FcϵRI and the T cell receptor for antigen activate similar signalling pathways in T cell-RBL cell hybrids

Cellular Signalling Vol. 5. No. 2, pp. 155-167, 1993. Printedin Great Britain.

Fc~RI A N D SIMILAR

0898-6568/93$6.00 + 0.00 PergamonPressLtd

THE T CELL

SIGNALLING

RECEPTOR

PATHWAYS

FOR ANTIGEN

IN T CELL-RBL

ACTIVATE CELL HYBRIDS

NADIA MARANO,*Jf MARY ANNE LIOTTA,~'~ JAMES P. SLATTERY,§ DAVID

HOLOWKA*I[

and BARBARABAIRDII¶ *Department of Biology, Middlebury College, Middlebury, VT 05753, U.S.A., :~Section of Biochemistry, Molecular and Cell Biology, §Biotechnology Program Flow Cytometry and Imaging Facility, and IlDepartment of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301, U.S.A. (Received 22 June 1992; and accepted 1 October 1992)

Abstract--In order to investigate the functional similarities of the high affinity receptor for IgE (Fc~RI) and the T cell receptor for antigen, we have developed a high efficiency polyethylene glycol-mediated fusion method to make somatic hybrids between cells from a mast cell line (RBL-2H3) and cells from T lymphoma cell lines (Jurkat and HPB-ALL). Using flow cytometry to select for the heterologously fused cells, we demonstrated that aggregation of the T cell receptor results in the efficient secretion of [3H]5-hydroxytryptamine from RBL cell-derived granules. In addition, both receptors mediate Ca 2÷ mobilization in the hybrid cells that is insensitive to inhibition by the protein kinase C activator phorbol-I 2-myristoyl-13-acetate (PMA). In contrast, Ca 2÷ mobilization caused by aggregation of Fc~RI in the parent RBL cells is completely inhibited by PMA. The results indicate that these two different receptors for foreign antigen can substitute for each other to trigger responses in the hybrid cells that are unique to each cell type. The methodology employed has general utility for studying signal transduction mediated by mammalian cell surface receptors. Key words: IgE receptor, mast cells, signal transduction, Ca2÷ mobilization, cell fusion.

Fc, RI. The TCR complex consists of at least six different polypeptides. The variable ~t-fl heterodimer binds peptide antigens in association with MHC molecules on the surface of target or accessory cells [2, 3]. These variable chains of the TCR are associated with three invariant polypeptides, ~, 6 and e, collectively referred to as CD3 and a disulphide-linked dimer of either two ~ chains or ( and ~/(an alternatively spliced form of () [4]. Antibodies to TCR subunits are able to stimulate the signals that lead to T cell activation, probably as the result of receptor aggregation [1, 5]. The early events that can be triggered by soluble antibodies include increased hydrolysis of phosphatidylinositol phosphates, increased concentration of cytoplasmic calcium and increased activity of both serine/threonine and tyrosine kinases [6]. RBL-2H3 (RBL) cells are from a mucosal mast cell line and express Fc~RI on the cell

INTRODUCTION RECEPTORS in the immune response that mediate cellular activation by foreign antigens have recently been recognized as having some common structural and functional characteristics that suggest a much closer relationship than previously appreciated [1]. Two such receptors of the immune response are the T cell receptor for antigen (TCR) and the high affinity receptor for immunoglobulin E (IgE),

"['The first two authors in this study contributed equally to the work described. ¶Author to whom correspondence should be addressed. Abbreviations: BSS---buffered salt solution; DNP-BSA-dinitrophenyl conjugated to bovine serum albumin; FITC--fluorescein isothiocyanate; 5HT--5-hydroxytryptamine; IgE--immunoglobulin E; PE--phycoerythrin; PEG--polyethylene glycol; PMA--phorbol-12-myristoyl13-acetate; T C R - - T cell receptor for antigen. 155

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N. MAI~Noet al.

