Cytosolic free calcium concentration in the mitogenic stimulation of T lymphocytes by anti-CD3 monoclonal antibodies

Cell Calcium (1994) 16, 167-180 Q Longman Group Ltd 1994

Cytosolic free calcium concentration in the mitogenic stimulation of T lymphocytes by anti-CD3 monoclonal antibodies M. MURGIA’, M. MION’, L. VERONESE2, M. PANOZZ02, V. COPPOLA2, R. RIZZUTO’, M. BRINI’, F. MALAVAS13, A. AMADOR12, L. CHIECO BlANCHI and T. POZZAN’ ‘Department Biomedical Sciences, CNR Unit for the Study of the Physiology of Mitochondria, University of Padova, 21nsfifute of Oncology, lnteruniversify Center for Cancer Research, University of Padova, and 3Department of Genetics and Medical Chemistry, CNR Unit for immunology and Histocompatibility, University of Torino, ha/y The effects of anti-CD3 monoclonal antibodies on cytosolic free Ca*+ Abstract concentration, [&Ii, were investigated in freshly isolated lymphocytes, T cell lines, T clones and the leukemic T cell line Jurkat with three different methodologies, i.e. classical cuvette experiments, cytofluorimetry and videoimaging. With any technique, concentrations of anti-CD3 antibodies optimal for stimulation of DNA synthesis were completely ineffective at inducing early increases of [Ca2’]i in freshly isolated lymphocytes. At supraoptimal mitogenic concentrations: (i) anti-CD3 mAb induced negligible increases of [Ca*+]i when tested in suspensions of freshly isolated lymphocytes, but the response increased progressively during in vitro culturing with 112; (ii) most, but not all, T clones, when tested in suspension, were responsive to these concentrations of anti-CD3 antibodies in terms of [Ca2’]i; (iii) using the videoimaging technique at the single cell level, It was demonstrated that the anti-CD3 antibodies induced large increases of [Ca2’]i in lymphocytes only under conditions which allowed adherence of the antibodies (and of the cells) to the glass surface. In all T cell types investigated, the [Ca’+]i increases were most often composed by multiple, asynchronous oscillations. The buffering of [Ca*+]i increases, obtained by loading the cells with membrane permeant esters of Quin-2 and Fura-2, inhibited anti-CD3 mAb induced DNA synthesis, but this appeared entirely attributable to a toxic side effect of the ester hydrolysis. The relevance of these data is discussed in terms of their methodological and functional implications for the understanding of the role of Ca*+ in mitogenic stimulation of T cells.

Activation by properly presented antigens of resting T lymphocytes is a highly complex phenomenon which eventually results in activation of DNA synthesis and/or expression of specialized cell functions

[ 11. These events are the consequence of a multistep signal transduction pathway controlled both by the T cell receptor (TCR) and by coreceptors and costimulatory molecules (CD4, CD& CD28. LFA-1) [2,3] 167

168

whose paramount importance in T cell activation has been recently recognized [4]. Protein tyrosine phosphorylation is the very first intracellular signal following TClUCD3 activation [5]; it involves receptor-mediated triggering of various protein tyrosine kinases belonging to the src and SyklZAP70 families, and of at least one tyrosine phosphatase, CD45, which has a fundamental role in modulating downstream intracellular events [6,7]. These latter include activation of Ras and the tyrosine phosphorylation of phospholipase Cyl [2], which leads to InsP3 production and [Ca*+]i increases as well as stimulation of the ubiquitous enzyme protein kinase C [8]. Dozens of papers have been published in recent years about these problems, using different experimental approaches, stimuli and cellular models (for recent review see IS]). In particular, as far as Ca*+ signalling is concerned, the general consensus appears to be that activation of the T cell receptor elicited by antigen or monoclonal antibodies (mAb) elicit large increases in [Ca*+]i, dependent on both Ca*+ mobilization from intracellular stores and increased influx through plasma membrane channels [9]. In addition, several other membrane antigens, when crosslinked by specific antibodies, appear endowed with the capacity to activate a [Ca2+]i increase [IO . Almost invariably these antibodies also increase [4HI-thymidine incorporation in peripheral blood mononuclear cells (PBMC) [ 1I]. Indeed, the measurement of [Ca*+]i increases has become a routine experiment in cellular immunology [12]. The increase of [Ca*+]i elicited by mAb against specific surface antigens has been utilized not only as an easy and reproducible test of T cell activation, but it has also been considered an essential component of the signalling pathway that from the T cell receptor leads to IL2 production, DNA synthesis and cell division [ 131. A closer analysis of the literature, however, reveals that the situation is far more complex and that clearly contradictory results have been obtained in this field. For example, only a few antibodies against CD3 are efficient in triggering [Ca2+]i increases in freshly isolated PBMC [12], despite their overall efficacy as mitogenic stimuli [I I], while further crosslinking by anti antibodies does indeed cause [Ca2+]i increases but results in inhibition of DNA synthesis [ 131. In this contribution we have critically recon-

