Transferrin receptor induction in mitogen-stimulated human T lymphocytes is required for DNA synthesis and cell division

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1 Proc. Natl Acad. Sci. USA Vol. 80, pp , June 1983 Medical Sciences Transferrin receptor induction in mitogen-stimulated human T lymphocytes is required for DNA synthesis and cell division and is regulated by interleukin 2 (cell cycle/monoclonal antibodies/serum factors/t-cell growth factor) LEONARD M. NECKERS AND JEFFREY COSSMAN Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland Communicated by Peter C. Nowell, February 28, 1983 ABSTRACT Transferrin is required by many cells for growth. Mitogen-induced T lymphocyte proliferation is dependent on the presence of both interleukin 2 (IL-2; T-cell growth factor) and transferrin, even though resting lymphocytes do not have receptors for either. Exposure to mitogen (phytohemagglutinin) alone is sufficient to induce transient appearance of IL-2 receptors on lymphocytes. Using monoclonal antibodies to the IL-2 receptor and to the transferrin receptor, we examined those signals required for transferrin receptor induction during T lymphocyte proliferation. Our study has revealed that (i) monocytes, or a monocyte substitute such as the phorbol ester tetradecanoylphorbol 13-acetate, are required for transferrin receptor expression after mitogen exposure; (ii) the presence of IL-2 receptors is necessary for transferrin receptor induction; (iii) antibody to the IL-2 receptor inhibits thymidine incorporation (DNA synthesis) in lymphocytes, but only if administered before transferrin receptors have appeared; and (iv) antitransferrin receptor antibody inhibits DNA synthesis but has minimal effect on IL-2 receptor expression. Thus, IL-2 receptor induction leads to transferrin receptor induction and subsequent initiation of DNA synthesis. These data indicate that IL-2 stimulates T lymphocyte proliferation, at least in part, by induction of transferrin receptors on these cells. 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 solely to indicate this fact. Transferrin, the predominant serum iron-binding glycoprotein, is an essential component of serum-free media and is required for cell growth (1). Mitogen-induced lymphocyte proliferation is transferrin dependent, even though resting lymphocytes do not possess detectable transferrin receptors (2-4). The signal responsible for transferrin receptor induction in these cells is not known. T lymphocyte proliferation is known to be dependent on the presence of interleukin-2 (IL-2; T-cell growth factor) even though resting T cells lack receptors for this substance and do not respond to it (5-9). IL-2 has been shown to function via specific cell surface membrane receptors (10). These receptors are induced in mitogen-stimulated cells which then can utilize IL-2 (in the presence of transferrin) to undergo many rounds of cell division. A relationship between IL-2 and transferrin in this phenomenon would appear likely. Recently, monoclonal antibodies directed against the transferrin receptor and the IL-2 receptor have become available (11-16). In the present study, these reagents were used to characterize the kinetics of transferrin receptor appearance in mitogen-stimulated lymphocytes and to investigate the role of IL- 2 in this process. Our results demonstrate an interdependence of receptors for IL-2 and transferrin because the presence of IL-2 receptors was required for the appearance of transferrin receptors on T cells, thereby permitting DNA synthesis. Furthermore, blockade of the expression of transferrin receptors eliminated the mitogenic capability of IL-2. MATERIALS AND METHODS Cell Preparation. Human peripheral blood mononuclear cells were obtained by Ficoll-Hypaque separation of heparinized venous blood drawn from healthy volunteers. For macrophage/ monocyte depletion, cells were resuspended at 5 X 106/ml in RPMI 1640 (GIBCO) containing 10% heat-inactivated fetal calf serum and 1% penicillin/streptomycin solution. Twenty milliliters of this suspension was placed in Corning 75-cm2 culture flasks and incubated for 1 hr at 370C in 5% C02/95% air. At the end of 1 hr, nonadherent cells were removed and reincubated in a fresh flask as above. This process was repeated three to five times until surface marker analysis using monoclonal anti-monocyte antibody (63XD3, Bethesda