surface. Fc~RI consists of an ~t subunit which contains a binding site for the Fc portion of IgE, a fl subunit and two disulphide-linked ( subunits which show some sequence homology to the ( subunit of TCR [7]. Upon aggregation, these receptors initiate cellular degranulation that results in the release of mediators of the immediate allergic response, such as histamine and 5-hydroxytryptamine (serotonin). As with the TCR, the early events in signal transduction of RBL cells include increased hydrolysis of phosphatidylinositol phosphates, increased cytoplasmic calcium and increased kinase activity, as well as stimulated production of arachidonic acid and eicosanoid metabolites [8, 9]. Recently, it has become clear that the ( subunit of T C R and the 7 subunit of Fc~RI play important roles in signal transduction mediated by these receptors [10-12]. In addition, these subunits are required for their respective receptors to be efficiently expressed on the cell surface. Furthermore, it has been shown that TCR ( can substitute for Fc~RI ~ and vice versa in allowing assembly and transport of these receptors to the cell surface [13]. In previous experiments, we used a high efficiency polyethylene glycol (PEG)-mediated membrane fusion method to demonstrate that IgE receptors that had been dissociated from their native cellular interactions retain the ability for signal transduction in other RBL cells [14]. Similar cell fusion methods have also been used by Goldsmith et al. [15, 16] to demonstrate complementation between different signal transduction mutants of Jurkat cells. In the present study, we demonstrate that it is possible to trigger the efficient release of [3H]5-hydroxytryptamine ([3H]5HT) from RBL cell-derived granules of T ceI1-RBL hybrids by stimulation of TCR with the anti-CD3 monoclonal antibody OKT3[17]. Furthermore, we find that the Ca ~÷ response stimulated by aggregation of Fc,RI in these hybrid cells has lost its normal sensitivity to inhibition by PMA. The results suggest that the signal transduction pathways stimulated by these two receptors are highly complementary, but not identical.

MATERIALS AND METHODS Antibodies and triggering reagents

Ascites fluid containing the anti-CD3 monoclonal antibody OKT3 [17] was a gift of Dr Stephen Shaw (National Institutes of Health). OKT3 was purified from the ascites fluid as described [18] or by ion exchange chromatography using a Pharmacia Mono Q column. For this purification, the antibody was loaded on to the column in low ionic strength buffer consisting of 20mM Tris sulphate, pH 7.5, with 0.02% NaN 3. The column was eluted with a gradient of 0-250 mM Na2SO 4 in the same buffer with OKT3 eluting at approximately 42mM. The anti-CD2 monoclonal antibody (clone G42-211) was obtained from Pharmingen. Phycoerythrin (PE)-labelled goat anti-mouse IgG antibody was obtained from Southern Biotechnology. Mouse monoclonal anti2,4-dinitrophenyl IgE (H1 DNP-e-26-82) was purified from ascites [19] as previously described [20]. The purified monoclonal anti-Fc,-RI antibody CD3 [21] was the gift of Dr Reuben Siraganian (National Institutes of Health). Fluorescein isothiocyanate (FITC)-IgE was prepared as previously described [22]. Bovine serum albumin conjugated with an average of 15 dinitrophenyl groups (DNP-BSA) was prepared as previously described [23]. Ce//s

The human T cell leukemia line Jurkat was a gift of Dr Phillip Rosoff (Tufts University School of Medicine) and was maintained in RPMI 1640 medium supplemented with 5% foetal bovine serum, 0.5ml/l Mito+ serum extender (Collaborative Research), 4 mM L-glutamine, 10 U/ml penicillin and 10 U/ml streptomycin. The HPB-ALL human T cell line was obtained from Becton Dickinson and was maintained in the same medium except with 100 foetal bovine serum, 1 ml/I Mito+ serum extender and 0.1 mM sodium pyruvate. Both T cell lines were grown in a humidified atmosphere containing 5% CO 2 and harvested by centrifugation. RBL cells (subline 2H3, [24]) were grown adherent in sealed flasks in Minimal Essential Medium (MEM) supplemented with 10% foetal bovine serum, I ml/I Mito + serum extender, 5raM L-glutamine and 10#g/ml gentamicin (complete medium). Confluent adherent cells were harvested after 5-6 days in culture with trypsin-EDTA (Gibco). RBL cells were replated to a density of 7x 104 cells/cm 2, 12-18h prior to cell fusion.

