Proc. Natl. Acad. Sci. USAVol. 81, pp. 4169-4173, July 1984Immunology

Role of T3 surface molecules in human T-cell activation:T3-dependent activation results in an increase incytoplasmic 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*ttThe 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 aT3-negative mutant of Jurkat (S.5) were used to study the roleof T3 in human T-cell activation. Incubation of Jurkat withphytohemagglutinin (PHA) resulted in the production of inter-leukin 2, which was markedly increased by the addition ofphorbol 12-myristate 13-acetate (PMA). Antibodies reactivewith T3 could activate Jurkat only if added together withPMA. However, S.5 cells failed to produce interleukin 2 inresponse to PHA and produced 1/16th the interleukin 2 activi-ty that Jurkat produced in response to PHA and PMA. Incu-bation of S.5 cells with the calcium ionophore A23187 andPMA resulted in the production of interleukin 2 activity com-parable to that produced by Jurkat. Like antibodies reactivewith T3, A23187 demonstrated an obligate requirement forPMA in order to activate Jurkat or S.5. These observationssuggested that T3 might participate in T-cell activationthrough mechanisms that increase intracellular Ca2+. Thiswas examined by using the Ca2+ sensitive fluor, quin-2, tomeasure levels of cytoplasmic free Ca2+ ([Ca2+]). Addition ofPHA, A23187, or monoclonal antibodies reactive with T3 toJurkat cells resulted in substantial increases of [Ca2+]1. In con-trast, only A23187 could induce an increase in [Ca2+]1 in S.5cells. Three other monoclonal antibodies reactive with othermembrane antigens expressed on Jurkat or S.5 did not in-crease [Ca2+]. These results suggest that T3 and/or associatedmolecules participate in T-cell activation through mechanismsthat lead to increases in [Ca2+] and that their expression is arelative requirement for T-cell activation by PHA.

T-cell-specific membrane antigens have been implicated inthe functional activities of T cells. The appearance of onesuch antigen, T3, during thymocyte ontogeny has beenclosely linked to the acquisition of immunological compe-tence (1). T3 consists of three polypeptides of 19-26 kDa (1-3). Several observations have suggested that the T3 complexis associated noncovalently with the putative T-cell receptorfor antigen. First, immunoprecipitates of T3 molecules, un-der appropriate conditions, contained 43 and 47 kDa poly-peptides that exhibited antigenic and structural polymor-phism among different T-cell clones and were presumed torepresent antigen-binding molecules (4). Second, T3 anti-gens comodulated with antigens of these polymorphic pep-tides on T-cell clones (5). Third, a stoichiometric relation-ship between these antigens has been demonstrated on suchT-cell clones, with 30,000 binding sites of each antigen com-plex being expressed (6).The functional role of T3 is not clear, but studies have

suggested that it may be involved in T-cell activation. Forexample, antibodies reactive with T3 could activate restingperipheral blood T lymphocytes, as measured by prolifera-

tive assays and by the secretion of y-interferon (IFN-y) orinterleukin 2 (1L2) (7-9). However, activation of T cells withanti-T3 monoclonal antibodies, as in the case of antigen andlectins, required a second signal (10, 11). Thus, cultures ofadherent cell-depleted peripheral blood leukocytes failed toproliferate or produce IFN-y in response to monoclonal anti-bodies reactive with T3, suggesting that these adherent cellsprovided a second signal for T-cell activation (10, 11). Re-cent studies from this laboratory using the human T-cell leu-kemia, Jurkat, demonstrated that Jurkat cells stimulatedwith anti-T3 monoclonal antibodies also failed to produceIL2 unless a second stimulus, phorbol 12-myristate 13-ace-tate (PMA), was added (12). Furthermore, Jurkat cells re-quired both stimuli for the appearance of IL2 or IFN-y RNA,indicating that both activation signals exerted their effectson pretranslational events (13).The mechanism by which T3 or the associated putative

antigen receptor transmit an intracellular activation signalhas not been studied. In the studies reported here, the hu-man T-cell leukemia Jurkat and a T3-negative mutant of Jur-kat are used to study the requirement for the T3 antigen in T-cell activation and the mechanism by which interactions withT3 are transmitted to the interior of the cell. These studiesdemonstrate a relative requirement for the expression of theT3 complex in phytohemagglutinin (PHA)-induced activa-tion of Jurkat and suggest that the T3 complex participates inactivation by mechanisms that lead to an increase in cyto-plasmic free calcium concentration ([Ca2+]1).