Research Laboratories) revealed <1% monocytes. Initial analysis routinely found 7-10% monocytes in our preparations. T cells comprised 70-80% of peripheral blood mononuclear cells [detection by LYT-3, a monoclonal anti-t cell reagent (New England Nuclear)] and 80-90% of peripheral blood lymphocytes left after monocyte depletion (PBL). Culture Methods. Nonadherent cells were plated in 24-well microtiter plates (Costar) at 1 x 106/ml (2 ml per well). Phytohemagglutinin (PHA; Sigma) was used at a final concentration of 2 pkg/ml. Tetradecanoylphorbol 13-acetate (TPA) was used at a final concentration of 50 ng/ml (81 nm). Monoclonal antibody to the IL-2 receptor (anti-tac, a gift from Thomas Waldmann, National Institutes of Health, Bethesda, MD) was used at a final ascites fluid dilution of 1:500. Monoclonal antibody to the transferrin receptor (42/6, a gift from Ian Trowbridge, Scripps Clinic, San Diego, CA) was used at a final concentration of 2,4g of purified antibody per ml. 42/6, a mouse IgA antibody, blocks the transferrin binding site and only minimally interferes with the binding of OKT9 (12). OKT9 (Ortho Pharmaceutical, Raritan, NJ) is directed against the transferrin receptor (11, 13) but does not block transferrin binding (12). In this study, OKT9 was used to detect receptors by immunofluorescence (see below). Anti-Leul, a pan T-cell reagent, was purchased from Becton Dickinson and used at a final concentration of 2.,ug/ml. All reagents were added to cultures as described in the text. Antibodies containing azide were dialyzed overnight at 40C against sterile phosphate-buffered saline containing 2% albumin and were filtered through 0.2-,Am filters before use. IL-2-dependent human T cells were cultured at 1 X 105/ml in RPMI 1640 containing 10% heat-inactivated hu Abbreviations: IL-2, interleukin 2; PBL, peripheral blood lymphocytes; PHA, phytohemagglutinin; TPA, 12-0-tetradecanoylphorbol 13-acetate; FACS, fluorescence-activated cell sorter.

2 Medical Sciences: Neckers and Cossman man AB serum, 1% penicillin/streptomycin solution, and 10% partially purified IL-2 concentrate (17). This amount of IL-2 has been shown to sustain the growth of these cells for 1 week (17). Immunofluorescence. IL-2 receptors and transferrin receptors were detected by indirect immunofluorescence using fluorescence-activated cell sorter (FACS) analysis. Cells were prepared by incubation of 2-4 x 105 cells with an appropriate dilution of primary antibody or mouse ascites control for 30 min on ice. After two washes, fluorescein-labeled goat anti-mouse IgG antibody (Kirkegaard and Perry, Gaithersburg, MD) was added and the cells were incubated an additional 30 min on ice in the dark. After three final washes, the cells were resuspended in medium and analyzed on a FACS II (Becton Dickinson) by using the 488-nm line of an argon ion laser at 500 mw power. Data analysis was based on the collection of 10,000-50,000 cells per sample. Dual staining of cells for transferrin receptor by using OKT9 and for DNA content by using propidium iodide (Calbiochem) was performed as described by Braylan et al. (18). Two-color fluorescence studies were performed by using an avidin-texas Red conjugate (Molecular Probes, Junction City, OR) together with biotinylated goat antimouse immunoglobulin (TAGO, Burlingame, CA) and OKT9 to tag transferrin receptors; directly fluoresceinated anti-tac (a gift from Thomas Waldmann) was used to tag IL-2 receptors. FACS analysis was performed by using the 568-nm line of a krypton laser at 170 mw and the 488-nm line of an argon.laser at 500 mw. Data analysis was carried out on 50,000 cells. DNA Synthesis. At appropriate times, 1,uCi (1 Ci = 3.7 X 1010 Bq) of [methyl-3h]thymidine (Amersham, 5 Ci/mmol) was added to 1 x 10' cells in 1 ml of culture medium. After 4 hr at 370C, tube contents were filtered, washed, and analyzed by liquid scintillation spectrometry. Results are expressed as a stimulation index = cpm in stimulated culture/cpm in unstimulated culture. RESULTS Requirements for in Vitro Expression of Receptors for Transferrin and IL-2 and DNA Synthesis in PHA-Stimulated Lymphocytes. The mononuclear cell population contained <1% monocytes (63XD3-positive cells) after adherent cell depletion. These PBL showed negligible thymidine incorporation after a 4-day exposure to PHA compared to nondepleted controls containing at least 