Cell fusion The fusion procedure was adapted from previous work [14] and was carried out with adherent R B L

Signalling in T celI-RBL cell hybrids cells in MEM buffered with 20 mM HEPES, pH 7.4, at ambient temperature. The cells were rinsed with the buffered MEM and treated for 5 min with wheat germ agglutinin (7.5 ttg/ml for Jurkat cells, 1.5/~g/ml for HPB-ALL cells) to promote adherence of the T cells. 2 x 107 Jurkat cells or 1 x 107 H P B - A L L cells per 25 cm 2 flask were added to the RBL cells and incubated for 20 min at room temperature. Then nonadherent T cells were decanted and 50% polyethylene gylcol (PEG) 1540 (Polysciences, Warrington, PA) in buffered MEM was added. After 1 min the supernatant was diluted about 10-fold with complete medium; cells were washed and fresh complete medium with DNase I (50/tg/ml) was added. After a recovery period of 1 h at room temperature followed by 1 h at 37°C, non-adherent and dead cells were decanted. Then the adherent cells were harvested with trypsin-EDTA and washed by sedimentation and resuspension in fresh medium. For control samples, RBL cells alone (no T cells added to the flask) or T cells alone (wheat germ agglutinin added to an empty flask before addition of T cells) were taken through the same fusion procedure.

Flow cytometry A Coulter EPICS 753 (Hialeah, FI) flow cytometer was used to separate R B L - T cell hybrids from other cells for degranulation assays and also to monitor changes in intracellular calcium. After the fusion procedure, the harvested cells were incubated with 2/~g/ml anti-CD2 for 20 min at 4°C. Cells were then centrifuged, resuspended in balanced salt solution (BSS; 135mM NaCI, 5 m M KCI, l m M MgCI2, 1.8mM CaCI2, 5.6mM glucose, 20mM HEPES, pH7.2, and 0.1% gelatin) and incubated with 4/~g/ml PE-labelled goat anti-mouse IgG antibody for 20 min at 4°C to label cell-bound anti-CD2. Cells were sedimented for 5 min at 200 x g through a cushion of 10% sucrose in BSS and resuspended for sorting in BSS without protein. Sorting was carried out using two parameters: PE fluorescence and forward angle light scattering. RBL and hybrid cells are larger than the T cells and thus scatter more light. CD2 is present only on T cells and hybrid cells. Therefore, RBL-T cell hybrids have a large forward angle light scatter and high PE fluorescence. The PE was excited with the 514-nm emission from an argon ion laser (Coherent Innova 90-4). Fluorescence emission from PE was selected with a 575-nm band pass filter (Omega Optical, Brattleboro, VT). For degranulation assays, the T celI-RBL hybrids were collected under sterile conditions and recultured overnight in complete medium containing [3H]5HT and IgE (see below). Cells to be used for calcium measurements were loaded after the fusion procedure CELLS S:2-E

157

with indo-1 [25] as follows: cells (1 x 107/ml) were incubated for 1 min at 37°C with 8/~M of the acetoxymethyl ester of indo-I (Molecular Probes, 50/tg in 50 pl DMSO; 25/~1 pleuronic acid (stock = 135mg/ml) and 750#1 newborn calf serum in BSS without gelatin and containing 1 mg/ml BSA and 25 mM sulphinpyrazone to prevent leakage of the dye [26]. The suspension was then diluted 10-fold in the same buffer and incubated for another 30 min at 37°C. Cells were sedimented, resuspended in BSS at 4°C and labelled with anti-CD2 and PE-labelled second antibody as described above. Cells were kept at 4°C until analysis in the flow cytometer, where they were warmed to 37°C. For these experiments indo-I was excited by the 360 nm light from an argon ion laser (Coherent Innova 90-5). Fluorescence emission that passed through a 560-nm dichroic short pass filter was further selected for emission from the indo-l-Ca 2÷ complex with a 395-nm band pass filter, or emission from the un-complexed form of indo-I with a 440-nm dichroic long pass filter, followed by a 408-nm long pass and a 525-nm band pass filter. Fluorescence reflected from the 560-nm dichroic filter was further selected with the 575-nm band pass filter for PE emission (all filters from Omega Optical, Brattleboro, VT). The ratio of the analogue signals from the short- and long-wavelength emissions of the indo-1 was obtained using the Analogue Function 3 card (Coulter Electronics) and plotted as a function of time. Signals were appropriately gated and triggered using the 360-nm forward angle light scatter detected through a 365-nm band pass filter. After a baseline was obtained, cells were triggered with OKT3 or the anti-Fc, RI monoclonal antibody CD3 [21]. For some experiments, cells were treated for 1520 min at 37°C with 70 nM PMA (Sigma, St Louis, MO) prior to triggering.