MATERIALS AND METHODSCells. 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, CTLL-20, was obtained from Frank Fitch and was maintained inIL2 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 MOPC195 (IgG 2b; nonreactive with Jurkat) were obtained frompreviously listed sources (12).Generation of S.5, a T3-Negative Mutant of Jurkat. E6-1

cells were cultured in the presence of the mutagen ethylmethanesulfonate (Sigma) at 200 ,sg/ml for 24 hr. After 5additional days of culture, T3-bearing cells were selectedagainst by treatment with OKT3 (final dilution, 1:200) andrabbit complement (final dilution, 1:8). Surviving cells weresubsequently treated with OKT3 and complement 3 addi-tional times during the next 2 weeks of culture. Indirect im-munofluorescence using OKT3 was used to identify cells

Abbreviations: PMA, phorbol 12-n~yristate 13-acetate; IL2, inter-leukin 2; IFN-y, y-interferon; [Ca 'i, cytoplasmic free calcium;PHA, phytohemagglutinin.

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that expressed T3 antigens, and negative cells were sortedon a FACS IV fluorescence-activated cell sorter (see below).These T3-negative cells were expanded and cloned by limit-ing dilution. S.5, which is described in detail in these studies,was derived from cultures that were seeded at 0.3 cells perwell in which 7.3% of cultures were positive for growth.

Production of IL2 and Determination of IL2 Activity. Stim-ulation of Jurkat or S.5 cells was done as described (12). Cul-tures were prepared with the indicated monoclonal antibod-ies (final dilution, 1:100 to 1:400); PHA (Burroughs Well-come, Research Triangle Park, NC) at 1 ,tg/ml; the calciumionophore A23187 (Calbiochem-Behring) at 1 ,ug/ml; and/orPMA (Sigma) at 50 ng/ml. After 24 hr of culture, the IL2activity was assessed using the IL2-dependent mouse CTLline, .CTLL-20, as described (12).Flow Cytometric Analysis. Indirect immunofluorescence

was determined using cells stained with the indicated mono-clonal antibody followed by fluoresceinated-F(ab')2 goatanti-mouse IgG (Cappel Laboratories, Cochranville, PA).Cells were analyzed on a FACS IV cell sorter (Becton Dick-inson, Sunnyvale, CA) using a wavelength of 488 nm and 400mW of power for excitation. Fluorescence was assessed us-ing a Long Pass 515 filter (Becton Dickinson).

Determination of [Ca21]1. Jurkat cells were loaded with theacetoxymethyl ester of quin-2 according to the methods ofTsien 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 aPerkin-Elmer 650-40 spectrofluorimeter (excitation, 339 nm;emission, 492 nm) in ratio mode. The signals were calibratedin each experiment by lysing the quin-2-loaded cells with Tri-ton X-100 (0.0259-0.1%) for the maximum fluorescence inthe presence of excess Ca2+ (>1 mM). The minimum fluo-rescence was determined after the addition of 10 mM EGTAand sufficient 1 M Tris-base to raise the pH to >8.3. [Ca2+],was calculated as described (14, 15).

RESULTSPrevious experiments by others and from this laboratoryshowed that activation of Jurkat cells resulting in the produc-tion 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 monoclonalantibodies reactive with T3 or the plant lectin PHA (Table 1).Although PHA alone could induce the production of someIL2 activity, maximal levels required the addition of PMA.To determine the role that the T3 complex played in this T-cell activation, a T3-negative mutant of Jurkat was generat-ed.These mutant cells were derived in this laboratory by ex-

posing Jurkat cells to ethyl methanesulfonate followed bynegative selection with OKT3 and complement. Negativecells were sorted on FACS IV and subsequently cloned bylimiting dilution. Three assay systems were used to assessthe T3 negativity of such clones: indirect immunofluores-cence, quantitative absorption, and antibody/complement-mediated cytotoxicity. The phenotype and functional capa-