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sidered the problem of Ca2+ signalling elicited by mitogenic anti CD3 antibodies. We demonstrate that three well characterized anti-CD3 mAbs induced negligible increases in [Ca*+]i in suspensions of freshly isolated PBMC. Under the same conditions, the same mAbs were highly effective on [Ca2+]i in Jurkat cells. Of interest, during in vitro culturing with IL2, the same antibodies became effective in causing [Ca2+]i increases, but this was not due to an increased expression of CD3 molecules on the plasma membrane. When [Ca2+]i was studied at the single cell level by means of a computerized image analysis system, not only was it found that the increase in [Ca*+]i induced by anti-CD3 mAb in T lymphocytes was most often composed of multiple, asynchronous oscillations, but it was also demonstrated that the ability of the mitogenic mAb to modulate [Ca*+]i in PBMC depends on the binding of the stimulus (and of the cells) to a solid substratum. The methodological implications of these observations and the role of [Ca*+]i increases in the process of antigen stimulation in T cells is briefly discussed.

Materials and methods Cell source and preparation

PBMC were isolated from preservative-free heparin (or citrate) anticoagulated venous samples of healthy volunteers by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient centrifugation, as detailed elsewhere [14]. T lymphocytes were obtained by double-rosetting with neuraminidase treated sheep red blood cells [ 151 and were ~99% pure, as judged by cytofluorographic analysis with specific mAb. To obtain T cell lines, PBMC were resuspended in RPM1 medium supplemented with 10% fetal calf serum (FCS, Gibco, Grand Island, NY, USA) and cultured (1 x lo6 cells/ml) in the presence of phytohemagglutinin (PHA, Gibco; 1 pglml) or anti-CD3 mAb (100 ng/ml). 3 days later, the cells were supplemented with 10 U/ml recombinant IL2 (Eurocetus, Milan, Italy) and maintained in culture for up to 5 weeks. T cell clones were obtained from the T cell lines by limiting dilution at 3, I and 0.3 cells/well, as detailed elsewhere [ 161.

169

Jurkat cells, a kind gift of Dr F. Gerosa (University of Verona), were maintained in culture as described previously [ 171. Cell proliferation in response to PHA and antiCD3 mAb was assessed as detailed elsewhere [IS]. C~tojhorinietric

analysis

Cytofluorimetric analysis was performed with an Epics C cytofluorimeter (Coulter Electronics. Hialeah, FL, USA) as previously reported [19]. To determine the number of cells in the different phases of the cell cycle, 48 h after addition of the mitogen (PHA or anti-CD31 3 x 10” cells were incubated in complete RPM1 in the presence of IO PM 5-bromo2-deoxyuridine (BrdU. Sigma Chemicals, St Louis, MO. USA): 45 min later, the cells were harvested, washed with phosphate-buffered saline, PBS, pH 7.4 and fixed overnight in ice-cold 70% ethanol. The cells were then washed. resuspended in 2 N HCI for 20 min at room temperature, washed again and finally resuspended in 200 pl of PBS containing 0.5% Tween-20 and 0.5% serum albumin, in the presence of a I : I00 dilution of a FITC-labelled anti-BrdU mAb. After 2 h at room temperature, the cells were washed with PBS and incubated overnight at 4°C with propidium iodide (10 pg/ml). The cells were then analyzed cytofluorographically for double red and green fluorescence after excitation with a 488 nm argon ion laser (for other details see [ 181). [CCr”+]i measnrenrerrt in cell susperision [Ca”]i was measured with the intracellular indicators Furaand Indo-l as described [20]. After loading with the Ca2+ indicator, the cells were washed, resuspended in a modified Krebs Ringer (HEPES buffered) medium, KRH (I 25 mM NaCI. 5 mM KCI, I mM Na3P04, ImM MgS04. lmM CaCl2, 5.5 mM glucose, 20 mM HEPES, pH 7.4 at 37°C) and analyzed either in a cuvette with a standard fluorimeter or with a fluorescence activated cell sorter. The [Ca*+]i increases, qualitatively and quantitatively, measured in the cuvette with Furaand Indo-l were very similar. In cytofluorimetric analysis, only Indo-l loaded cells were employed and [Ca”]; increases were measured essentially as described by Alexander et al. (211.

The basic principle of the methodology is described in detail in (221. Briefly, Furaloaded cells were incubated in a thermostated chamber (Medical System Corp., NY, USA) mounted in the stage of an inverted fluorescence microscope (Olympus IMT2). The cells were allowed to sediment at the bottom of the chamber (a glass coverslip) and then illuminated alternatively by monochromatic light (340 and 380 nm) through a 40x objective (Nikon). The emitted fluorescence was filtered (cut-off filter 450 nm) and captured by an intensified CCD camera coupled to an image analysis system (FL4000, Analytical Imaging Concepts, Georgia, USA). Average images (usually 16) were either divided in real time and visualized on a color monitor or stored on an optical disk and analyzed quantitatively off line. The ratio ima es (340/380) represent a direct measure of f [Ca’+]i. The calibration of the ratio values in terms of [Ca*+]i was obtained as described 120). Other materials

Fura-2, Indo-I, Quin-2 and BCECF acetoxymethylesters and free acids were obtained from Molecular Probes (Eugene, OR, USA), [“HI-thymidine from Amersham. 0KT3 from Who Diagnostics, Leu-4 from Bekton & Dickinson; CBT3 was obtained as previously reported [23]. All other materials were of analytical or the highest available grade. Unless otherwise specified all experiments are representative of at least 5 independent trials.