7% monocytes (Table 1). Concurrently, only low levels of transferrin receptors and IL-2 receptors were expressed on PBL when monocytes were depleted. By contrast, in the PHA-stimulated monocyte-containing population, 76% of the cells expressed transferrin receptors and 84% expressed IL-2 receptors. DNA synthesis was maximally stimulated in this Table 1. Requirements for expression of transferrin receptors, IL-2 receptors, and DNA synthesis in PHA-treated lymphocytes Receptor-positive cells, % Transferrin stimulation Additive IL-2 (TAC+) (OKT9+) index Monocytes + serum Monocytes Serum None Receptor-positive cells and thymidine incorporation were measured 4 days after PHA treatment (2 Ag/ml). PBL were depleted of monocytes. Monocytes (7%) or serum (10%) or both were added back to these PBL. The stimulation index was calculated based on cells freshly prepared from peripheral blood with no further incubation (80-90 cpm). Proc. NatL Acad. Sci. USA 80 (1983) 3495 group. However, when serum was not added to the cultures, DNA synthesis was markedly decreased, even though the percentage of OK7T9 and TAC+ cells remained essentially unchanged (Table 1). Thus, PHA-induced expression and maintenance of transferrin and IL-2 receptors required that monocytes but not serum be present, whereas PHA-stimulated DNA synthesis required the presence of both monocytes and serum. Substitution of Phorbol Ester for Monocytes. Because the phorbol ester TPA can substitute adequately for monocytes in mitogen-driven lymphocyte activation (19), TPA (81 nm) was selected as a substitute for monocytes in the remainder of our studies. The ability of TPA to promote mitogenesis, although not mitogenic itself, is shown in Table 2. Monocyte-depleted cells exposed to PHA for only 6 hr (PHA then being washed out), then to TPA, still synthesized DNA maximally. Thus, TPA did not have to be present together with PHA to replace monocytes in this system. However, one cannot rule out the continued presence of membrane-bound PHA affecting the cellular response to TPA. Kinetics of Induction of IL-2 and Transferrin Receptors in PHA-Stimulated Lymphocytes. PBL were treated with PHA and then, 6 hr later, with 81 nm TPA. In one group, PHA was removed after 6 hr and TPA was added to one-half of the cells. The expression of IL-2 receptors and transferrin receptors was determined at several times after PHA addition. Cells became IL-2-receptor positive in the presence of PHA alone, but receptor appearance was unstable and did not persist for 72 hr after addition of PHA (Fig. 1A). In the presence of PHA alone, cells never became transferrin-receptor positive (Fig. 1B). When TPA was added to the cultures 6 hr after PHA, both IL-2 receptors and transferrin receptors were induced in the majority of the cells. Removal of PHA 6 hr after its addition had no effect on receptor expression as long as TPA was added. IL-2 receptors were expressed prior to transferrin receptors. Transferrin receptors did not become maximally expressed until 72 hr after PHA addition. Two-color fluorescence studies demonstrated that, 72 hr after PHA addition, the majority of cells possessed both transferrin receptors and IL-2 receptors. A large proportion were positive only for IL-2 receptors but <1% of the cells were transferrin-receptor positive and IL-2-receptor negative (Fig. 2). When cells were treated with TPA alone, <25% were positive for both IL-2 and transferrin receptors 48 hr later (data not shown). When transferrin-receptor expression was examined as a function of the cell cycle, receptor-positive cells were found in all phases, including Go/G1. Forty-eight hours after PHA addition, only 25% of GO/GI cells were transferrin-receptor positive but nearly all cells in S and G2+ M were receptor positive (84% and 88%, respectively; data not shown). Table 2. TPA replaces monocytes in PHA-induced lymphocyte DNA synthesis Addition* stimulation indext PHA + monocytes (10%) 33 PHA + TPA 23 PHA (0-6 hr) + TPA (added at 6 hr) 24 PHA (0-6 hr) 8 TPA 5 * PHA (2 pug/ml) was present throughout the experiment unless indicated otherwise. TPA (50 ng/ml) was either added with PHA or after PHA had been removed. tdna synthesis was measured 72 hr after the beginning of the experiment. The stimulation index was calculated based on cells depleted of monocytes and kept in culture for 4 days with no other additions ( cpm).