Purification of T celI-RBL hybrids using magnetic anti-CD2 beads Prior to fusion, RBL cells were sensitized with IgE and granules loaded with [3H]5HT in an overnight incubation. After the fusion procedure, cells were harvested and resuspended at a concentration of 107/mi. Dynabeads M-450 coated with anti-CD2 (Dynai) were added at a ratio of 3:1 to the hybrid cells estimated to be present in the fused cell mixture (approximately 20% of the total) and incubated on a rotator at 4°C for 30 min. Beads with attached cells were collected by applying a magnetic particle concentrator (Dynal) to the side of the tube for 1 min. Cells not attached to the side of the tube were aspirated along with the supernatant. Cells attached to beads were washed three times and used immediately for a [3H]5HT release assay.

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Degranulation assays

After sorting by flow cytometry, T celI-RBL hybrids, or RBL cells and T cells taken independently through the fusion procedure were incubated overnight with 30#Ci [3H]5HT and 5/zg IgE per 10ml. Cells were harvested as above, washed in MEM with 5% foetal bovine serum and 20mM HEPES, pH 7.4, incubated for 30 min at 37°C, then centrifuged and resuspended in BSS at 0.5-2 × 10 6 cells/ml. Cells were plated into 96-well assay plates and triggered for l h at 37°C with 0.01-1 #g/ml OKT3 or 0.1/zg/ml DNP-BSA. Supernatants were harvested using a Supernatant collection system (Skatron Inc., Sterling, VA) and 3H-radioactivity counted as previously described [27] using a Beckman LS no. 1801 liquid scintillation counter. lmmunofluorescence

RBL cells were labelled overnight with FITC-IgE, then fused with Jurkat cells as described above. After the fusion procedure, cells were incubated with 1 #g/ml OKT3 for 20 min at 4°C, then centrifuged, resuspended in fresh buffer and incubated with 2 pg/ml PE-labelled secondary antibody for 20 min at 4°C. Cells were then washed, incubated for 15 min at 37°C to allow for internalization of the receptorbound OKT3 and examined using a Leitz Ortholux II fluorescence microscope with fluorescein and rhodamine optics as previously described [28]. Both PE and FITC can be seen using the fluorescein optics, but they appear as different colours (yellow and green, respectively). Photography was with Fujicolor 1600 ASA film, using the automatic exposure setting of an Olympus OM-2 camera mounted on the microscope. RESULTS Using the PEG fusion method described in Materials and Methods we were able to obtain efficient fusion between the human T cell lines Jurkat or HPB-ALL and the rat mucosal mast cell line RBL-2H3. As seen in the fluorescence micrographs of Fig. 1, the hybrid cells are large (>/ 10#m diameter) and stain positively for both IgE receptors and TCR. In Fig. IA, the uniform peripheral stain seen on all of the cells in fluorescein optics is green and is due to FITC-IgE bound to RBL-derived Fc, RI receptors. The bright internal fluorescence seen in two of the cells is yellow in fluorescein optics and is due to PE-anti-mouse IgG staining of

O K T 3 - T C R complexes. After labelling at 4°C, these complexes are initially seen as patches at the cell periphery, but quickly become internalized when the slide warms to room temperature on the microscope stage. We allowed this internalization because the resulting PE fluorescence was most easily distinguished from the surface fluorescein fluorescence. Figure I B shows the fluorescence image of the same field of cells viewed in rhodamine optics. The two doubly stained cells show the intracellular fluorescence due to PE-labelled TCR; fluorescence from the FITC-IgE is not visible with these optics. The fused cells can be sorted preparatively by flow cytometry. For identification, the cells were incubated with an anti-CD2 monoclonal antibody and PE-labelled anti-mouse IgG such that the T cells and T cell-containing hybrids would be labelled [29]. Then the cells were sorted for both PE fluorescence and size, as determined by forward angle light scatter. The fused cells distribute into two major populations (Fig. 2A): one that is large and positive for the CD2 marker (quadrant 2) and one that is large but negative for the CD2 marker (quadrant 1). The latter are RBL cells and R B L - R B L fusion products, as judged by the similar distribution that is obtained when RBL cells are taken through the fusion procedure in the absence of T cells (not shown). There is a small population of unfused T cells and T cell-T cell hybrids as judged by T cells that have been taken through the fusion procedure in the absence of RBL cells (not shown); this is seen as the small cells that stain positively for CD2 (quadrant 4). These cells are rare in the fused population, because they do not adhere to plastic and are mostly removed after the fusion step prior to sorting. Together, these results indicate that the population of cells in quadrant 2 is highly enriched in T cell-RBL hybrids. These hybrid cells represent 7-26% of the total cells sorted in six different fusion experiments using either Jurkat or HPB-ALL cells as the T cell partner (Table 1, column 5). Figure 2B shows the analysis of this hybrid cell population represented by quadrant 2. In

