ARTHUR WEISS*t, JOHN IMBODEN*, DOLORES SHOBACK*, AND JOHN STOBO*t. tThe Howard Hughes Medical Institute and the *Department of Medicine, ...
Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4169-4173, July 1984
Immunology
Role of T3 surface molecules in human T-cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium (Jurkat cell/T3 mutant/Ca2+/interleukin 2/activation of T cells by two signals)
ARTHUR WEISS*t, JOHN IMBODEN*, DOLORES SHOBACK*,
AND JOHN
STOBO*t
tThe Howard Hughes Medical Institute and the *Department of Medicine, University of California, San Francisco, CA 94143
Communicated by H. Sherwood Lawrence, February 27, 1984
ABSTRACT The human T-cell leukemia, Jurkat, and a T3-negative mutant of Jurkat (S.5) were used to study the role of T3 in human T-cell activation. Incubation of Jurkat with phytohemagglutinin (PHA) resulted in the production of interleukin 2, which was markedly increased by the addition of phorbol 12-myristate 13-acetate (PMA). Antibodies reactive with T3 could activate Jurkat only if added together with PMA. However, S.5 cells failed to produce interleukin 2 in response to PHA and produced 1/16th the interleukin 2 activity that Jurkat produced in response to PHA and PMA. Incubation of S.5 cells with the calcium ionophore A23187 and PMA resulted in the production of interleukin 2 activity comparable to that produced by Jurkat. Like antibodies reactive with T3, A23187 demonstrated an obligate requirement for PMA in order to activate Jurkat or S.5. These observations suggested that T3 might participate in T-cell activation through mechanisms that increase intracellular Ca2+. This was examined by using the Ca2+ sensitive fluor, quin-2, to measure levels of cytoplasmic free Ca2+ ([Ca2+]). Addition of PHA, A23187, or monoclonal antibodies reactive with T3 to Jurkat cells resulted in substantial increases of [Ca2+]1. In contrast, only A23187 could induce an increase in [Ca2+]1 in S.5 cells. Three other monoclonal antibodies reactive with other membrane antigens expressed on Jurkat or S.5 did not increase [Ca2+]. These results suggest that T3 and/or associated molecules participate in T-cell activation through mechanisms that lead to increases in [Ca2+ ] and that their expression is a relative requirement for T-cell activation by PHA.
tive assays and by the secretion of y-interferon (IFN-y) or interleukin 2 (1L2) (7-9). However, activation of T cells with anti-T3 monoclonal antibodies, as in the case of antigen and lectins, required a second signal (10, 11). Thus, cultures of adherent cell-depleted peripheral blood leukocytes failed to proliferate or produce IFN-y in response to monoclonal antibodies reactive with T3, suggesting that these adherent cells provided a second signal for T-cell activation (10, 11). Recent studies from this laboratory using the human T-cell leukemia, Jurkat, demonstrated that Jurkat cells stimulated with anti-T3 monoclonal antibodies also failed to produce IL2 unless a second stimulus, phorbol 12-myristate 13-acetate (PMA), was added (12). Furthermore, Jurkat cells required both stimuli for the appearance of IL2 or IFN-y RNA, indicating that both activation signals exerted their effects on pretranslational events (13). The mechanism by which T3 or the associated putative antigen receptor transmit an intracellular activation signal has not been studied. In the studies reported here, the human T-cell leukemia Jurkat and a T3-negative mutant of Jurkat are used to study the requirement for the T3 antigen in Tcell activation and the mechanism by which interactions with T3 are transmitted to the interior of the cell. These studies demonstrate a relative requirement for the expression of the T3 complex in phytohemagglutinin (PHA)-induced activation of Jurkat and suggest that the T3 complex participates in activation by mechanisms that lead to an increase in cytoplasmic free calcium concentration ([Ca2+]1).