Results E’ects

art [Ca2’]i

of mitogenic

artri-CD3

mAb in

mixed lymphocyte populations

Figure IA-C shows the effects on [Ca2+]i elicited, in suspensions of freshly isolated human PBMC, by three mitogenic anti-CD3 mAbs. OKT3 and Leu4 are commercially available, while CBT3 was generated in the laboratory of one of the authors and has been characterized in detail [23]. For comparison, the results obtained in the T cell line Jurkat, a

170

CELL CALCIUM

A -1111111,

I

1

I

I

OK13

CD15

PHA

PHA

I

,.QU4

Fig. 1 Effects of three anti-CD3 mAb on [Ca”]i in PBMC and Jurkat cells in suspension. Aliquots of Fura-24oaded cells were resuspendedin the fluorimeter cuvette containing 2 ml of KRH medium. Where indicated, 2 pg/ml of anti-CD3 mAb and 3 @ml

of

PHA were added under continuousstirring. The calibrated [Ca*‘]i is reported on the right hand side. A-C = PBMC; D-F = Jurkat cells. Inset: effect of crosslinking by anti mouse Ig on [Ca”]i in PBMC suspensions;where indicated, 8

pg/mlrabbitanti mouse antibody

were added.

widely employed T cell model, are also presented (Fig. ID-E). In the Jurkat cells the three anti-CD3 mAbs increased [Ca2+]i with slightly different kinetics and amplitudes, while in PBMC the same mAb caused no, or marginal, increases in mean [Ca”]i. The lack of response to the anti-CD3 mAb in PBMC could not be attributed to technical artifacts or to the incapacity of these cells to respond with a [Ca*+]i rise to any stimulus, since: (i) the mitogenic lectin PHA (Fig. IA-C), or the Ca2+ ionophore ionomycin (not shown) caused a large and sustained [Ca*Q increase; (ii) identical results were obtained with PBMC from 10 different donors; and (iii) as shown previously (see, for example, Wolff and coworkers [24]), further crosslinking with oat 9+ anti-mouse Ab resulted in a rapid increase in [Ca ]i (Fig.1 inset). As typical anti-CD3 mAb in all the following experiments, CBT3 and OKT3 were rou-

tinely employed, though at times also Leu-4 was used with identical results. An even more striking discrepancy between the effects on [Ca2+]i and the mitogenic efficacy was observed when the dose dependence for the two effects was compared (Fig. 2). Concentrations of CBT3 which were optimal for [3H]-thymidine incorporation were completely ineffective on [Ca*+]i not only in PBMC, but also in Jurkat cells. Only at very high antibody concentrations (> 10 pg/ml), also in PBMC, could an increase in [Ca2+]i be detected. Similar results were obtained with 0KT3 (not shown). Effects on [Ca2+]i of anti-CD3 lymphocyte populations

mAb in purified

T

Experiments aimed at determining whether the lack of response to the anti-CD3 mAb in terms of [Ca2+]i

ANTI-CD3

171

INDUCED [Ca’+l, INCREASE

OL

Fig. 2

CBT3 concentration dependence of [‘HI-thymidine

incorporation

in PBMC (A)

incorporation and [Ca2’], increase. The dose-dependence for [‘HI-TdR

is compared to that of [Ca”]i

increase in PBMC (0)

and Jurkat cells (m). Conditions for [Ca”],

measurements as in Figure I. [Ca’+]i increase is expressed as the difference (A) between basal level and the maximum increase caused by the stimulus, while 13H]-TdR incorporation as % of the maximal effect. Note that [3H]-thymidine incorporation was carried out in the presence of FCS. i.e. the condition which prevents the [Ca”]i

was attributable to cell heterogeneity were carried out on purified T cell populations. T cells were obtained by: (i) rosetting with sheep erythrocytes PBMC preparations; (ii) stimulating PBMC with polyclonal mitogens (PHA or anti-CD3 mAb) and maintaining the cells in culture with IL2 (IL2-dependent T cell lines); and (iii) isolating from the T cell lines IL2-dependent T cell clones. The effects on [Ca*+]i of CBT3 and OKT3 on purified T lymphocytes are summarized in Figure 3. Figure 3A shows that CBT3 hardly increased [Ca’+]i in T lymphocytes purified by rosetting and a similar effect was obtained with OKT3 (not shown). The results obtained with T cell lines and clones were quite different. Figure 3B shows the time dependence of the maximal [Ca*+]i response to CBT3 of two T cell lines, generated with CBT3 and PHA, respectively. In the inset, the kinetics of the [Ca*+]i increases. elicited by CBT3 at different culture times, are also shown. A significant response was observed already after I week in culture with IL2 and the peak [Ca2+]i reached its maximum after 4-5 weeks. Similar results were obtained with 0KT3 as the stimulating antibody. Out of 7 T cell lines analyzed,

oscillations in single cells (Fig. 6A)

