using submitogenic concentrations of an anti-T3 monoclonal antibody, have suggested that IL-2 receptor expression is itself positively influenced by IL-2 secretion. We have taken advantage of the physiological decline in IL-2 receptor
4234
Immunology: Depper et A
expression in lectin-activated lymphoblasts maintained in culture to demonstrate that purified IL-2 alone can upregulate IL-2 receptor expression. IL-2 receptor upregulation was maximal after 12 hr and required new RNA and protein synthesis, thus excluding the possibility that mobilization of cryptic preformed receptors to the cell surface accounted for the increase in receptor number. Furthermore, RNA blots hybridized with an IL-2 receptor cDNA probe demonstrated increased IL-2 receptor mRNA accumulation after incubation in purified IL-2. Transcription assays performed with isolated nuclei further demonstrated that, at least in part, the increase in IL-2 receptor mRNA resulted from IL-2 activation of IL-2 receptor gene transcription. Upregulation of cell-surface receptors by specific ligands is certainly not without precedent in other experimental systems. For example, insulin induces a specific, reversible, and dose-dependent increase in high-affinity insulin receptors on rat chondrosarcoma cells (19). Manipulations that increase prolactin levels in hypophysectomized rats increased prolactin receptors on rat hepatocytes (20). Finally, receptors for the Fc region of IgE (21) and IgA (22) appear to be induced by incubation with their specific ligands. IL-2-mediated effects were not confined solely to induction of its own receptor. Both c-myc and transferrin receptor mRNA levels were increased after stimulation with IL-2, suggesting that IL-2 initiates a program of transcription of other genes involved in lymphocyte activation. Kelley et al. have previously shown an early increase in c-myc mRNA after mitogen activation of PBL (23). Neckers and Cossman have suggested that IL-2 receptor expression is required for transferrin receptor expression after lymphocyte activation (24). Consistent with this, the increase in transferrin receptor mRNA temporally occurred after the increase in IL-2 receptor mRNA. In contrast to these antigens selectively expressed during cell activation and growth, IL-2 did not induce increases in the expression of mRNA for the 83 chain of the T-cell-antigen receptor. Robb et al. have reported the presence of two affinity classes of IL-2 receptors (10), which are not distinguished by anti-Tac binding. While the growth-promoting effects of IL-2 appear to be mediated via the high-affinity receptors, the biological role of the more numerous low-affinity receptors in T- and B-cell activation remains undefined. We found that both the increase in IL-2 receptor number and cell proliferation were mediated by concentrations of IL-2 sufficient only to saturate the high-affinity class of receptors. Furthermore, addition of IL-2 in concentrations necessary to titrate the low-affinity receptors did not further augment receptor number or cellular proliferation. Thus, we would conclude that receptor reexpression is mediated by an interaction of IL-2 with its high-affinity receptor. IL-2 receptor number can be increased on activated lymphocytes maintained in culture by single unique signals, including reexposure to mitogen or antigen (3-5), PMA and other activators of protein kinase C (3), and IL-2. While IL-2 alone does not induce IL-2 receptor expression in freshly isolated lymphocytes, it appears to be required for optimal receptor expression when such cells are activated with PHA (6) or anti-T3 (18). Thus, unstimulated cells require two or more signals in combination in order to express IL-2 receptors. In previously activated cells, we observed a loss of responsiveness to IL-2 alone in some cell populations. The reconstitution of IL-2 responsiveness in these cells with small concentrations of PHA suggests that the continued presence of a competence signal, whether mitogen or antigen, may be required for IL-2 receptor upregulation by IL-2. This synergy of IL-2 and a second signal is further demonstrated by large increases in IL-2 receptor mRNA accumulation after stimulation with both IL-2 and submitogenic concentrations of PHA.