3 3496 Medical Sciences: Neckers and Cossman Proc. Nad Acad. Sci. USA 80 (1983) 100 A B 100 Anti-TAC added e- a) 0c 60 PHA + TPA PHA (0-6 hr) TPA (6 hr-y) X) /Control 60 -._ Go 20 - PHA (0-6 hr) Time after PHA, hr FIG. 1. PHA (2 pg/ml) was added to PBL and then 6 hr later it was washed out of some cultures and not others. TPA (50 ng/ml) was added at this time. Cells expressing IL-2 receptors (TACk) (A) and transferrin receptors (OKT9 ) (B) were examined at various time points after PHA addition. Dependence of Transferrin Receptor Induction on the Presence of IL-2 Receptors and IL-2. Two pieces of circumstantial evidence suggest that IL-2 receptors may be involved in transferrin receptor induction. First, in the presence of PHA alone, PBL temporarily expressed IL-2 receptors but these receptors soon disappeared. In this case, no transferrin receptors appeared. Second, in the presence of PHA and TPA, IL-2 receptors were maximally expressed at least 1 day before transferrin receptors, even though both receptors were eventually expressed on the same cells. If functional IL-2 receptor expression is required for transferrin receptor expression, then blockade of IL-2 receptors should inhibit appearance of transferrin receptors. When anti-tac antibody (1:500 dilution of ascites) was added to PHA/TPA-stimulated PBL before transferrin receptors were expressed (6 hr after PHA addition), transferrin receptor acquisition was completely inhibited (Fig. 3). When E0 To OKT9 TAC- 9% FIG. 2. PBL were treated with PHA (2 1g/ml) and TPA (50 ng/ml) for 3 days. The cells were then simultaneously stained for transferrin receptors by OKT9 and for IL-2 receptors by anti-tac and subjected to FACS analysis. The data (collected for 50,000 cells) are expressed as a contour plot with OKT9 fluorescence intensity increasing on they axis and anti-tac fluorescence intensity increasing on the x axis. The contours represent cell numbers. Greater than 50% of the cells had both transferrin and IL-2 receptors. Forty percent of the cells had only IL- 2 receptors, but <1% of the cells had only transferrin receptors. 20 Anti-TAC added Time after PHA, hr FIG. 3. Anti-TAC antibody (anti-il-2 receptor) was added to cultures at either 6 hr or 48 hr after PHA addition. Transferrin-receptor positive cells were measured at various time points after PHA addition in antibody-treated and control cultures. Expression of transferrin receptors was decreased only if anti-tac antibody was added to cultures prior to the expression of these receptors. anti-tac was added after transferrin receptors could be detected (48 hr after PHA addition), the antibody had no effect on continued transferrin receptor expression. Brief exposure of PBL to mitogen has been reported to result in IL-2 receptor induction. To test the requirement for IL- 2 in transferrin receptor induction, we added PHA to PBL for 3 hr, washed the lymphocytes twice, and resuspended them in fresh medium in the presence or absence of lectin-free IL-2 (a gift of S. A. Rosenberg and E. A. Grimm). Three days later, the cells were analyzed for the presence of transferrin receptors, IL-2 receptors, and DNA synthesis (Table 3). After a brief pulse of PHA, a significant portion of the cells cultured in IL- 2-containing medium for 3 days were positive for IL-2 and transferrin receptors; those cells cultured in the absence of IL- 2 were essentially transferrin receptor negative. DNA synthesis was markedly enhanced in the IL-2-treated cells but not in those cells to which IL-2 was not added. Effect of Antibodies to the