2 remained almost unresponsive to the anti-CD3 mAb, when tested for up to 5 weeks after the initial challenge with the mitogen. The time-dependent increase of the response was slightly different among the various responding lines, but no correlation was found with the type of stimulus (anti-CD3 mAb or PHA) initially employed to generate the cell lines. Furthermore, the response to PHA, quantitatively and qualitatively, did not change appreciably during the culture period, indicating that the ‘maturation’ of the [Ca*+]i response was specific for anti-CD3 mAb. 27 T cell clones were tested, 15 generated initially by CBT3 and I2 by 0KT3. The amplitude of the response in terms of [Ca’+]i appeared to be clone specific, but independent of the challenging or generating anti-CD3 mAb. Figure 4 shows that out of 27 clones, 2 did not respond in terms of [Ca2+Ji, while in the remaining 25 the peak response ranged from 20 to more than 200 nM; the distribution between high and low responders, however, did not vary whether CBT3 or 0KT3 were used as the challenging anti-CD3 mAb (not shown). No correlation was found between the amplitude of the response to anti-CD3 mAb and the phenotype (CD4 or CDS) of

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CELL CALCIUM

the clones. Indeed, of 3 highly responding clones (peak [Ca2+Ji increase 200 nM),

whose phenotype

was analyzed, 2 were CD4+ and I CD8’,

and of the

50 nM) analyzed, 2 were CD4+ and 2 CD8”. Interestingly, the amplitude of the [Ca2+]i response to PHA was rather constant among the clones (data not shown). The above mentioned results demonstrate that while most T cell clones and T lines, as well as Jurkat cells, were capable of responding in suspension with a [Ca2+J rise to mitogenic anti-CD3, PBMC 4 low responders (peak [Ca2+]i increase

responded poorly and only at very high doses. A possible explanation of these results would be a variable density of CD3 on the plasma membrane of the different lymphocyte populations. This appeared not to be the case, since cytofluorimetric analysis of CD3 expression revealed that the density of CD3 was similar in PBMC and in several of the responding clones or lines. In fact, after labelling with fluorescein-conjugated anti-CD3 mAb, the mean fluorescence intensity (in arbitrary units) of PBMC was 446 f 132 (n = 4 _+SD), while that of responding T

1 1

A

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1

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CBT3

1 min

d&k 4

-300 -110

a

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16

min

24

32

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Fig. 3 Effects of anti-CD3

mAb in purified T lymphocyte populations.

(A) Fluorimetric analysis of the effect of CBT3 on [Ca”Ji in

suspensions of T lymphocytes purified by rosetting with sheep erythrocytes. (B) Maximum increase in [Ca*+]i elicited by 2 pg/ml CBT3 in two T cell lines. The cells were stimulated at day 0 with CBT3 (W) or PHA (0) and then maintained in culture with IL2 as described in Materials and methods. At various days from the initial stimulation, an aliquot of the cells was removed, washed, loaded with Furaand analyzed in a cuvette. Other conditions as in Figure I. In the inset, the kinetics of [Ca”]i generated with CBT3, at day 0 (a). 14 (b) and 24 (c) are shown.

rise in a representative T cell line,

ANTI-CD3

INDUCED

173

ICa?‘]i INCREASE

value not very different from that observed with PHA as the mitogen (from 25-350/o), despite the grossly different effects of the two stimuli on [Ca2+]i (not shown). Thus the lack of response in terms of [Ca2’]i increase to anti-CD3 mAbs is not attributable to a low number of PBMC activated to enter the cell cycle.

-

-

-

[Ca2’li rises in single T cells [Ca2’li

-

5 3 F 8 Q

5 0

-

s

7

v-

G

-

-

Fig. 4 Distribution of the [Ca”]i

‘; P

-

responsein

T cell clones. The

clones were generated as described in Materials and methods with either CBT3 or 0KT3

mAb. Aliquots of cells from each

clone were loaded with Fura-

and stimulated with a fixed (2

pg/ml) CBT3 concentration. Other conditions as in Figure I. The clones were then grouped on the basis of their peak [Ca”]i response. The bars represent the number of clones showing a definite range of [Ca*‘]i. as defined in Figure 2. Similar results were obtained using OKT3 as the stimulating mAb.