FIG. 1. Photomicrographs of T celI-RBL hybrids stained with FITC-IgE and anti-CD2 followed by PE-labelled goat anti-mouse IgG. (A) Fluorescein optics: both fluorescein and PE are visible; and (B) rhodamine optics (only PE is visible). Scale bar = l0/~m.

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160

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~Per cent release of [3H]5HT

*RBL cells were fused with T cells; hybrid cells were selected via flow cytometry; Fc~RI was sensitized with lgE, and granules were loaded with [3H]5HT for 18 h. The cells were then stimulated with OKT3 or DNP-BSA and [3H] released was assayed as a measurement of degranulation. tPer cent of each cell population was determined via quadrant analysis of the cell sorting profiles (see text).

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this experiment, about 80% of the cells re-sort to quadrant 2, indicating that a large percentage of these preparatively sorted cells are the T cell-RBL hybrids. To confirm the identity of the population of cells in quadrant 2, the hybrid cells separated in the preparative sort were incubated with FITC-IgE and OKT3, followed by PE-labelled goat anti-mouse IgG and > 75% were found to be labelled with both IgE and OKT3 by fluorescence microscopy. The T cell-RBL hybrids enriched by flow cytometry were used to test whether the TCR complex could activate the cellular systems necessary to stimulate release of [3H]SHT from RBL-derived granules. Following sorting, RBL-derived granules of the T cell-RBL hybrids were loaded with [3H]SHT and the Fc~RI was sensitized with IgE during an overnight incubation. Following washing to remove unincorporated [3H]5HT and IgE, the release of [3H]SHT in response to cross-linking the IgE receptor with DNP-BSA was compared to the release in response to cross-linking TCR with the anti-TCR monoclonal antibody OKT3. The T cell-RBL hybrids released [3H]SHT in response to OKT3 in a dose-dependent manner (Fig. 3A). Release of [3H]5HT at the maximum OKT3 dose tested in this experiment, 1 #g/ml, was similar to that obtained with an optimal dose of DNP-BSA. Antigen-stimulated release of [3H]SHT from RBL cells taken through the fusion procedure separately was 35% in this experiment, similar to the value for the hybrid cells in Fig. 3A. T cells taken through this fusion procedure take up very little [3H]5HT compared with RBL cells (or the hybrids) and insignificant [~H]5HT is released by the T cells in response to OKT3 or DNP-BSA (Fig. 3B). Figure 3B also shows that RBL cells taken through the fusion procedure in the absence of T cells readily take up [3H]SHT and this is efficiently released by DNP-BSA, but not released in response to OKT3. Hybrid cells that are loaded with [3H]SHT give similar values for total cell-associated [3H]-c.p.m./105 cells as shown for RBL cells in Fig. 3B, indicating that cell-bound anti-CD2 plus PE-anti-IgG does not significantly trigger degranulation under the

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FIG. 3. Stimulation of degranulation from the T celi-RBL hybrids. (A) Jurkat-RBL hybrids are selected by flow cytometry and triggered with 0,01-1 #g/ml OKT3 or with 0.1/ag/ml DNP-BSA. Results are from a single experiment (no. 4 in Table 1). The ordinate represents percentage of [3H]5HT released after subtraction of spontaneous release (4.4% in this experiment). Error bars represent the range of duplicate samples for these measurements. (B) Controls for RBL and Jurkat cells were incubated overnight with [3H]5HT, then separately taken through the fusion procedure and treated with l#g/ml OKT3 or with 0.1 #g/ml DNP-BSA. The ordinate represents average [3H]5HT c.p.m./1 x 105 cells in three experiments. Error bars represent the standard deviation of triplicate samples from each of the three experiments. conditions of these experiments (data not shown). Results from four different experiments with Jurkat-RBL hybrids and two with HPB-ALL-RBL hybrids are summarized in