Table 1. Production of IL2 by Jurkat cells stimulated with PHAor OKT3

IL2 activity, units/mlStimulus Without PMA With PMA

None 0 0PHA 14.9 ± 4.4 206 ± 24.6OKT3 0 88.4 ± 15.7

bility of one of these clones, S.5, are described in furtherdetail.The lack of expression of T3 surface molecules on S.5 and

the specificity of this mutation were assessed using indirectimmunofluorescence analyzed by flow cytometric analysis.Fig. 1 represents fluorescent histograms of Jurkat cells or

S.5 cells (T3-negative mutant) stained with the indicatedmonoclonal antibodies or an irrelevant mouse myeloma,MOPC 195, followed by fluoresceinated anti-mouse IgG. S.5cells had no detectable T3 surface antigen as assessed by any

of the three independently derived monoclonal antibodies re-

active with the T3 antigen complex-OKT3, anti-Leu 4, or

64.1.1. Furthermore, quantitative absorption studies re-

vealed that S.5 expressed less than 1/64th the quantity of T3determinants that were expressed on Jurkat (data notshown). The failure of S.5 to express T3 was further con-

firmed by antibody/complement-mediated cytotoxicity(data not shown). The failure to detect T3 antigens with any

of the three monoclonal antibodies used suggests, but doesnot prove, that the mutation in S.5 cells leads to the lack ofexpression of T3 surface molecules as opposed to an alter-ation of the determinant(s) recognized by these antibodies.Competitive binding experiments suggest that these antibod-ies recognize spatially close, if not identical, determinants(unpublished data). However, two additional independentlyderived mutants of Jurkat also fail to express T3 as assessedby all three monoclonal antibodies reactive with T3 (data notshown). In further support of the likelihood that S.5 repre-

sents a deletion mutant, a monoclonal antibody derived inthis laboratory, which appears to react with an antigen re-

ceptor-like molecule on Jurkat, also failed to react with S.5or the other two independently derived T3-negative mutantsof Jurkat (unpublished data). The specificity of the mutationin S.5 was demonstrated by the nearly equivalent histogramsobtained by staining Jurkat or S.5 with anti-Leu 1 (reactivewith the T1 antigen), anti-Leu 5 (reactive with the T11 anti-gen), or anti-HLA (reactive with HLA-A, -B, and -C anti-gens).The functional ability of this T3-negative mutant to re-

spond to a variety of stimuli was used to assess the require-ment for the participation of the T3 complex in human T-cellactivation. When S.5 cells were stimulated with PHA only,no detectable IL2 was produced (Fig. 2 and Table 2). In con-

trast, the wild-type Jurkat cells produced a mean of 15.1 un-

its/ml of IL2 in response to PHA. Stimulation of S.5 by PHA

5)E

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T3 negativeJurkat OKT3 mutant

A IG

Leu4B / H

64.1.1

11 .~~~~~~~~~~~~~....

T3 negativeJurkat mutant

Leu1D

Leu5AE iK

Anti-HLA

~~L

Fluorescence intensity (log scale)

FIG. 1. Fluorescent histograms of Jurkat (A-F) and S.5 (T3-neg-ative mutant; G-L) cells. Cells were stained with monoclonal anti-

bodies of the indicated reactivities (solid lines) or a nonreactive

mouse myeloma protein MOPC 195 (dotted lines) followed by a

fluoresceinated-anti-mouse IgG and analyzed on a FACS IV cell

sorter. Data are represented as relative cell number on the vertical

axis versus the log of the fluorescence intensity on the horizontal

axis.

Cultures of Jurkat cells were stimulated with the indicated stimu-lus without PMA or with PMA at 50 ng/ml for 24 hr, and IL2 activityin the resultant supernates was determined. Results represent mean+ SEM of three experiments.

