Proc. Natl. Acad. Sci. USA Vol. 85, pp. 9689-9693, December 1988 Immunology Activation of human T lymphocytes: Differential effects of CD3- and CD8-mediated signals (protein phosphorylation/CD3 modulation/interleukin 2 receptor/interleukin 2/interferon 'y) YVONNE SAMSTAG*, FRANK EMMRICHt, AND THEOPHIL STAEHELINt Max-Planck-Institut fur Immunbiologie, 7800 Freiburg, Federal Republic of Germany Communicated by Niels K. Jerne, July 18, 1988 (received for review March 9, 1988)

ABSTRACT T cells are activated physiologically by trig- Ca2", activation of protein kinase C (PKC), phosphorylation gering the T-cell receptor-CD3 complex. There is evidence that of components of the CD3 complex, and endocytosis (mod- invariant accessory molecules on the T-cell membrane (CD8 ulation) of TCR-CD3 (for review, see refs. 10-15 and 16). and CD4) are involved in the major histocompatibility com- However, neither the correct sequence of these events nor plex-restricted recognition process. Moreover, binding and their absolute necessity are known for effects related to T-cell crosslinking ofthese accessory molecules to the T-cell receptor- function, such as expression of growth factor receptors and CD3 complex exerts a positive synergistic signal, as has been lymphokine secretion. shown by stimulation with crosslinked antibodies. Here we Here we characterize and compare the polyclonal stimu- demonstrate that stimulation mediated by immobilized lation of purified resting T cells by immobilized anti-CD3 anti-CD3/CD8 antibodies differs from stimulation mediated versus stimulation by immobilized anti-CD3 plus anti-CD8 solely by anti-CD3. Whereas interleukin 2 receptor expression monoclonal antibodies (mAbs) with respect to TCR-CD3 and interferon y production are seen to a similar extent in both modulation and CD3 phosphorylation as well as the induction cases, a second signal provided by the additional involvement of specific functions [interleukin 2 (IL-2) receptor (IL-2R) of CD8 seems to be essential for interleukin 2 production and expression and IL-2 and interferon y (IFN-y) secretion]. full interleukin 2 responsiveness in CD8' T cells. This second Further, we asked whether the effects of the CD3- or signal is much more sensitive to inhibition by 1-(5- CD8-mediated stimuli depend selectively on specific protein isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of pro- kinases, including PKC. tein kinase C and cGMP/cAMP-dependent kinases. Our results also show that substantial modulation of the T-cell MATERIALS AND METHODS receptor complex and most likely CD3 phosphorylation are not essential for initiating the activation of resting T cells. Instead, T-Cell Preparation. Peripheral blood T lymphocytes from we found a 22- to 24-kDa phosphoprotein whose strong healthy adult volunteers were purified according to Garotta phosphorylation correlated reliably with T-cell activation. and Neri (17) as modified by Emmrich et al. (6). Briefly, mononuclear cells were isolated by Ficoll/metrizoate (Phar- Although B-cell receptors (antibodies) bind antigen indepen- macia) density gradient centrifugation. Monocytes were dently of accessory structures, T-cell receptors (TCR) rec- depleted by adherence to a plastic surface at 37°C for 2 hr. T ifpresented in with self cells were separated from B cells and residual monocytes by ognize antigen only association major rosette formation with (2-aminoethyl)isothiouronium bro- histocompatibility complex (MHC) antigens. T cells bearing mide (hydrobromide, Sigma)-treated sheep erythrocytes fol- the CD4 surface molecule recognize antigen in association lowed by Ficoll/metrizoate gradient centrifugation and lysis with MHC class II structures on the presenting cell, while of erythrocytes in 0.02 M Tris-buffered (pH 7.2) 0.87% CD8-bearing T cells are restricted to antigen recognition in NH4Cl. the context of MHC class I target structures. Coating of Culture Wells with Anti-CD3 and Anti-CD8 To explain this striking dichotomy in MHC restriction, it Monoclonal Antibodies for T-Cell Activation. Flat-bottom 24- was suggested that the CD8 and the CD4 structures them- or 96-well culture plates (Nunc) were first coated with selves, in