T-cell-specific membrane antigens have been implicated in the functional activities of T cells. The appearance of one such antigen, T3, during thymocyte ontogeny has been closely linked to the acquisition of immunological competence (1). T3 consists of three polypeptides of 19-26 kDa (13). Several observations have suggested that the T3 complex is associated noncovalently with the putative T-cell receptor for antigen. First, immunoprecipitates of T3 molecules, under appropriate conditions, contained 43 and 47 kDa polypeptides that exhibited antigenic and structural polymorphism among different T-cell clones and were presumed to represent antigen-binding molecules (4). Second, T3 antigens comodulated with antigens of these polymorphic peptides on T-cell clones (5). Third, a stoichiometric relationship between these antigens has been demonstrated on such T-cell clones, with 30,000 binding sites of each antigen complex being expressed (6). The functional role of T3 is not clear, but studies have suggested that it may be involved in T-cell activation. For example, antibodies reactive with T3 could activate resting peripheral blood T lymphocytes, as measured by prolifera-
Cells. E6-1 is an IL2 producing clone of Jurkat-FHCRC obtained from Kendall Smith and was passaged as described (12). An 1L2-dependent mouse cytolytic T-cell line, CTLL20, was obtained from Frank Fitch and was maintained in IL2 supplemented medium as described. Monoclonal Antibodies. Monoclonal antibodies OKT3 (IgG 2a; anti-T3), anti-Leu 1 (IgG 2a; anti-Ti), anti-Leu 4 (IgG 1; anti-T3), anti-Leu 5 (IgG 1; anti-T11), 64.1.1 (IgG 2; anti-T3), anti-HLA antigens (IgG 1; anti-HLA), and MOPC 195 (IgG 2b; nonreactive with Jurkat) were obtained from previously listed sources (12). Generation of S.5, a T3-Negative Mutant of Jurkat. E6-1 cells were cultured in the presence of the mutagen ethyl methanesulfonate (Sigma) at 200 ,sg/ml for 24 hr. After 5 additional days of culture, T3-bearing cells were selected against by treatment with OKT3 (final dilution, 1:200) and rabbit complement (final dilution, 1:8). Surviving cells were subsequently treated with OKT3 and complement 3 additional times during the next 2 weeks of culture. Indirect immunofluorescence using OKT3 was used to identify cells
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Abbreviations: PMA, phorbol 12-n~yristate 13-acetate; IL2, interleukin 2; IFN-y, y-interferon; [Ca 'i, cytoplasmic free calcium; PHA, phytohemagglutinin.
MATERIALS AND METHODS
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Proc. Natl. Acad. Sci USA 81 (1984)
Immunology: Weiss et aL
that expressed T3 antigens, and negative cells were sorted on a FACS IV fluorescence-activated cell sorter (see below). These T3-negative cells were expanded and cloned by limiting dilution. S.5, which is described in detail in these studies, was derived from cultures that were seeded at 0.3 cells per well in which 7.3% of cultures were positive for growth. Production of IL2 and Determination of IL2 Activity. Stimulation of Jurkat or S.5 cells was done as described (12). Cultures were prepared with the indicated monoclonal antibodies (final dilution, 1:100 to 1:400); PHA (Burroughs Wellcome, Research Triangle Park, NC) at 1 ,tg/ml; the calcium ionophore A23187 (Calbiochem-Behring) at 1 ,ug/ml; and/or PMA (Sigma) at 50 ng/ml. After 24 hr of culture, the IL2 activity was assessed using the IL2-dependent mouse CTL line, .CTLL-20, as described (12). Flow Cytometric Analysis. Indirect immunofluorescence was determined using cells stained with the indicated monoclonal antibody followed by fluoresceinated-F(ab')2 goat anti-mouse IgG (Cappel Laboratories, Cochranville, PA). Cells were analyzed on a FACS IV cell sorter (Becton Dickinson, Sunnyvale, CA) using a wavelength of 488 nm and 400 mW of power for excitation. Fluorescence was assessed using a Long Pass 515 filter (Becton Dickinson). Determination of [Ca21]1. Jurkat cells were loaded with the acetoxymethyl ester of quin-2 according to the methods of Tsien et al. (14). The fluorescence of the quin-2-loaded cells (1.2-1.5 x 107 cells per ml in saline) was monitored with a Perkin-Elmer 650-40 spectrofluorimeter (excitation, 339 nm; emission, 492 nm) in ratio mode. The signals were calibrated in each experiment by lysing the quin-2-loaded cells with Triton X-100 (0.0259-0.1%) for the maximum fluorescence in the presence of excess Ca2+ (>1 mM). The minimum fluorescence was determined after the addition of 10 mM EGTA and sufficient 1 M Tris-base to raise the pH to >8.3. [Ca2+], was calculated as described (14, 15).