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Jurkat@0

50- A PH* PMA

30 ~ \o PHA+PMA

10- \son onI T I1 1I

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10-T T

50- C * Leu 4

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+ PMA

+ PMA

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co I~ i 06411~QL * 64.1.13 50 0D 0 64.1 1 + PMA

a) 30-

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I 50 ELeu 5Jul c) ILP.,, -4+ PMA

F * A,

O A,

k23187k23187+ PMA

II2 3 4 5 6 7 8 9

T3 negative mutant0 0

_G 0 PHA_GAd * PMA_ PHA +PMA

Ho

OKT3_ OKT3 + PMA

0 Leu40 Leu4+PMA

* 64.1 1_J ~~~~064.1. +PMA

i'_F_ FrrEKz * Leu 5_ Leu 5+ PMA

,~~~ F_ L my0 A231870A23087tPMA

2r 3 4 6 7T8 92 3 4 5 6 7 8 9

Dilution (1092)

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 indicatedstimulus 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 ofIL2 produced by Jurkat cells (means, 12.9 and 208 units/ml,respectively). When S.5 cells were stimulated with any ofthe three monoclonal antibodies reactive with T3 and PMA,IL2 was either undetectable or barely detectable (<0.2 un-its/ml). In the four experiments presented in Table 2, OKT3and PMA failed to induce any detectable IL2 activity in twoexperiments but did induce low levels in the other two ex-periments. Such results indicate that there might be a de-creased level, rather than a complete absence, of T3 antigenson S.5. Alternatively, such results could reflect the presenceof a small number of contaminating T3-bearing cells, possi-bly representing revertants. Attempts to distinguish betweenthese two possibilities have failed to identify any T3-positivecells within the S.5 cell population but may reflect an insen-

Table 2. IL2 production by Jurkat and T3-negative mutant cellsin response to various stimuli

IL2 activity, units/mlStimulus Jurkat T3-negative mutant

None 0 0PHA 15.1 ± 5.0 0PMA 0 0PHA and PMA 208 ± 34.6 12.9 ± 4.6OKT3 0 0OKT3 and PMA 100 ± 14.9 0.1 ± 0.05A23187 0 0A23187 and PMA 183 ± 57.1 134 ± 35.7

Cultures were prepared with Jurkat or S.5 cells (T3-negative mu-tant) and stimulated as indicated for 24 hr. Results represent mean ±SEM of four experiments.

sitivity of the assays used. In more recent studies using thetwo additional independently derived T3-negative mutants ofJurkat, no detectable IL2 was produced in response to PHAand PMA (data not shown). Thus, the above observationsconfirm a role for T3 in the activation of Jurkat and furthersuggest that activation of Jurkat by PHA requires T3 expres-sion.To exclude the possibility that S.5 cells were incapable of

producing normal levels of IL2, experiments were undertak-en to bypass the T3 antigen using a different activating sig-nal, the calcium ionophore A23187. When S.5 cells werestimulated with A23187 and PMA, IL2 levels of comparablemagnitude to those seen in cultures of Jurkat stimulated withA23187 and PMA were observed (means, 134 and 183 un-its/ml, respectively; Figs. 2 F and L; Table 2). These levelsof IL2 activity were comparable to those seen when Jurkatwas stimulated with either PHA or anti-T3 and PMA. Thus,these results demonstrated that S.5 cells had the functionalability to produce normal levels of IL2 if the T3-mediatedactivation signal could by bypassed. Activation of Jurkat byA23187 also exhibited the requirement for a second signal,PMA.The observation that A23187 could substitute for T3 anti-

bodies suggested that activation of Jurkat via the T3 complexmight involve changes in [Ca2+]j. This possibility was ex-plored in a series of experiments using quin-2, a recently de-veloped Ca2+-sensitive fluorescent probe (14, 15). Quin-2has been used to determine [Ca2+], in a variety of cell typesas well as to measure changes in [Ca2+], in response to exog-

enous stimuli (15, 17, 18).In unstimulated Jurkat cells, [Ca2+], was 111 4 nM

(mean + SEM). [Ca2+], increased to 287 ± 26 nM after theaddition ofPHA (Figs. 3A and 4). These results are similar to

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previously reported data for resting and for mitogen-activat-ed pig lymphocytes and murine thymocytes (15, 19). Also inagreement with previous reports (15), PMA alone decreased[Ca2+1 by up to 30 nM (data not shown), and A23187, in aconcentration (0.1 pug/ml) that gave submaximal activationof Jurkat, increased peak [Ca2+], to >450 nM (ref. 14; datanot shown). The appreciable fluorescence of A23187 pre-cluded 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 thendecreased to a plateau level, remaining above basal levelsthroughout observation periods of up to 30 min. A substan-tial increase was observed in all six experiments in whichOKT3 was used; a typical tracing is shown in Fig. 3B. Simi-lar results were obtained with anti-Leu 4, although meanpeak 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, anti-Leu 1, anti-Leu 5, and an anti-HLA monoclonal antibody didnot'change [Ca2+], (Figs. 3C and 4); these monoclonal anti-bodies bind to Jurkat but do not activate it (12).