Signallingin T celI-RBLcell hybrids Table 1. In order to compare the amount of [3H]5HT release from T ceI1-RBL hybrids triggered via TCR vs Fc,RI, the amount of [3H]5HT release stimulated by DNP-BSA was corrected for the amount of release due to the contaminating unfused RBL cells and RBL-RBL hybrids. The percentages of these two cell populations were determined in the analytical sort of the preparatively sorted cells (Table 1, columns 7, 8) and the percentage release due to DNP-BSA was assumed to be the same in both populations. The last column in Table 1 shows the ratio of [3H]5HT release mediated by TCR compared to that mediated by FC~RI in the T ceI1-RBL hybrids. The relative efficiencies of TCR-Fc~RI stimulation range from 0.4 to 2.2 in these experiments, indicating that the TCR is similar to Fc, RI in its capacity to trigger [3H]5HT in these T cellRBL hybrids. To address the possibility that the OKT3induced release of [3H]5HT from the T cellRBL hybrids could be due to loading of [3H]5HT into T cell granules during the overnight incubation of the fused cells, a separate experiment was carried out in which the RBL cells were loaded with [3H]5HT prior to fusion with HPB-ALL cells. In this experiment, hybrids were separated with anti-CD2-conjugated magnetic beads and tested for release of [3H]5HT within 3 h after fusion. OKT3-stimulated release of [3H]5HT in this experiment was found to be comparable to the experiments shown in Table 1, which rely on preparative flow cytometry, followed by overnight loading with [3H]5HT (data not shown). To characterize further the signal transduction properties of these hybrid cells, we used flow cytometry to test the ability of TCR and Fc, RI to mediate an increase in the concentration of cytoplasmic Ca 2+. After fusion and recovery, cells were loaded with the calciumsensitive dye indo-I [25] and labelled with antiCD2 followed by a secondary antibody conjugated with PE. By using the flow cytometer and quadrant analysis we could monitor simultaneously the calcium response from the three different cell populations present after the

163

fusion: T cells (or T-T hybrids), RBL cells (or RBL-RBL hybrids) and T celI-RBL hybrids. Under these conditions, the T cell marker antibodies (anti-CD2 plus PE-anti-mouse IgG) did not stimulate a Ca 2+ response in any of the cell populations. Figure 4 shows the Ca 2+ responses of hybrid cells, RBL cells and T cells in the absence (A-D) and presence (E-H) of PMA. As seen in Fig. 4A, the hybrid cells show a strong, transient Ca 2+ response to the monocional anti-Fc, RI antibody that follows a lag phase of approximately 1 min. Figure 4B shows a similar response of the hybrid cells to OKT3. As expected, the T cells respond to OKT3, but the anti-Fc, RI does not stimulate a response in the T cells when added either after (Fig. 4C), or before OKT3 (data not shown). Although the T cells in the fusion mixture responded poorly to OKT3 in the experiment shown, in other experiments their response to OKT3 was more typical of that in untreated T cells and similar to that for hybrid cells (Fig. 4B). Figure 4D shows that RBL cells in the fusion mixture do not respond to OKT3, but are stimulated by the anti-Fc~RI as expected. The strongly biphasic nature of the responses seen for T cells and T ceI1-RBL cell hybrids in these experiments is generally more exaggerated than that for RBL cells (Fig. 4D), or for T cells not prelabelled with anti-CD2 plus PE-anti-IgG. In control experiments, prebound anti-CD2 plus anti-IgG does not stimulate a significant Ca 2+ response in the T cells in the absence of OKT3, but it appears to cause a larger initial response to OKT3 that is followed by a rapid decline to a plateau level (Fig. 4B) or occasionally to baseline (Fig. 4A). The results in Fig. 4A-D demonstrate that, as for degranulation, the T ceI1-RBL cell hybrids can mobilize Ca 2+ in response to aggregation of either TCR or IgE receptors. Additional insight into the mechanism of receptor-stimulated Ca 2+ responses in the hybrid cells was gained by investigating the sensitivity of this response to inhibition by a brief pre-treatment with PMA. The Ca 2+ response of Fc~RI is known to be completely