lines and clones was 349 f 104 (n = 10 + SD) and 479 + 64 (n = 15 + SD), respectively. The differences in the capacity of anti-CD3 mAb to increase [Ca2+]i in T cell lines compared to PBMC and the progressive increase of the response during in vitro culturing with IL2 raised the possibility that the cells capable of proliferating and of responding with [Ca2+]i rises to anti-CD3 mAb represented a minor proportion of the CD3 positive lymphocytes; during weeks in culture with IL2 the highly responding clones would be selected. This possibility was investigated by determining the number of cells in S phase by ethidium bromide staining of DNA. The fraction of cells that 48 h after stimulation with anti-CD3 mAb were in S phase ranged between 16-30% of the total PBMC, a

was measured by cytofluorimetric analysis in Indo-l loaded cells. While a clear response in terms of [Ca2+]i in freshly isolated PBMC was easily detected with PHA or ionomycin, no measurable effect was observed with CBT3 and a very small increase was obtained with 0KT3 at high concentrations (> 10 j_tg/ml). Neither with 0KT3 nor with CBT3 was there any evidence for the existence of a highly responding subpopulation of cells (data not shown). In the last few years, it has been demonstrated that in a number of cell types the [Ca2+]i response elicited by agonists linked to PtInsP2 hydrolysis is often characterized by asynchronous oscillations of [Ca2+]i in single cells [25]. Were this the case with the anti-CD3 mAb, even the cytofluorimetric analysis might underestimate the [Ca2+]i response of a large proportion of the cells. The response to antiCD3 mAb was thus investigated at the single cell level by a computerized image analIsis system. By this technique, the changes of [Ca +]i can be followed continuously in each individual cell. Figure 5 shows the time course of [Ca2+]i in lymphocytes from a typical responding clone, named B3, challenged with CBT3. Similar results were obtained with the three other clones studied and with 0KT3 as the stimulating antibody (not shown). For the sake of simplicity the results obtained in three cells, typical of the behavior of at least 30 cells in the same field, are presented. Upon touching the glass surface most cells showed spontaneous, small, asynchronous oscillations of [Ca2+]i which tended to fade with time and eventually almost ceased within 3-5 min. Spontaneous oscillations of [Ca2+]i in cells adhering to glass have been previously observed in macrophages and neutrophils and attributed to stimulation of adherence receptors [26,27]. Addition of CBT3, after a lag phase of a few minutes, caused

174

CELL CALCIUM

an often abrupt, large rise of [Ca2’]i. After the initial spike, [Ca2+]i started to oscillate. The peak amplitude and the frequency of the [Ca2+]i spikes were

Fig. 5 Single cell analysis of the [Ca*+]i increases in anti-CD3 were loaded with Fun-2

quite variable from cell to cell and tended to decrease in frequency and amplitude with time. Two IL2 dependent T cell lines were also investigated: again, most cells responded to anti-CD3 mAb addition with an abrupt increase in [Ca2+]i, followed by a series of oscillations. Based on the results obtained on sus ensions of the same cell preparations, a rise Y+ in [Ca ]i was expected from single cell analysis of T cell lines and clones. Clearly, however, the asynchronous response between cells and the oscillatory behaviour may lead to a gross underestimation of the real peak of the [Ca2+]i increases not only when the signal is averaged over several thousands of cells, but also when single cells are analyzed for a brief period of time (as in cytofluorimetry, see Discussion). The single cell response of freshly isolated PBMC and purified T cells was then investigated. Similarly to the T cell clones and lines, a proportion of the cells showed one or more spontaneous small oscillation of [Ca2+]i which, in most cases, ceased within 3-5 min (not shown). Figure 6A shows the result of a typical experiment performed with PBMC and T cells. Quite unexpectedly, a large proportion of PBMC and of purified T cells responded with a [Ca2+]i increase to CBT3 (and 0KT3, not shown). In a number of similar experiments the proportion of responding cells plated on glass was 36 + 6% (n = 4 f SEM) and 51 + 14% (n = 4 f SEM) in PBMC and purified T cells, respectively. The kinetics of two responsive and one unresponsive cell from a population of freshly prepared PBMC is presented in Figure 6B. As with T cell clones and lines, the [Ca2+]i increased abruptly and slightly asynchronously, after a lag phase, followed by [Ca2+]i oscillations or waves of decreasing peak amplitude. These results appear in clear contradiction with those obtained in cell populations or at the single cell level using the cytofluorimetric analysis. A similar result, i.e. an oscillatory response of [Ca2+]i in PBMC treated by anti-CD3, was reported

mAb generated T cell clones. Cells from the CBT3-generated

clone 83

and allowed to sediment on a glass coverslip in a thermostatted chamber placed on an inverted fluorescence

microscope stage. The experiments were performed in KRH medium. Where indicated. 2 pg/ml CBT3 mAb were added. The responses of 3 different cells (panel I. 2 and 3) from the same clone a~ shown. For other conditions see Materials and methods. Although cells adhere to glass. vigorous mixing of the medium results in movemen! or detachment of the cells from the surface. Thus. in order to avoid addition artifacts, the antibody was diluted in 50 p1 of medium and the solution added to the chamber containing 500 ~1 of KRH without further stirring. Part of the lag phase is likely due to diffusion of the antibody in the chamber.