addition to the TCR, interact with MHC class I and purified goat antimouse IgG [Dianova, Hamburg; 5 ,ug/ml in class II molecules, respectively (1-5). This would imply a isotonic phosphate-buffered saline (PBS)]. The stimulating fixed juxtaposition of TCR-CD3 and CD8 or CD4 on the T antibodies were diluted in 0.5% bovine serum albumin/PBS cell upon antigen presentation. This situation can be mim- at the concentrations indicated for each experiment: anti- icked by presenting to the T cell crosslinked antibodies CD3 at 2-1500 ng/ml (BMA030/IgG2a; a gift of R. Kurrle, against the membrane molecules involved (TCR-CD3 and Behringwerke) and anti-CD8 at 1500 ng/ml (T811/IgGl; a gift CD8 or CD4). of P. Rieber, Munich). When anti-CD3 or anti-CD8 mAbs A pronounced synergism was observed (6-9) between were used alone, the second antibody was replaced by a anti-CD3 and anti-CD4 or between anti-CD3 and anti-CD8 in nonspecific antibody of the same isotype. The stimulating the activation ofthe corresponding T-cell subpopulation. The antibodies were allowed to bind for 4 hr at 37°C. Wells used molecular mechanisms responsible for this synergism have not yet been elucidated. Abbreviations: mAb, monoclonal antibody; SDS, sodium dodecyl Numerous studies, mostly done with T-cell clones or sulfate; TCR, T-cell receptor; MHC, major histocompatibility com- malignant T-cell lines, describe initial events for the activa- plex; IL-2, interleukin 2; IL-2R, IL-2 receptor; IFN-y, interferon y; tion by antigen, by antibodies to the receptor complex, or by PKC, protein kinase C mitogens and/or phorbol esters. These events involve ino- *Present address: Deutsches Krebsforschungszentrum, Im Neuen- sitol phospholipid breakdown, a rapid rise in cytoplasmic free heimer Feld 280, D-6900 Heidelberg 1, F.R.G. tPresent address: Max-Planck-Gesellschaft, Klinische Arbeits- gruppe fur Rheumatologie, Schwabachanlage 10, D-8520 Erlangen, The publication costs of this article were defrayed in part by page charge F.R.G. payment. This article must therefore be hereby marked "advertisement" tPresent address: Ciba-Geigy AG, Pharmaceuticals Research Divi- in accordance with 18 U.S.C. §1734 solely to indicate this fact. sion, R-1056.4.13, CH-4002 Basel, Switzerland.

9689 Downloaded by guest on September 25, 2021 9690 Immunology: Samstag et al. Proc. Natl. Acad. Sci. USA 85 (1988) for culturing unstimulated T cells as controls were coated IL-2 Activity. IL-2 in cell-culture supernatants was mea- with goat anti-mouse IgG and nonspecific IgG2a and IgG1 sured by the ability of the supernatant to promote the mAbs. The plates were rinsed three times with 0.01 M proliferation ([3H]thymidine incorporation) of a cloned T-cell Tris-buffered (pH 7.5) 0.9% NaCl before use. line, CTLL (20). CTLL were grown in serial 1:2 dilutions of Cell Culture for Stimulation of T Cells, 32p Incorporation, test supernatants, and [3H]thymidine incorporation was mea- and Functional Analysis. According to flow cytofluorometric sured over the last 12 hr of a 48-hr culture period. All values analysis, the purified T cells contained no MO1' cells and shown are mean values of triplicate samples and were within were 95-97% CD3-positive. They were suspended at 2.8 x the linear range of the standard curve obtained with human 106 cells per ml in 25 mM Hepes (pH 7.2)-buffered phosphate- recombinant IL-2 (gift of Hoffmann-La Roche). free RPMI 1640 medium containing 2 g of glucose per liter, 2 mM fresh glutamine, 1 mM sodium pyruvate, 50 uM 2- RESULTS mercaptoethanol, penicillin at 100 units/ml, streptomycin at 100 gg/ml, 2% (wt/vol) nondialyzed heat-inactivated human Phosphorylation of a 22- to 24-kDa Protein Not Associated serum, and, ifprotein phosphorylation was to be studied, 25- with CD3 (y, 8, and E Chains) Is Characteristic of T-Cell 30 ,uCi of [32P]orthophosphate (Amersham) per ml (1 Ci = 37 Activation. Our first experiments explored whether TCR- GBq). The T cells were incubated over night in a culture flask CD3 complex phosphorylation and modulation are important humidity to remove signals for activation of resting T cells. In most experiments, at 37TC in 5% C02/95% air and 96% using different donors, CD3 