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FIG. 4. Calcium responses of T celI-RBL hybrids (A, B, E, F), T cells (C, G) and RBL cells (D, H) in the fusion mixture. Fused cells were loaded with indo-1 and labelled with anti-CD2 and PE-goat anti-mouse IgG prior to analysis. (A-D) Aliquots of cells were warmed to 37°C and triggered with 1 #g/ml OKT3 or 6 #g/ml anti-Fc,RI receptor monoclonal antibody at the times indicated by the arrows. (E-H) Same as A-D, but cells were treated with 70 nM PMA for 15 min at 37°C prior to stimulation.

inhibited by this treatment for RBL cells that are maintained in suspension [30, 31] and this is seen with the RBL cells in the fusion mixture in Fig. 4H. Ca 2+ responses to aggregation of TCR in Jurkat cells are little affected by a brief pretreatment with PMA [32, 33, N. Marano, unpublished results] and the data in Fig. 4G show that the T cells from the fusion mixture give a strong Ca 2+ response to OKT3 under these conditions. As expected, subsequent addition of anti-Fc,RI gave no further response in these cells. As shown in Fig. 4F, pre-treatment of the hybrid cells with PMA did not significantly inhibit their Ca 2÷ response to OKT3 (cf. Fig. 4B). Figure 4E shows that pre-treatment of the hybrid cells with PMA does not inhibit the IgE receptor-mediated response (cf. Fig. 4A), even though the response in RBL cells from the same mixture is completely inhibited (Fig. 4H). Similar results were obtained in three different experiments and they indicate that components from the T cells are able to complement the RBL components in cells that are inactivated by treatment with PMA. These results provide further evidence that these two different immu-

noreceptors, TCR and Fc, RI, have common signalling mechanisms that utilize similar pathways to generate similar (but not identical) biological responses. DISCUSSION The present study utilized a high efficiency cell fusion method to generate a population of hybrid cells in sufficient quantity to assess the ability of receptors from one cell type to stimulate a functional response that is specific for a second cell type. In this study, human T lymphoma cells (Jurkat and HPB-ALL) were fused to rat cells from a mucosal mast cell lineage (RBL cells) [34]. Receptors that normally respond to foreign antigen in each cell type were examined for their ability to trigger a response requiring components from the other cell type. This approach is potentially applicable to a wide variety of receptors in diverse cell types. It should provide a useful complement to molecular genetic approaches, when it is difficult to obtain functional expression of the transfected genes of a cloned receptor in a

Signallingin T CelI-RBLcell hybrids particular foreign cell type. Fc~RI, for example, can be transiently expressed on the surface of COS cells, but lacks the ability to mediate signal transduction in this cell type [35, 36]. It should be possible to test mutants of Fc~RI for function by efficient fusion of COS cells with the T cell lines, followed by assessment of the ability of these hybrids to respond to ligands which aggregate this receptor. The key to obtaining a large percentage of heterohybrids in the present study (7-26%, Table 1) is the fusion of one population in monolayer culture (the RBL cells) to the other population (the T cells) that could settle on top of the adherent cells and become attached to them via a non-stimulating lectin. This procedure minimizes RBL-RBL cell fusion induced by PEG and allows for the removal of most of the T cells not fused to RBL cells. Flow cytometry following the fusion procedure permits preparative enrichment of the heterohybrid cells for the degranulation assay, or alternatively, simultaneous analysis of each subpopulation of cells for measurements of Ca 2÷ mobilization. The use of magnetic beads to separate the T cell-containing population from other cells provides an alternative to preparative flow cytometry; it is similarly efficient and permits the fractionation of a large cell population in a much shorter period of time. The results obtained with the T celI-RBL cell hybrids indicate that the aggregation of TCR with a soluble bivalent monoclonal antibody is sufficient to stimulate signals leading to the secretion of inflammatory mediators from RBL-derived storage granules. In contrast, soluble monoclonal anti-TCR antibodies are insufficient to trigger the production of IL-2 in the Jurkat T cell line without the addition of a second signal such as that provided by PMA [37]. It is noteworthy that, unlike Fc~RI-mediated degranulation of RBL cells, IL-2 production requires de novo synthesis of mRNA [38]. It will be interesting to see whether the T cell hybrids can be stimulated to produce IL-2 by aggregation of Fc~RI in the presence or absence of PMA. Our results imply that the signals generated