175

ANTI-CD.1 INDUCED [Ca’+], INCREASE

recently by Hess and coworkers [28], but in this last contribution no comparison with the data obtained in suspension was made. One major difference beA St-

1

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IIlooo~~

I

1

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OF

500..

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0

MO

400

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Fig. 6 PBMC

Single cell analysis of the [Ca”]i

response of PBMC

tween

experiments performed with imaging systems those carried out with cell suspensions is that in the first case the cells were stimulated while adhering to a solid substratum, glass. Two possible explanations of this discrepancy were thus considered: (i) activation of adherence receptors might prime T lymphocytes, permitting the activation of the [Ca*+], response by anti-CD3; and (ii) adherence of the antibody to the glass surface might result in an effective immobilization of the stimulus, increasing its efficiency of CD3 crosslinking and thus activation of the [Ca2+]i increase. The experiments presented in Figure 6A,C suggest that the latter possibility is the most likely. In fact, the percentage of CBT3 responding cells in a mixed PBMC population was almost identical whether glass or polylysine coated coverslips were employed (Figure 6A). On both surfaces the majority of the cells adhered. On the contrary, if the coverslip was pretreated with serum, or if serum was included in the buffer in order to saturate the protein binding sites on glass, cell adherence was dramatically reduced and most cells failed to respond with a [Ca*+]i increase to CBT3 (Figure 6A). However, if the cells were first allowed to adhere to glass or polylysine coated coverslips and then treated with serum and CBT3, a negligible response in terms of [Ca2+]i was observed (Figure 6A). Serum per se was not inhibitory, since the response of Jurkat cells in the presence of serum was indistinguishable from that of controls, both in cuvette and in single cell experiments. The above described experiments were carried out with 2 pg/ml CBT3. We also tried CBT3 concentrations I or 2 orders of magnitude lower, i.e those optimal for mitogenic stimulation (see Fig. 2). At these low antibody concentrations, oscillations of [Ca2+)i were observed, but their frequency and amplitude were indistinguishable from those occurring in untreated controls. In order to confirm that binding and immoand

(A) Percentage of antibody-responsive Where indicated. the coverslips were pretreated

and purified T lymphocytes.

(open bars) and T lymphocytes (hatched bars) in a representative experiment.

with simple medium, polylysine (IO Fg/ml) or FCS (3%). The cells were allowed to attach for 5 min and then stimulated with 2 pglml CBT3.

In the case of ‘polylys + FCS’

followed, 4 min later, by CBT3 increases caused by CBT3

,

(2 pg/ml).

the cells were first plated on polylysine and allowed to attach; 3% FCS was then added, No effect of this FCS concentration was observed in Jurkat cells.

(2 vg/ml) in three (I, 2 and 3) representative cells from freshly isolated PBMC.

PBMC plated on anti-CD3-coated

(B) Kinetics of [Ca2’]i

(C) [Cal’],

coverslips (cell 1.29). The coverslips were incubated in KRH medium containing 2 &ml

oscillations of of CBT3 at

20°C for I5 min. washed twice with fresh KRH and immediately used. The kinetics of [Ca*‘], increases of PBMC plated on untreated glass coverslips (cell 3.4. S) am shown for comparison.

176

CELL CALCIUM

0

0 Ouin2

A

A BCECF

o

l Fura-

B

100

200

300

400

100

p molele./lo6 cdls Fig. 7 Effects of loading with acetoxymethylestersof Quin-2, Fun-2 and BCECF on [‘HI-thymidine incorporation. (A) Loading with AM esters was carried out as described in Materials and methods.At the end of the loading procedure, aliquots of the cells were diluted in culture medium, incubated under standard conditions with CBT3 (IO0 ng/ml) and assayed for [3H]-thymidine incorporation, as described in Materials and methods, 72 h later. Results are expressedas % [‘HI-thymidine incorporation of controls, i.e. cells treated in the same way but without AM.

(B) The data of A were recalculated in terms of intracellular concentration of the dye; this was

determined on an aliquot of the cells used for [3H]-thymidine incorporationstudiesusing standardsof Quin-2, Fura-

and BCECF free

acid, Each point, for [3H]-thymidine incorporation. is the mean of triplicates and the data are pooled from 3 independent experiments and 5 different donors. Note that the scattering of data of A is larger than that of B, presumably due to the large variability in loading efficiency in different samplesof cells.

bilization of the stimulus on the coverslip surface was responsible for the different [Ca2+]i responses observed between cell suspension and single cell imaging, the glass coverslip was pretreated with anti-CD3. Figure 6C shows the kinetics of [Ca*+]i in two cells (traces I and 2) incubated on glass coverslips pretreated with anti-CD3. For comparison, the behaviour of three cells incubated on untreated glass coverslips (traces 3-5) is also presented. Cell 1 and 2 were chosen because they represent the two extreme responses under these conditions. In cell I, small waves of [Ca2’]i superimposed to a constantly elevated level; in cell 2, oscillations similar to those shown in Figures 5 62 6B were observed. The proportion of PBMC showing behaviours similar to those of cell 1 or 2 was 32 f 7% (n = 4 f SEM) of the total population analyzed. Also the cells plated on untreated glass underwent a series of oscillations (cell 3-5), but their amplitude was much smaller.