phosphorylation, as revealed by residual monocytes (and to equilibrate intracellular nucleo- SDS/PAGE of CD3 immunoprecipitates followed by auto- side phosphate pools with 32p for phosphorylation experi- radiography, was not detectable. However, total protein ments). For stimulation the cells were then seeded in 0.6-ml phosphorylation in postnuclear cell lysates analyzed by aliquots into the antibody-coated wells and incubated as autoradiography of SDS/PAGE gels showed a striking pre- indicated. Cells were harvested by vigorous pipetting in cold dominant phosphorylation of a 22- to 24-kDa protein (termed PBS. 23 kDa, possibly a double band) in anti-CD3- plus anti-CD8 32P-Labeled Protein Analysis. Approximately 107 labeled (anti-CD3/CD8)-stimulated T cells and less pronounced in cells from six wells were washed two times with 10 ml of only anti-CD3-stimulated T-cells. This 23-kDa protein phos- ice-cold PBS (pH 7.2) and once with 3 ml ofPBS (pH 8.0) and phorylation after 25 and 42 hr of stimulation is shown in Fig. lysed for 30 min at 4°C in 170 ,ul of 1% Nonidet P-40 (Sigma) 1. The preferential phosphorylation ofthe 23-kDa protein was in PBS (pH 8.0) containing leupeptin at 1 mg/ml, soybean quantified by determining the ratio of the 23-kDa protein to trypsin inhibitor at 20 ,g/ml, aproteinin at 10 Ag/ml, and 5 a 38-kDa protein phosphorylation from laser scans of the mM 4-nitrophenyl phosphate (all from Sigma). The lysates autoradiographs. The 38-kDa protein was chosen as refer- were centrifuged for 10 min at 100,000 X g. Fifteen microli- ence because it reflected the extent of overall protein phos- ters ofthe cleared lysate was mixed with 15 ,ll of2x reducing phorylation. After 25 hr of stimulation (Fig. la), this ratio was sample buffer (18) and electrophoresed in 8-18%/sodium 1.0 for unstimulated cells (lane 1), 2.8 for anti-CD3-stimulated dodecyl sulfate (SDS)/polyacrylamide gradient slab gels cells (lane 2), and 4.5 for anti-CD3/CD8-stimulated cells (lane according to Laemmli (18) followed by autoradiography. The 3). The 23-kDa protein phosphorylation was the same at both remaining 150 ,ul of lysate was immunoprecipitated with a high density (1500 ng/ml, lanes 2-4) and a low density (55 anti-CD3 mAb as described by Emmrich et al. (6) and ng/ml, lanes 5-7) of immobilized anti-CD3 mAb. The addi- analyzed by SDS/PAGE and autoradiography for detection tion of H7, a PKC and cAMP/cGMP-dependent kinase of phosphorylated CD3 (y, 8, and E chains). Ratios of the inhibitor (21), to anti-CD3/CD8-stimulated cells (lanes 4 and 23-kDa to the 38-kDa protein phosphorylation in cell lysates were determined by scanning the autoradiographs with a a b 1 2 3 4 5 6 7 1 2 3 4 LKB model 2202 Ultrascan laser densitometer connected to kD kD a Hewlett-Packard model 3390A integrator with a strip-chart 94 - , * W recorder. - _6794 67-6~~~~7-6 Determination ofCell Surface CD3, CD8, and IL-2R. (i) Cell 60- 60. surface antigens were identified by flow cytofluorometry with specific monoclonal antibodies and isotype-specific 45 - II -45 36-- Wie WO - 36 fluorescein isothiocyanate-conjugated anti-mouse F(ab')2 30- -30 (Southern Biotechnology, Birmingham, AL). Approximately 1 104 cells were analyzed per sample in an Ortho Cytofluoro- 2 0 - 0*_ -20 graph model 50 H. (ii) Alternatively, these antigens were measured by ELISA. Approximately 106 cells treated with 1.0 2.8 4.5 2.5 2.6 4.3 2.7 1.9 4.1 7.1 2.3 antigen-specific mAb and washed as for cytofluorometry P ratio 23 kD were lysed in 100 ,ul of 0.4% Nonidet P-40 in PBS containing 38 kD protease inhibitors. The lysates were centrifuged to remove FIG. 1. SDS/PAGE analysis of phosphorylated proteins in nuclei. Surface-bound mAb was measured in the lysate with postnuclear lysates of purified T cells stimulated in the presence of a sandwich-type ELISA in which each well was coated with [32P]orthophosphate at 25 ,tCi/ml. (a) A 25-hr stimulation with 50 ,u1 of goat anti-mouse IgG (5 ,ug/ml of PBS) and saturated immobilized anti-CD3 mAb BMA030. Lanes: 1, unstimulated con- with 1% bovine serum albumin in PBS. The lysates (50 ,ul) trol; 2-4, stimulation at high antibody density (1500 ng/ml); 5-7, were incubated at