Studies with quin-2-loaded S.5 cells provided an opportu-nity to study the role of the T3 complex in the PHA-inducedincrease in [Ca2+]i. As shown in Figs. 3D and 4, PHA did notchange [Ca2+], in the T3-negative mutant. These data furthersupport the hypotheses that the T3 complex and/or its asso-ciated structures are involved in the PHA-induced activationof Jurkat and that the mechanism by which they do this re-sults in an increase in [Ca2+]j. Although OKT3 increased[Ca21] by increments of up to 33 nM in some experimentswith S.5, an increase was not consistently observed, and theresponse was markedly less than the OKT3-induced changein [Ca2+]1 in the wild-type Jurkat (Figs. 3E and 4).

JurkatD

40011300

' -h -200

ia

0

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c

:)

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a)

cn

0)

U1)

._5c0

T3 negative mutantPHA A231871 -, -600

I , 0400300

200

-100

f "1 minE

10KT3

-4100

-i'9

80 400 - _ 30070 300 -_200

~.~200

50

0fi11 min 1 min

100 Leu5 OKT3 F Leu5

90 400_80 _ 11300 400

704 200 _ 030060r

50~~~~~~~~~~~~~~~~0

1mmQ 11 min 1 min

L-

CY0o

FIG. 3. Representative experiments measuring [Ca2"]J in Jurkat(A-C) and S.5 cells (T3-negative mutant; D-F) in response to vari-ous stimuli. Cells were loaded with quin-2, and the fluorescence ofthe cellular suspension was measured over the indicated time peri-od. Ca2+-sensitive fluorescence, as a percentage of the total, is dis-

played on the left-hand vertical axis, and [Ca2+]1 in nmol/liter is onthe 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 addi-

tion of A23187 was attributable to the fluorescence of A23187 itself.Each tracing is representative of at least four different experiments.

500 --

400

300-*

200

100 --

T3 noegtive mutant

0

Stimulus

FIG. 4. Basal and peak [Ca2"]J in Jurkat and S.5 cells (T3-nega-tive mutant). Jurkat and S.5 cells were loaded with quin-2, and thefluorescence of the cellular suspensions was measured before (basaldeterminations) and after the addition of PHA,'anti-Leu 4 (Leu 4),OKT3, or control antibodies (peak values). [Ca2+], is expressed innmol/liter as the mean ± SEM for n determinations. For Jurkatcells, basal (0) [Ca2+], was determined in 16 experiments, and peak[Ca2+], was determined after the additions of PHA (n = 4), anti-Leu4 (n = 3 ), OKT3 (n = 6), or control (C) antibodies (anti-Leu 1, anti-Leu 5, or anti-HLA; n = 6). For S.5 cells, basal (0) [Ca2+], wasmeasured in 9 experiments, and peak [Ca2+]1 was measured after theadditions 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 finalconcentration of 1 ,ug/ml, anti-Leu 4 and OKT3 were used at a finaldilution of 1:400, and control antibodies were at a final dilution of1:100.

DISCUSSION

Several lines of evidence presented in this study suggest thatthe role that the T3 complex plays in activation involves anincrease in [Ca2']i. First, monoclonal antibodies reactivewith T3 added to Jurkat cells could induce an increase in[Ca2+]l, whereas monoclonal antibodies reactive with othercell membrane molecules could not. Second, the calciumionophore A23187 could substitute for antibodies reactivewith T3 in the activation of Jurkat. Third, A23187 could by-pass the signal involving T3 in the T3-negative mutant S.5.Fourth, activation of Jurkat by both A23187 and antibodiesreactive with T3 exhibited an absolute requirement for thesecond signal (PMA).