Proc. Natl. Acad. Sci. USA 82 (1985) If IL-2 can upregulate its own receptor, why do receptor numbers fall when cells are grown in the continuous presence of IL-2-containing media? IL-2 concentrations in the tissue culture media used were 30-100 pM on the day of feeding, and declined thereafter. Hypothesizing that the trough concentrations of IL-2 were insufficient to maintain optimal IL-2 receptor number, we attempted to prevent the initial decline in cell-surface receptor expression by daily pulsing of freshly activated PBL with saturating concentrations of purified IL-2. However, cells treated in this manner continued to demonstrate a progressive decline in receptor number with time in culture. Thus, while IL-2 is capable of reinducing IL-2 receptor expression in cultured cells that have lost the majority of their surface receptors, this ligand cannot prevent the initial and physiological decline in receptor number that serves to terminate the T-cell proliferative response. We speculate that the initial decrease in IL-2 receptor number may reflect active but transient repression of receptor gene transcription. With the disappearance of this repression, IL-2-induced changes in receptor expression may emerge. Thus, IL-2 is required for optimal IL-2 receptor expression during the course of lymphocyte activation in the presence of lectin or antigen, and it can augment transcription of the IL-2 receptor gene in cultured cells maintained in a competent state of activation. However, IL-2 alone is insufficient to maintain a continuous state of maximal transcription of the IL-2 receptor gene. We would like to thank P. Svetlik and N. Peffer for excellent technical assistance. 1. Smith, K. A. (1980) Immunol. Rev. 51, 337-357. 2. Robb, R. J., Munck, A. & Smith, K. A. (1981) J. Exp. Med. 154, 1455-1474. 3. Depper, J. M., Leonard, W. J., Kronke, M., Noguchi, P., Cunningham, R., Waldmann, T. A. & Greene, W. C. (1984) J. Immunol. 133, 3054-3061. 4. Cantrell, D. A. & Smith, K. A. (1983) J. Exp. Med. 158, 1895-1911. 5. Hemler, M. D., Brenner, M. B., McLean, J. M. & Strominger, J. L. (1984) Proc. Natl. Acad. Sci. USA 81, 2172-2175. 6. Reem, G. & Yeh, N. H. (1984) Science 225, 429-430. 7. Wang, A., Lu, S.-D. & Mark, D. (1984) Science 224, 1431-1433. 8. Uchiyama, T. S., Broder, S. & Waldmann, T. A. (1981) J. Immunol. 126, 1393-1397. 9. Leonard, W. J., Depper, J. M., Uchiyama, T., Smith, K. A., Waldmann, T. A. & Greene, W. C. (1982) Nature (London) 300, 267-269. 10. Robb, R. J., Greene, W. C. & Rusk, C. M. (1984) J. Exp. Med. 160, 1126-1146. 11. Depper, J. M., Leonard, W. J., Kronke, M., Waldmann, T. A. & Greene, W. C. (1984) J. Immunol. 133, 1691-1695. 12. Chirgwin, J. M., Przybyla, A. E., Macdonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299. 13. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 14. Leonard, W. J., Depper, J. M., Crabtree, G. R., Rudikoff, S., Pumphrey, J., Robb, R. J., Kronke, M., Svetlik, P. B., Peffer, N. J., Waldmann, T. A. & Greene, W. C. (1984) Nature (London) 311, 626-631. 15. McKnight, G. S. & Palmiter, R. D. (1979) J. Biol. Chem. 254, 9050-9058. 16. Kronke, M., Leonard, W. J., Depper, J. M., Arya, S. K., Wong-Staal, F., Gallo, R. C., Waldmann, T. A. & Greene, W. C. (1984) Proc. Natl. Acad. Sci. USA 81, 5214-5218. 17. Pledger, W. J., Stiles, C. D., Antoniades, H. N. & Scher, C. D. (1977) Proc. Natl. Acad. Sci. USA 74, 4481-4485. 18. Welte, K., Andreeff, M., Platzer, E., Holloway, K., Rubin, B., Moore, M. & Mertelsman, R. (1984) J. Exp. Med. 160, 1390-1403. 19. Stevens, R. L., Austen, K. F. & Nissley, S. P. (1983) J. Biol. Chem. 258, 2940-2944. 20. Shiu, R. P. C. & Friesen, H. G. (1981) in Receptors and Recognition, ed. Lefkowitz, R. J., (Chapman and Hall, London), Ser. B, Vol. 13, pp. 67-81. 21. Yodoi, J. & Ishizaka, K. (1980) J. Immunol. 124, 934-938. 22. Hoover, R. G., Dieckgraefe, B. K. & Lynch, R. G. (1981) J. Immunol. 127, 1560-1563. 23. Kelley, K., Cochran, B. H., Stiles, C. D. & Leder, P. (1983) Cell 35, 603-610. 24. Neckers, L. M. & Cossman, J. (1983) Proc. Natl. Acad. Sci. USA 80, 3494-3498.