three dilutions in duplicate wells for 2 hr at stimulation at low antibody density (55 ng/ml). Cells of lanes 3, 4, 6, 37°C, and wells were rinsed, incubated with isotype-specific and 7 had been costimulated with anti-CD8 mAb, coated at 1500 goat anti-mouse IgG-alkaline phosphatase conjugate ng/ml. Cultures shown in lanes 4 and 7 also contained 12 A&M H7 Biotechnology) for 2 hr at 37°C, and developed (Sigma), the PKC/cAMP/cGMP kinase inhibitor. (b) A 42-hr stim- (Southern ulation. Lanes: 1, control; 2, anti-CD3 stimulated; 3, anti-CD3/CD8 with 4-nitrophenyl phosphate (Sigma). Absorption at 405 nm stimulated; 4, anti-CD3/CD8 stimulated plus 50 ,uM nifedipine was measured in a Titertek ELISA reader. Reference curves (Sigma) in the culture medium. Anti-CD3 or anti-CD8 mAb was for each mAb used were established in all experiments. coated at 1500 ng/ml. Lysate equivalent to 9 x 101 cells was analyzed IFN-y. IFN-y was determined by a sandwich-type ELISA per sample. Autoradiography was at -800C on Kodak X-Omat S film with mAbs according to Gallati et al. (19). All the reagents without intensifying screen for 75 hr (a) and 27 hr (b). Star, 38-kDa were a generous gift of H. Gallati (Hoffmann-La Roche). protein; double-headed arrow, 23-kDa protein. Downloaded by guest on September 25, 2021 Immunology: Sarnstag et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9691

7) reduced both the 23-kDa and general protein phosphoryl- Stimul.: anti-CD3 anti-CD3+CD8 ation to the level of anti-CD3 stimulation alone. After 42 hr I (Fig. lb), the costimulation with anti-CD8 (lane 3) resulted CD3 not only in the marked increase of the 23-kDa protein 100 phosphorylation but also in a stronger overall protein phos- 100 phorylation. This coincided with the observed blast trans- o 80 80 formation of these cells, which was much less pronounced in U 60 60 cells stimulated only with anti-CD3 (lane 2). The addition of 0 the calcium channel blocker nifedipine (lane 4) reduced the be 40 40 phosphorylation of all proteins to control level without 20 20 affecting the viability of the cells. In contrast to the strong phosphorylation of this 23-kDa o 2 6 16 55165 500150 0 2 6 18 55 165 5001500 protein and the expression of IL-2R, IFN-y, and IL-2, all of 50 IL-2R 50 which were observed in every experiment, phosphorylated y and 8 chains of CD3, as first described by Cantrell et al. (13), 40 40 were detected only on rare occasions and never before 20 hr U 30 30 1._1 of stimulation. . 20 20 CD3 Modulation Is Not a Consequence of CD3 Phosphoryl- 0co ation. Since Cantrell et al. (13, 15) and Krangel (14) had C. 10 10 observed that phorbol ester stimulation of human T cells at leads to a concomitant phosphorylation and modulation of 0 2 6 18 55 165 500 1500 0 2 6 18 55 165 500 1500 CD3, we tested whether there might be a causal relationship - 60 IFN 60 between these two phenomena. E In experiments where CD3 phosphorylation was detect- 1 40 40 able, we found that CD3 modulation consistently preceded 0 CD3 phosphorylation (for kinetics of CD3 modulation see 20 20 Fig. 3). Furthermore, the calcium channel blocker nifedipine as 2Miu0LA~tt well as the PKC and cyclic nucleotide-dependent kinase 0 6 18 55 165 500 1500 0 6 18 55 165 500 1500 inhibitor H7 abolished CD3 phosphorylation without influ- anti-CD3 conc. (ng/ml) encing CD3 modulation. Thus our results failed to confirm that CD3 phosphorylation is an obligatory event for TCR- FIG. 2. CD3 modulation, IL-2R expression, and IFN-y produc- CD3-mediated activation and exclude that it is essential for tion by stimulated T cells are dependent on the density of immobi- lized anti-CD3 mAbs and affected by costimulation with anti-CD8 CD3 modulation. mAbs. Wells had been coated with anti-CD3 mAb BMA030 at the T-Cell Activation Does Not Require Extensive TCR-CD3 concentrations indicated on the abscissa and supplemented with an Modulation. When we explored TCR-CD3 modulation during irrelevant IgG1 mAb at 1500 ng/ml (Left) or with anti-CD8 mAb T-cell activation, we found that CD3 modulation depended on T811/IgGl at 1500 ng/ml (Right). Stimulation was for 36 hr. CD3 was the density of the immobilized anti-CD3 antibody used for determined