It appears likely that PHA-induced activation of Jurkat oc-curs via an interaction with T3 or associated structures forthe following two reasons. First, the' T3-negative mutant S.5exhibited no detectable increase in [Ca2+] when exposed toPHA. Second, S.5 did not produce IL2 in response to PHAalone and made only a small amount of IL2 activity in re-sponse to the combination of PHA plus PMA. PHA binds toa large number of membrane glycoproteins on T cells (20).However, given the selective nature of the mutation in S.5cells, it is probable that the basis by whlch PHA fails to acti-vate S.5 is the result of the failure of S.5 to express the T3complex. No direct biochemical data are available demon-strating the binding of PHA to T3. The production of someIL2 by S.5 stimulated with PHA and PMA without any de-tectable change in [Ca2+1, suggests that this combination ofstimuli may be able to activate the cell by pathways indepen-dent of changes in [Ca2+]i. Alternatively, there may be a sub-population within S.5 whose increase in [Ca2+], cannot bedetected by this technique because of the dilutional effect ofthe larger unactivated population. The more recent data ob-

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tained with two additional independently derived mutants ofJurkat that fail to produce detectable IL2 in response to PHAand 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 heresuggests two models for the T3/antigen receptor complex inhuman T-cell activation (4-6). In the first, T3 could serve aneffector function in transmitting the activation signal. Inter-actions between the antigen receptor and the appropriate fig-and (antigen and major histocompatibility complex determi-nants) could lead to subsequent perturbations of T3 andtransmission of a signal that causes an increase in [Ca2 ],.This perturbation could be bypassed by anti-T3 antibodies orby PHA binding to T3. In the second model, T3 may functionin some structural capacity necessary for the expression orstabilization of the antigen receptor. In this model, the anti-gen receptor, and not T3, serves as the effector moleculecapable of signaling increases in [Ca2+]i. Antibodies reactivewith T3 could indirectly affect appropriate perturbations;PHA, by binding to the antigen receptor, could have a directeffect. The failure of S.5 cells to respond to PHA could re-sult from the absence of one, two, or all three components ofT3 and/or the antigen receptor. In this regard, recent investi-gations have shown that Jurkat cells bear antigen receptor-like molecules that are not expressed on S.5 (unpublisheddata). Thus, the results of this study do not distinguish be-tween these two models.Two stimuli or signals are required for the activation of

resting human peripheral blood T cells and Jurkat cells (11-13). In the case of Jurkat cells, one signal can be provided byperturbation of T3 and/or associated structures resulting inan increase in [Ca2+]i. The second signal can be provided byPMA and may involve protein phosphorylation, because re-cent studies have demonstrated that PMA specifically bindsto and activates protein kinase C (21, 22). A requirement fortwo similar signals has been demonstrated in the activationof other cells, including platelets (22, 23). Thus, an increasein both [Ca2+] (which was mediated by A23187) and PMAwere necessary for full activation of platelets as measured byrelease of ATP. Of interest, a physiologic stimulus, throm-bin, can mimic these two signals by increasing [Ca2+]i andactivating protein kinase C in platelets (23). This is similar tothe observation that one signal, PHA, can by itself induce alow level of IL2 production by Jurkat cells. In experimentspresented elsewhere, these same two signals are shown toalso be required for the production of another lymphokine,IFN-y, by Jurkat (13). Furthermore, both of these two acti-vation signals are required for the appearance of IL2 andIFN-y-specific RNA, demonstrating that these two activa-tion signals are operating at a pretranslational level (12, 13).These studies showed that one of these signals, mediatedthrough 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 ac-tivation signals. How an increase in [Ca2+] and the activa-tion of a protein kinase result in such gene regulation is un-clear, but it provides a basis for further experiments.

We would like to thank Drs. Kendall Smith, Frank Fitch, and Jef-frey Ledbetter for providing reagents and cells used in these studies;Dr. Carl Grunfeld for providing access to the fluorimeter used inthese studies; and Dr. Claude Arnaud for support. The excellenttechnical assistance of Ms. Karen Chinn is gratefully acknowledged.Flow cytometry was carried out on the FACS IV with the technicalassistance of Ms. Araxy Bastian of the Howard Hughes MedicalInstitute (University of California, San Francisco). We thank PhyllisCameron for help in the preparation of this manuscript. A.W. is anassociate and J.S. is an investigator in the Howard Hughes MedicalInstitute. This work was supported in part by U.S. Public HealthService Grant Al 14104 and National Institutes of Health Grant AM21614.

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