RESULTS Previous experiments by others and from this laboratory showed that activation of Jurkat cells resulting in the production of maximal amounts of IL2 activity required two signals (12, 16). The phorbol ester PMA provided one of the signals. The other signal could be provided by either monoclonal antibodies reactive with T3 or the plant lectin PHA (Table 1). Although PHA alone could induce the production of some IL2 activity, maximal levels required the addition of PMA. To determine the role that the T3 complex played in this Tcell activation, a T3-negative mutant of Jurkat was generated. These mutant cells were derived in this laboratory by exposing Jurkat cells to ethyl methanesulfonate followed by negative selection with OKT3 and complement. Negative cells were sorted on FACS IV and subsequently cloned by limiting dilution. Three assay systems were used to assess the T3 negativity of such clones: indirect immunofluorescence, quantitative absorption, and antibody/complementmediated cytotoxicity. The phenotype and functional capaTable 1. Production of IL2 by Jurkat cells stimulated with PHA or OKT3 IL2 activity, units/ml With PMA Without PMA Stimulus 0 0 None 206 ± 24.6 14.9 ± 4.4 PHA 88.4 ± 15.7 0 OKT3 Cultures of Jurkat cells were stimulated with the indicated stimulus without PMA or with PMA at 50 ng/ml for 24 hr, and IL2 activity in the resultant supernates was determined. Results represent mean + SEM of three experiments.
bility of one of these clones, S.5, are described in further detail. The lack of expression of T3 surface molecules on S.5 and the specificity of this mutation were assessed using indirect immunofluorescence analyzed by flow cytometric analysis. Fig. 1 represents fluorescent histograms of Jurkat cells or S.5 cells (T3-negative mutant) stained with the indicated monoclonal antibodies or an irrelevant mouse myeloma, MOPC 195, followed by fluoresceinated anti-mouse IgG. S.5 cells had no detectable T3 surface antigen as assessed by any of the three independently derived monoclonal antibodies reactive with the T3 antigen complex-OKT3, anti-Leu 4, or 64.1.1. Furthermore, quantitative absorption studies revealed that S.5 expressed less than 1/64th the quantity of T3 determinants that were expressed on Jurkat (data not shown). The failure of S.5 to express T3 was further confirmed by antibody/complement-mediated cytotoxicity (data not shown). The failure to detect T3 antigens with any of the three monoclonal antibodies used suggests, but does not prove, that the mutation in S.5 cells leads to the lack of expression of T3 surface molecules as opposed to an alteration of the determinant(s) recognized by these antibodies. Competitive binding experiments suggest that these antibodies recognize spatially close, if not identical, determinants (unpublished data). However, two additional independently derived mutants of Jurkat also fail to express T3 as assessed by all three monoclonal antibodies reactive with T3 (data not shown). In further support of the likelihood that S.5 represents a deletion mutant, a monoclonal antibody derived in this laboratory, which appears to react with an antigen receptor-like molecule on Jurkat, also failed to react with S.5 or the other two independently derived T3-negative mutants of Jurkat (unpublished data). The specificity of the mutation in S.5 was demonstrated by the nearly equivalent histograms obtained by staining Jurkat or S.5 with anti-Leu 1 (reactive with the T1 antigen), anti-Leu 5 (reactive with the T11 antigen), or anti-HLA (reactive with HLA-A, -B, and -C antigens). The functional ability of this T3-negative mutant to respond to a variety of stimuli was used to assess the requirement for the participation of the T3 complex in human T-cell activation. When S.5 cells were stimulated with PHA only, no detectable IL2 was produced (Fig. 2 and Table 2). In contrast, the wild-type Jurkat cells produced a mean of 15.1 units/ml of IL2 in response to PHA. Stimulation of S.5 by PHA Jurkat A
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Immunology: Weiss et al.