IL-2 Receptor and the Transferrin Receptor on DNA Synthesis and Cell Growth. Although functionally active IL-2 receptors must be present before transferrin receptors will appear, this in itself does not demonstrate that transferrin receptors are required for mitogenesis. To test this directly, we measured thymidine incorporation (DNA synthesis) in cells to which anti-tac antibodies were added either before or after transferrin receptor expression (6 and 48 hr after PHA addition, respectively) (Table 4). DNA synthesis was significantly reduced (15% of control) only when anti-tac was added before transferrin receptors were expressed. When anti- Table 3. IL-2 is required to induce transferrin receptors on PHAtreated lymphocytes Receptor-positive cells, % Additive IL-2 (TAC+) Transferrin (OKT9+) stimulation index PHA + IL-2 PHA PHA (2,g/ml) was added to the cultures at the initiation of the experiment and removed 3 hr later. IL-2 was added to half the cells at this point. The cultures were maintained for 3 days, and then receptor-positive cells and thymidine incorporation were determined. The stimulation index was calculated based on cells depleted of monocytes and kept in culture for 3 days with no further treatment ( cpm).

4 Medical Sciences: Neckers and Cossman Table 4. Effect of time of addition of anti-tac on PHAtreated lymphocytes Time of addition Transferrin receptor- stimulation of anti-tac positive cells, % index None hr hr Anti-TAC (1:500 dilution of ascites) was added at the times indicated after PHA addition. Transferrin receptor-positive cells and DNA synthesis were determined 72 hr after PHA was added to the cultures. The stimulation index was based on cells depleted of monocytes and kept in culture for 3 days with no further treatment ( cpm). TAC was added 48 hr after PHA addition, DNA synthesis was decreased by only 20%. Thus, the ability of anti-tac antibody to inhibit DNA synthesis was closely correlated with its ability to block transferrin receptor induction. When antitransferrin receptor antibody (42/6) was added to these cultures, it consistently decreased DNA synthesis by approximately 75% (Table 5). These antibodies also appeared to modulate transferrin receptors off the surface of cells because the receptors could no longer be detected by OKT9. IL-2 receptor expression was not significantly decreased under these conditions. As a control, a pan T-cell antibody, anti-leul, was added to cultures; it had no effect on either receptor expression or DNA synthesis. Thus, 42/6 antibody caused the disappearance of transferrin receptors from the cell surface and greatly inhibited DNA synthesis but left IL-2 receptors essentially unaffected. To test the growth-regulating potential of the transferrin receptor in the presence of excess IL-2, we added antitransferrin receptor antibody (42/6) to cultures of IL-2-dependent T cells seeded at 1 x 105 cells per ml in the presence of excess partially purified IL-2. Cells were counted 3 days later and antitransferrin receptor antibody was found to have nearly completely inhibited their growth (by 68% and 81% in two experiments) (Table 6). Thus, even in the presence of excess IL-2, blockade of transferrin receptors inhibits the growth of IL-2-dependent T cells. DISCUSSION During the lectin-induced in vitro mitogenesis of PBL, receptors for IL-2 are generated. The present study demonstrates that this event is a prerequisite for the induction of transferrin receptors. Furthermore, the sequential induction of IL-2 receptor expression and transferrin receptor expression