[Ca2’]i buffering and DNA synthesis Taken together, the above mentioned data demonstrate that the [Ca2+]i response of T cells to antiCD3 depends on the differentiation stage of the cells. In particular, freshly explanted T cells require either high doses of antibody or further crosslinking (by anti-antibodies or by adherence to glass) in order to elicit a Ca2+ response to anti-CD3 mAb. T lines and clones or the model cell Jurkat, on the contrary, respond to soluble anti-CD3. Since soluble anti-CD3 are highly efficacious as mitogenic stimuli, the question then arises as to whether, during several hours in culture, an efficient crosslinking, even at low mAb concentration, might occur either spontaneously or via interaction with Fc receptor bearing cells. A kinetic study of [Ca2’]i for periods longer than 60 min appears, however, hampered by a number of technical problems (photo-

177

ANTI-CD3 lNDUCED[Ca*+~lNCREASE

bleaching, cell damage by UV light, etc.). More important, the key question is not only whether late [Ca*+]i increases might be triggered even in PBMC, but also to what extent these increases, if they occur, are important in the stimulation of DNA synthesis. In order to answer this question, we tried to buffer intracellular Ca*+ by loading the cells with high concentrations of Ca*+ chelators. If [Ca*+]i increases do occur during culture and they play a major role in triggering DNA synthesis, it is predicted that increasing the soluble cytosolic Ca*+ buffering capacity would result in blunting of the [Ca”]i increases and inhibition of [“HJ-thymidine incorporation. Figure 7A shows that loading the cells with the Ca*+ indicator and buffer Quin-2 caused a dose dependent inhibition of anti-CD3 mAb-induced [“HI-thymidine incorporation. Quin-2 was preferred to BAPTA, usually employed in this type of experiment, because the latter is not fluorescent and its cellular concentration is hard to determine (see also below). Although the experiment of Figure 7A might indicate that indeed [Ca2’]i increases are vital for stimulating DNA synthesis, it should be taken into account that Quin-2 can also bind other cations in the cytosol (heavy metals in particular) and that the hydrolysis of Quin-2/AM releases in the cytoplasm two compounds (acetate and formaldehyde) which by themselves might be inhibitory [29]. In order to distinguish among these possibilities, the cells were loaded with Fura-2/AM and his-carboxyethyl-carboxy-fluorescein (BCECF). Furais a Ca2* chelator, like Quin-2, but has a lower affinity for heavy metals [30], while BCECF is a pH indicator and binds neither Ca*+ nor heavy metals [22,3 I]. Figure 7A shows that for the same concentration of extracellular acetoxymethylester. Quin-2 was similar to BCECF and more effective at inhibiting DNA synthesis than Fura-2. However, when the same data were replotted as a function of the intracellular concentration of dye eventually achieved, the three compounds were almost equally effective. The discrepancy between the concentration of acetoxymethylester added and the final intracellular concentration achieved is likely attributable to the well known differences in loading efticiency of the various dyes [32]. We also confirmed that, under our ex erimental conditions, removal of exY+ tracellular Ca with EGTA results in complete sup-

pression of DNA synthesis. However, Ca*+ removal is a drastic condition, which may affect other cellular parameters (e.g. viability, adherence receptors, etc.) and the interpretation of this type of experiment solely in terms of [Ca*+]i highly problematic.

Discussion Understanding at the cellular and molecular level the signalling cascade initiated by antigen-T cell receptor interaction is not only of paramount importance in basic immunology, but also in the understanding of immunopathological processes. For example, compelling evidence has been obtained indicating that during HIV infection not only does the number of helper lymphocytes decrease, but also the capacity of T cells to be activated in vitro is reduced [33-351. Some authors have even suggested that AIDS is, at least in part, due to a pathological derangement in T cell signal transduction [35]. Polyclonal mitogens such as plant lectins or mAb directed against the T cell receptor have been widely employed as convenient in vitro models to study the process of signal transduction in T lymphocytes [28,36]. This approach offers a number of experimental advantages compared to the physiological stimulation by specific antigens: (i) it does not require the use of antigen-presenting cells; (ii) it can be applied both to freshly isolated cells and to cell clones; and (iii) it is more amenable to biochemical investigation. The implicit assumption is that the intracellular events activated by polyclonal mitogens closely mimic what happens in vitro and in vivo when the appropriate antigen is presented to T lymphocytes. The demonstration that in T cell clones specific antigens also induce tyrosine phosphorylation, PtInsP2 hydrolysis and [Ca*+]i increases, further strengthened this hypothesis [ 371. Given the heterogeneity of freshly isolated lymphocytes, in the last few years T cell clones (or T cell lines) have become the most popular model for these studies, based on the assumption that the response of clones to antigens and mitogens, in general, faithfully reproduce the response of ‘normal’ lymphocyte populations. Our data demonstrate that both these assumptions should be taken with caution, at least as far as