Proc. Natl. Acad. Sci. USA 81 (1984) T3 negative mutant
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FIG. 2. IL2 production by Jurkat or S.5 (T3-negative mutant cells). Cultures of Jurkat (A-F) or S.5 (G-L) were prepared with the indicated stimulus for 24 hr and the resultant cell-free supernates were collected. Antibodies of the indicated reactivities were used at a final dilution of
and PMA resulted in the production of 1/16th the quantity of IL2 produced by Jurkat cells (means, 12.9 and 208 units/ml, respectively). When S.5 cells were stimulated with any of the three monoclonal antibodies reactive with T3 and PMA, IL2 was either undetectable or barely detectable (450 nM (ref. 14; data not shown). The appreciable fluorescence of A23187 precluded studies at higher concentrations. When OKT3 was added to Jurkat, [Ca2+], increased to a peak of 388 ± 25 nM within 90 sec (Figs. 3B and 4) and then decreased to a plateau level, remaining above basal levels throughout observation periods of up to 30 min. A substantial increase was observed in all six experiments in which OKT3 was used; a typical tracing is shown in Fig. 3B. Similar results were obtained with anti-Leu 4, although mean peak values were lower (286 ± 25 nM; Fig. 4). Moreover, addition of both OKT3 and PMA resulted in an increase in [Ca2+]1 of 130 nM from basal levels. On the other hand, antiLeu 1, anti-Leu 5, and an anti-HLA monoclonal antibody did not'change [Ca2+], (Figs. 3C and 4); these monoclonal antibodies bind to Jurkat but do not activate it (12). Studies with quin-2-loaded S.5 cells provided an opportunity to study the role of the T3 complex in the PHA-induced increase in [Ca2+]i. As shown in Figs. 3D and 4, PHA did not change [Ca2+], in the T3-negative mutant. These data further support the hypotheses that the T3 complex and/or its associated structures are involved in the PHA-induced activation of Jurkat and that the mechanism by which they do this results in an increase in [Ca2+]j. Although OKT3 increased [Ca21] by increments of up to 33 nM in some experiments with S.5, an increase was not consistently observed, and the response was markedly less than the OKT3-induced change in [Ca2+]1 in the wild-type Jurkat (Figs. 3E and 4).
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FIG. 4. Basal and peak [Ca2"]J in Jurkat and S.5 cells (T3-negative mutant). Jurkat and S.5 cells were loaded with quin-2, and the fluorescence of the cellular suspensions was measured before (basal determinations) and after the addition of PHA,'anti-Leu 4 (Leu 4), OKT3, or control antibodies (peak values). [Ca2+], is expressed in nmol/liter as the mean ± SEM for n determinations. For Jurkat cells, basal (0) [Ca2+], was determined in 16 experiments, and peak [Ca2+], was determined after the additions of PHA (n = 4), anti-Leu 4 (n = 3 ), OKT3 (n = 6), or control (C) antibodies (anti-Leu 1, antiLeu 5, or anti-HLA; n = 6). For S.5 cells, basal (0) [Ca2+], was measured in 9 experiments, and peak [Ca2+]1 was measured after the additions of PHA (n = 4), OKT3 (n = 5), or control (C) antibody (anti-Leu 5; n = 4). For both Jurkat and S.5, PHA was used at a final concentration of 1 ,ug/ml, anti-Leu 4 and OKT3 were used at a final dilution of 1:400, and control antibodies were at a final dilution of 1:100.
DISCUSSION Several lines of evidence presented in this study suggest that the role that the T3 complex plays in activation involves an increase in [Ca2']i. First, monoclonal antibodies reactive
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FIG. 3. Representative experiments measuring [Ca2"]J in Jurkat (A-C) and S.5 cells (T3-negative mutant; D-F) in response to various stimuli. Cells were loaded with quin-2, and the fluorescence of the cellular suspension was measured over the indicated time period. Ca2+-sensitive fluorescence, as a percentage of the total, is displayed on the left-hand vertical axis, and [Ca2+]1 in nmol/liter is on the right-hand vertical axis. OKT3 (final dilution, 1:400), anti-Leu 4 (Leu 4; 1:400), anti-Leu 5 (Leu 5; 1:100), PHA (final concentration, 1 ,ug/ml), and A23187 (final concentration, 0.1 ,ug/ml) were added at the times indicated. Approximately 10% of the increase after addition of A23187 was attributable to the fluorescence of A23187 itself. Each tracing is representative of at least four different experiments.