on the same cell is essential for DNA synthesis in this system. However, once transferrin receptors appear, the continued pres- Table 5. Effect of antitransferrin receptor antibody (42/6) on PHA-treated lymphocytes Receptor-positive cells, % Transferrin stimulation Antibody added IL-2 (TACk) (OKT9+) index None Anti-Leul Antitransferrin receptor (42/6) Antibodies (2 gg/ml) were added 15 hr after PHA addition. Receptorpositive cells were determined 48 hr after PHA addition. DNA synthesis was measured 72 hr post PHA. The stimulation index was based on cells depleted of monocytes and kept in culture for 3 days with no further additions ( cpm). Proc. Natd Acad. Sci. USA 80 (1983) 3497 Table 6. Antitransferrin receptor antibody (42/6) inhibits growth of IL-2-dependent T cells Cells, no. X 105/ml* Addition Exp. 1 Exp. 2 None Mouse ascites Antitransferrin receptor (42/6) /6 or nonreactive mouse ascites (2 Mg/ml) was added to 1 x 105 cells in 1 ml. Cell counts were made 72 hr later. * Increase in cell number per ml above 1 x 106. ence of functional IL-2 receptors is not required for the cell to proceed with at least one round of DNA synthesis. The presence of monocytes or a monocyte-replacing factor [TPA (19)] is absolutely required for the induction and maintenance of receptors for both IL-2 and transferrin (Table 1; Fig. 1). However, cells possessing these receptors will not synthesize DNA maximally in the absence of serum (Table 1). Because transferrin can replace serum in mitogen-induced lymphocyte proliferation (3), the requirement for serum in our system is presumably a requirement for transferrin (a major serum component). The low level of DNA synthesis observed in the absence of serum (but in the presence of monocytes) may be dependent on transferrin produced and secreted by the cultured lymphocytes themselves (20, 21). We have shown that anti-tac antibody prevents the appearance of transferrin receptors on- PHA-stimulated lymphocytes. Similarly, Tsudo et al. (22) recently reported that addition of anti-tac antibody to allo-activated T cells blocks the appearance of Ia antigen on these cells. Ia antigens are not normally present on resting T cells but appear after activation, probably during or after S phase of the cell cycle (23, 24). Anti- Tac antibody has no effect on Ia antigen when added to T cells already expressing Ia, even though it appears to modulate the IL-2 receptor off the cell surface (22). In our study, anti-tac had no effect on transferrin receptors once the latter were detectable. Recently, it has been proposed that IL-2 provides a signal necessary for activated lymphocytes to enter S phase (25). Our data would support the idea that IL-2 production is part of a cascade of biochemical events initiated by mitogen (or antigen) stimulation. In this cascade, the appearance of functional IL-2 receptors is an early and pivotal step. After the appearance of IL-2 receptors, and in the presence of IL-2, transferrin receptor expression is induced and the cells enter S phase (Table 3). If transferrin receptors are blocked, DNA synthesis and cell growth are inhibited. Furthermore, IL-2-dependent T cells also require transferrin receptor expression prior to DNA synthesis. Transferrin receptors have been shown to appear on cells before they enter S phase (4, 26, 27). Our data demonstrate that transferrin receptors appear on those cells already possessing IL-2 receptors and are not lost by cells in S and G2+M; on the contrary, the vast majority (>80%) of cells in these two cycle phases still possess transferrin