178

the coupling of CD3 to [Ca’+]i increases is concerned. In fact, the three anti-CD3 mAb employed in our study, all mitogenic for PBMC and capable of activating early gene transcription (data not shown), were quite ineffective on [Ca2+]l when tested under standard conditions, i.e. with cytofluorimetry or in fluorimeter cuvettes. All these anti-CD3 mAbs, on the contrary, caused large [Ca2+]i increases under the same conditions in Jurkat cells and in a large proportion of T cell clones. We believe that these observations might explain some of the numerous contradictions existing in the literature concerning this issue. For example, when using the same mAb, OKT3, Hess and coworkers showed that [Ca*‘]l increases required further crosslinking [28], while Oettgen and coworkers [38] demonstrated that large responses were elicited by soluble mAbs. In the first case, freshly explanted T cells were employed, in the second a T cell clone. Similarly, Wacholtz and Lipsky [ 131 reported a strong response of PBMC to soluble OKT3 in imaging experiments, while this antibody failed to elicit significant [Ca2+]i rises when tested in soluble form (present results). In theory, cuvette, cytofluorimetric and image analysis experiments should give qualitatively, though not quantitatively, similar results. This is clearly not the case. In particular, the use of an apparently ‘neutral’ substratum, such as glass, in the imaging experiments drastically alters the response to anti-CD3, because it efficiently binds the antibody, thus mimicking the effect of an anti-antibody in cell suspensions. Confirmation that the binding and immobilization of the antibody onto glass is the cause of the qualitative change in the response of PBMC in this type of experiments comes from the experiment carried out in the presence of serum. The serum proteins presumably saturate the binding sites on glass, thus preventing immobilization of the stimulatory antibody. In other words, in the presence of serum, the imaging data more faithfully represent the condition in suspension. A third important variable is the nature of the immunological epitope recognized by anti-CD3. For example, the anti-CD3 mAb Gl9-4 (see for instance [ 121) has been reported to cause large increases of [Ca2+]l in PBMC suspensions, while the commercially available OKT3 and Leu4 are quite ineffective. The reason for this qualitative difference is presently unknown. The different re-

CELL CALCIUM

quirement of PBMC and IL2-dependent blasts in terms of coupling to the Ca2+ pathway is unexplained in molecular terms, but it indicates a major role of the differentiation stage on the Ca2+ signalling mechanisms. In this respect, our data nicely complement those of Steinman and coworkers [39,40], who clearly demonstrated that the requirement of accessory signals for effective stimulation of freshly explanted T cells and of IL2 dependent clones are quite distinct. A last point of great relevance, not only from the methodological point of view, needs stressing: as observed here and by Hess and coworkers [28], the [Ca2+]l increases induced in T lymphocytes (PBMC, T lines and clones) are oscillatory in nature. In the last few years, measurement of [Ca2+]i by cytofluorimetric analysis has become the most popular method among immunologists. With this technique cells are analyzed not only in suspension, but also for a very brief period of time (a few ms/cell). Thus, if oscillations occur also in suspension, the time course of [Ca2+]l increases, the number of responding cells and the amplitude of the response, as measured by cytofluorography, are all subjected to large errors. A cell may in fact be analyzed before the onset of the [Ca2+]i increase, at the peak of the response, during the falling phase or at the bottom of a [Ca2+]l spike. In other words, cytofluorimetric analysis of [Ca2+]i could easily generate false negatives or suggest the existence of cell subpopulations simply on the basis of the asynchronous [Ca2+]l oscillations in each individual cell. From a physiological point of view, the main question raised by the present data concerns the role, if any, played by [Ca2+]l increases in the signalling cascade initiated by T cell receptor stimulation and eventually leading to DNA synthesis and cell division. Several arguments can be used in favor of [Ca2+]i increases as necessary components of the mitogenic signal: (i) Immobilized anti-CD3 mAb (on plastic, sepharose beads, etc) are known to be more efficient as mitogenic stimuli compared to the same mAb in soluble form [4l]. Effective immobilization of anti-CD3 mAb is expected to result from interaction of the antibody with the Fc receptors on antigen presenting cells. (ii) The requirement for antibody-monocyte

ANTI-CD?

INDUCED

[Ca”],

179

INCREASE

interaction for effective mitogenic stimulation of T cells by anti-CD3 the

IgGl

individuals

subtype

are

expressing

mAb

non mitogenic

the isoform

in

of the FC

Research to TP, AA, FM and to LCB.

We are indebted to Mr G. Ronconi,

mAb is further supported by

the observation that monoclonal anti-CD3 of

Scientific

Santato for skillful Silvestro

technical

and P. Marson

General Hospital

G.A. Milani

and M.

assistance and to Dr G. De

of the Blood

for continuous

supply

Bank

of Padova

of blood

samples.

receptor which does not bind the mouse IgGl 1421.

(iii)

Last, but not least, removal of Ca*’ medium

in the first hours following

from the

addition invariably results in abortive stimulation of DNA

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: 6 April 1994 : I3 May 1994