with T3 added to Jurkat cells could induce an increase in [Ca2+]l, whereas monoclonal antibodies reactive with other cell membrane molecules could not. Second, the calcium ionophore A23187 could substitute for antibodies reactive with T3 in the activation of Jurkat. Third, A23187 could bypass the signal involving T3 in the T3-negative mutant S.5. Fourth, activation of Jurkat by both A23187 and antibodies reactive with T3 exhibited an absolute requirement for the second signal (PMA). It appears likely that PHA-induced activation of Jurkat occurs via an interaction with T3 or associated structures for the following two reasons. First, the' T3-negative mutant S.5 exhibited no detectable increase in [Ca2+] when exposed to PHA. Second, S.5 did not produce IL2 in response to PHA alone and made only a small amount of IL2 activity in response to the combination of PHA plus PMA. PHA binds to a large number of membrane glycoproteins on T cells (20). However, given the selective nature of the mutation in S.5 cells, it is probable that the basis by whlch PHA fails to activate S.5 is the result of the failure of S.5 to express the T3 complex. No direct biochemical data are available demonstrating the binding of PHA to T3. The production of some IL2 by S.5 stimulated with PHA and PMA without any detectable change in [Ca2+1, suggests that this combination of stimuli may be able to activate the cell by pathways independent of changes in [Ca2+]i. Alternatively, there may be a subpopulation within S.5 whose increase in [Ca2+], cannot be detected by this technique because of the dilutional effect of the larger unactivated population. The more recent data ob-
Immunology: Weiss et aL tained with two additional independently derived mutants of Jurkat that fail to produce detectable IL2 in response to PHA and PMA argues against pathways independent of changes in
[Ca2+ ].
The demonstrated association between T3 and the T-cell receptor for antigen coupled with the data presented here suggests two models for the T3/antigen receptor complex in human T-cell activation (4-6). In the first, T3 could serve an effector function in transmitting the activation signal. Interactions between the antigen receptor and the appropriate figand (antigen and major histocompatibility complex determinants) could lead to subsequent perturbations of T3 and transmission of a signal that causes an increase in [Ca2 ],. This perturbation could be bypassed by anti-T3 antibodies or by PHA binding to T3. In the second model, T3 may function in some structural capacity necessary for the expression or stabilization of the antigen receptor. In this model, the antigen receptor, and not T3, serves as the effector molecule capable of signaling increases in [Ca2+]i. Antibodies reactive with T3 could indirectly affect appropriate perturbations; PHA, by binding to the antigen receptor, could have a direct effect. The failure of S.5 cells to respond to PHA could result from the absence of one, two, or all three components of T3 and/or the antigen receptor. In this regard, recent investigations have shown that Jurkat cells bear antigen receptorlike molecules that are not expressed on S.5 (unpublished data). Thus, the results of this study do not distinguish between these two models. Two stimuli or signals are required for the activation of resting human peripheral blood T cells and Jurkat cells (1113). In the case of Jurkat cells, one signal can be provided by perturbation of T3 and/or associated structures resulting in an increase in [Ca2+]i. The second signal can be provided by PMA and may involve protein phosphorylation, because recent studies have demonstrated that PMA specifically binds to and activates protein kinase C (21, 22). A requirement for two similar signals has been demonstrated in the activation of other cells, including platelets (22, 23). Thus, an increase in both [Ca2+] (which was mediated by A23187) and PMA were necessary for full activation of platelets as measured by release of ATP. Of interest, a physiologic stimulus, thrombin, can mimic these two signals by increasing [Ca2+]i and activating protein kinase C in platelets (23). This is similar to the observation that one signal, PHA, can by itself induce a low level of IL2 production by Jurkat cells. In experiments presented elsewhere, these same two signals are shown to also be required for the production of another lymphokine, IFN-y, by Jurkat (13). Furthermore, both of these two activation signals are required for the appearance of IL2 and IFN-y-specific RNA, demonstrating that these two activation signals are operating at a pretranslational level (12, 13). These studies showed that one of these signals, mediated through an interaction with T3 and/or associated structures, results in an increase in [Ca2 +]. This suggests that Ca2+ functions as an intracellular messenger of one of the two activation signals. How an increase in [Ca2 +] and the activation of a protein kinase result in such gene regulation is unclear, but it provides a basis for further experiments.