receptors. The present results indicate that the growth-promoting effects of IL-2 on activated T cells are mediated via the induction of cell surface receptors for the serum glycoprotein transferrin. Transferrin plays an essential role in the maintenance of growth of continuous cell lines and is an essential component of serumfree media (1). Blockade of surface receptors for transferrin inhibits the growth of many cell lines (12, 27) and, as the present study indicates, DNA synthesis in mitogen-stimulated lymphocytes. The mechanism(s) by which transferrin exerts its ef-

5 3498 Medical Sciences: Neckers and Cossman fects on growing cells is not known. Because the serum concentration of transferrin is quite high (4 mg/ml), it is not unreasonable that expression of transferrin receptors would be under complex metabolic control. By studying how IL-2 interaction with its receptor leads to transferrin receptor expression, we will be able to better understand how cell growth is normally regulated. We thank Dr. T. A. Waldmann for a supply of anti-tac, Dr. I. Trowbridge for a supply of 42/6 antibody, and Dr. E. A. Grimm and Dr. S. A. Rosenberg for a supply of IL-2 and IL-2-dependent T cells. 1. Barnes, D. & Sato, G. (1980) Cell 22, Tormey, D. C., Imrie, R. C. & Mueller, G. C. (1972) Exp. Cell Res. 74, Dillner-Centerlind, M.-L., Hammarstrom, S. & Perlmann, P. (1979) Eur. J. Immunot 9, Larrick, J. W. & Cresswell, P. (1979)J. Supramol Struct. 11, Morgan, A. D., Ruscetti, F. W & Gallo, R. C. (1976) Science 193, Ruscetti, F. W., Morgan, A. D. & Gallo, R. C. (1977)J. Immunol. 119, Mier, J. W & Gallo, R. C. (1980) Proc. Natl. Acad. Sci. USA 77, Smith, K. A., Gillis, S. & Baker, P. E. (1979) in The Molecular Basis of Immune Cell Function, ed. Kaplan, J. G. (Elsevier/North- Holland, New York), pp Lotze, M. T., Strausser, J. L. & Rosenberg, S. A. (1980) J. Immunol. 124, Robb, R. J., Munck, A. & Smith, K. A. (1981)J. Exp. Med. 154, Goding, J. W & Burns, G. F. (1981)J. Immunol. 127, Trowbridge, I. S. & Lopez, F. (1982) Proc. Natl. Acad. Sci. USA 79, Proc. Natt Acad. Sci. USA 80 (1983) 13. Sutherland, R., Delia, D., Schneider, C., Newman, R., Kemshead, J. & Greaves, M. (1981) Proc. Natl, Acad. Sci. USA 78, Uchiyama, T., Broder, S. & Waldmann, T. A. (1981)J. Immunol 126, Uchiyama, T., Nelson, D. L., Fleisher, T. A. & Waldmann, T. A. (1981)]. Immunol 126, Leonard, W. J., Depper, J. M., Uchiyama, T., Smith, K. A., Waldmann, T. A. & Greene, W. C. (1982) Nature (London) 300, Grimm, E. A. & Rosenberg, S. A. (1982) in Isolation, Characterization and Utilization of T Lymphocyte Clones, eds. Fathman, G. & Fitch, F. (Academic, New York), pp Braylan, R. C., Benson, N. A., Nourse, V. & Kruth, H. S. (1982) Cytometry 2, Koretzky, G. A., Daniele, R. P. & Nowell, P. C. (1982)J. Immunot 128, Imrie, R. C. & Mueller, G. C. (1968) Nature (London) 219, Tormey, D. C. (1969) Dissertation (Univ. Wisconsin, Madison). 22. Tsudo, M., Uchiyama, T., Takatsuki, K., Uchino, H. & Yodoi, J. (1982) J. Immunot 129, Evans, R. L., Faldetta, T. J., Humphreys, R. E., Pratt, D. M., Yunis, E. J. & Schlossman, S. F. (1978) J. Exp. Med. 148, Ko, H.-S., Fu, S. M., Winchester, R. J., Yu, D. T. Y. & Kunkel, H. G. (1979) J. Exp. Med. 150, Maizel, A., Mehta, S. R., Hauft, S., Franzini, D., Lachman, L. B. & Ford, R. J. (1981)J. Immunol 127, Cotner, T., Williams, J. M., Christenson, L., Shapiro, H., Strominger, J. L. & Strom, T. B. (1982) Fed. Proc. Fed. Am. Soc. Exp. Biol, 41, 738 (abstr.). 27. Neckers, L. M. & Cossman, J. (1982) Fed. Proc. Fed. Am. Soc. Exp. Biol 41, 740 (abstr.).