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by this limited aggregation of TCR must contain components that are similar to the signals produced by the aggregation of Fc~RI complexes to stimulate cellular degranulation. These results are consistent with the recent study by Letourneur and Klausner [11], who showed that chimeric proteins containing the extracellular and transmembrane sequence from the IL-2 receptor ~ subunit and the cytoplasmic sequence of either the ( chain of the TCR or the 7 chain of Fc~RI can mediate degranulation in the RBL cells when aggregated by a soluble anti-IL-2 receptor antibody. It appears from their results that the cytoplasmic region of ( or y is sufficient to interact with other signal transduction components upon aggregation by external ligands. Recent evidence indicates that the activation of one or more tyrosine kinases may play an important role in the signalling process [6] and this activation is known to be mediated by Fc~RI [9] and TCR [39]. Activation of tyrosine kinase-mediated phosphorylation is not dependent o n C a 2+ mobilization and may represent the earliest event in the signal transduction cascade [9, 40, 41], but the mechanism of this process is not yet understood. In our experimental system, it is not yet possible to determine whether the components that interact most directly with activated TCR to trigger degranulation in the hybrid cells are derived from the T cells, or the RBL cells. In the case of the Ca 2+ response stimulated by aggregation of Fc~RI, it appears that a component from the T cells can overcome the inhibition caused by PMA in RBL cells. The site of this inhibition is not known with certainty, but one possibility is interference with the activation of PLC by a GTP binding protein (G protein) [42]. Antigen-stimulated production of inositol phosphates in RBL cells is sensitive to inhibition by PMA [43], and A I F ; stimulation of the Ca 2+ response in RBL cells is similarly inhibited by PMA (M. Weetall, D. Holowka and B. Baird, unpublished observations). A possible explanation for our results is that the T cells contribute a different PLC isozyme that can be activated by IgE

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receptor aggregation, but is not sensitive to PMA. There is recent evidence that T C R mediates the tyrosine phosphorylation of PLC 7-1 in T cells [44] and may, thereby, turn on this enzyme activity [45]. It is possible that such a pathway is not normally sufficient for Ca 2÷ mobilization in the RBL cells, but could become significant for Fc~RI-stimulated PLC activity in the hybrid cells if an appropriate component is supplied by the T cells. Alternatively, it is possible that another T cell component somehow prevents the inhibitory modification caused by PMA in the normal RBL pathway. In either case, it is apparent from our results that Fc~RI is capable of stimulating a response in the hybrid cells that involves one or more T cell components. Because the cytoplasmic segments of the TCR ( chain and the Fc~RI y chain can both mediate degranulation in RBL cells Ill], it is unlikely that the complementation observed in the present experiments is the result of exchange between these subunits. Such exchange is likely to be too slow to account for the results obtained in the Ca 2+ experiments, or in the degranulation experiments which were carried out after magnetic separation of cells and completed within several hours. In previous experiments, it was found that the exchange between ~ and fly subunits of Fc~RI on RBL cells occurs on a much longer time scale than these experiments [20]. Some T cells have been found to express TCR with endogenously synthesized Fc~RI 7 subunits [13], but the Jurkat cells express only (-containing TCR [46]. It is possible that the functional complementation we have observed does not depend entirely on the similarity of the ( and ~, subunits. T C R lacking (, q or Fc~RI y subunits can still respond to stimulation by anti-receptor antibodies, even though they are unable to respond to antigen/MHC [47, 48]. In addition, the loss of PMA sensitivity of the Ca 2+ response to Fc~RI that we have observed suggests a site of complementation 'downstream' from the receptors that is important in that response. Clearly, further experiments will be necessary to define the mechanisms of

complementation between these two different immune receptors in the hybrid cells. In other recent experiments, Beaven and colleagues have shown that a muscarinic acetylcholine receptor expressed in RBL cells can mediate degranulation [49]. This receptor directly interacts with G proteins to activate PLC and thereby led to Ca 2÷ mobilization and the activation of protein kinase C. These experiments indicate that degranulation is not dependent on signals that are unique to the family of immune receptors that are activated by foreign antigens. It is becoming increasingly evident that many different kinds of receptors can generate common signals that can stimulate a wide variety of cellular processes. The mechanisms by which these signals are regulated and channelled into responses appropriate for each cell type are just beginning to be understood. Acknowledgements--This work was supported by National Institutes of Health grants AI18306, AI07622 (postdoctoral NSRA; N.M.) and GM08210 (predoctoral training grant; M.A.L.).

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