Proc. Natl. Acad. Sci. USA 81 (1984)
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We would like to thank Drs. Kendall Smith, Frank Fitch, and Jeffrey Ledbetter for providing reagents and cells used in these studies; Dr. Carl Grunfeld for providing access to the fluorimeter used in these studies; and Dr. Claude Arnaud for support. The excellent technical assistance of Ms. Karen Chinn is gratefully acknowledged. Flow cytometry was carried out on the FACS IV with the technical assistance of Ms. Araxy Bastian of the Howard Hughes Medical Institute (University of California, San Francisco). We thank Phyllis Cameron for help in the preparation of this manuscript. A.W. is an associate and J.S. is an investigator in the Howard Hughes Medical Institute. This work was supported in part by U.S. Public Health Service Grant Al 14104 and National Institutes of Health Grant AM 21614. 1. van Agthoven, A., Terhorst, C., Reinherz, E. L. & Schlossman, S. F. (1981) Eur. J. Immunol. 11, 19-21. 2. Borst, J., Prendiville, M. A. & Terhorst, C. (1983) Eur. J. Immunol. 13, 576-580. 3. Kanellopoulos, J. M., Wigglesworth, N. M., Owen, M. J. & Crumpton, M. J. (1983) EMBO J. 2, 1807-1814. 4. Reinherz, E. L., Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Hodgdon, J. C., Acuto, 0. & Schlossman, S. F. (1983) Proc. Natl. Acad. Sci. USA 80, 4104-4108. 5. Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Hodgdon, J. C., Schlossman, S. F. & Reinherz, E. L. (1983) J. Exp. Med. 157, 705-719. 6. Meuer, S. C., Acuto, O., Hussey, R. E., Hodgdon, J. C., Fitzgerald, K. A., Schlossman, S. F. & Reinherz, E. L. (1983) Nature (London) 303, 808-810. 7. Van Wauwe, J. P., De Mey, J. R. & Goossens, J. G. (1980) J. Immunol. 124, 2708-2713. 8. Chang, T. W., Kung, P. C., Gingras, S. P. & Goldstein, G. (1981) Proc. Natl. Acad. Sci. USA 78, 1805-1808. 9. Venuta, S., Mertelsmann, R., Welte, K., Feldman, S. P., Wang, C. Y. & Moore. M. A. S. (1983) Blood 61, 781-789. 10. Chang, T., Testa, D., Kung, P. C., Perry, L., Dreskin, H. J. & Goldstein, G. (1982) J. Immunol. 128, 585-589. 11. Tax, W. J. M., Willems, H. W., Reekers, P. P. M., Capel, P. J. A. & Koene, R. A. P. (1983) Nature (London) 304, 445447. 12. Weiss, A., Wiskocil, R. L. & Stobo, J. D. (1984) J. Immunol. 133, 1-7. 13. Weiss, A., Imboden, J., Wiskocil, R. & Stobo, J. (1984) J. Clin. Immunol., in press. 14. Tsien, R. Y., Pozzan, T. & Rink, T. J. (1982) J. Cell Biol. 94, 325-334. 15. Tsien, R. Y., Pozzan, T. & Rink, T. J. (1982) Nature (London) 295, 68-71. 16. Gillis, S. & Watson, J. (1980) J. Exp. Med. 152, 1709-1719. 17. Gershengorn, M. C. & Thaw, C. (1983) Endocrinology 113, 1522-1524. 18. Charest, R., Blackmore, P. F., Berthon, B. & Exton, J. H. (1983) J. Biol. Chem. 258, 8769-8773. 19. Hesketh, T. R., Smith, G. A., Moore, J. P., Taylor, M. V. & Metcalfe, J. C. (1983) J. Biol. Chem. 258, 4876-4882. 20. Henkart, P. A. & Fisher, R. I. (1975) J. Immunol. 114, 710714. 21. Niedel, J. E., Kuhn, L. J. & Vandenbark, G. R. (1983) Proc. Natl. Acad. Sci. USA 80, 36-40. 22. Michell, B. (1983) Trends Biochem. Sci. 8, 263-265. 23. Rink, T. J., Sanchez, A. & Hallam, T. J. (1983) Nature (London) 305, 317-319.