by flow cytofluorometry with mAb BMA030. Measure- stimulation. The experiment shown in Fig. 2 compares the ments are expressed as mean channel fluorescence of all cells as extent of receptor modulation with the induction of IL-2R percent of unstimulated control cells. The mean channel fluores- expression and with IFN-y production. At the lower con- cence of all cells correlated well with CD3 determination by ELISA. centrations of immobilized anti-CD3 (<165 ng/ml), both in IL-2R was determined by cytofluorometry with an anti-Tac mAb (Clonab IL-2R; Biotest, Frankfurt) and with fluoresceinated isotype- the absence and presence of anti-CD8 (1500 ng/ml), IL-2R specific second antibody (Southern Biotechnology) and is expressed expression and IFN-y production had already reached their as percent of positive cells. The fluorescence intensity of positive maximum levels at low-receptor modulation. At the highest cells was constant within experimental error over the whole range of anti-CD3 concentration that resulted in .75% receptor mod- density of immobilized anti-CD3 mAb. IFN-y was determined by a ulation, IFN-y production was even reduced, while IL-2R sandwich-type ELISA. expression remained high. The enhancing effect by anti-CD8 stimulation on these two parameters of activation was only g and h). Thus, protein phosphorylation either by PKC or a moderate. cyclic nucleotide-dependent kinase is essential for the induc- Costimulation with Anti-CD8 Triggers Additional Protein tion of functions dependent on costimulation of CD8. Kinase Activation, IL-2 Production, and T-Cell Proliferation. With the experiment shown in Table 1, we tested the As demonstrated above, anti-CD antibody induced IL-2R proliferative response of anti-CD3- or anti-CD3/CD8- and IFN-y expression. However, as shown in Fig. 3, IL-2 stimulated T cells in the absence and presence of added production, essential for proliferation, depended strongly on exogenous IL-2. Surprisingly, anti-CD3 stimulation alone costimulation and crosslinking of CD8, which provided a resulted in a significant proliferative response that was, how- potentiating second signal. This experiment confirms again ever, 2.5-fold enhanced by costimulation with anti-CD8 and that low anti-CD3 density causes only minor CD3 modula- only 1.38-fold by saturating amounts of exogenous IL-2. It tion, but higher induction of IFN-y, IL-2R expression, appears, therefore, that costimulation of CD8 is required not and-most drastically-IL-2 production. only for production of large amounts of IL-2 but also for To further characterize the signals triggered by CD3 and functions other than IL-2R expression (as defined by the CD3-CD8 crosslinking, we asked whether the effects of one anti-Tac mAb) that are required for efficient proliferation. The of the two stimuli might depend selectively on specific kinase inhibitor H7 almost abolished proliferation and strongly protein kinases. The effects of H7, an inhibitor of PKC and impaired the ability to proliferate in response to exogenous cyclic nucleotide-dependent kinases (21), is shown in Fig. 4. IL-2. The inhibition by anti-IL-2R mAb shows that prolifera- IFN-y and IL-2R expression, induced also by CD3 stimula- tion depends fully on the IL-2-IL-2R interaction. tion alone (Fig. 4 c and e), and the 23-kDa protein phospho- rylation (Fig. 4 i and j) were only moderately inhibited (Fig. DISCUSSION 4 d, f, andj) whereas the IL-2 secretion, which depended on In this work we studied molecular and functional events costimulation of CD8, was totally suppressed by H7 (Fig. 4 resulting from the interactions of stimulating ligands with Downloaded by guest on September 25, 2021 9692 Immunology: Sarnstag et al. Proc. Natl. Acad. Sci. USA 85 (1988)

0 55 1500 0 55 1500 a -CD3 conc.(ng/ml) FIG. 4. Differential effects of the PKC and cAMP/cGMP- 8 26 48 8 26 48 dependent kinase inhibitor H7 on CD3 modulation, IFN-y produc- tion, IL-2R expression, IL-2 secretion, and 23-kDa protein phos- time (h) phorylation. T-cell stimulation with a low density (55 ng/ml) and a high density (1500 ng/ml) of immobilized anti-CD3 mAb alone (a, c, FIG. 3. Kinetics of CD3 modulation, IFN-y production, IL-2R e, g, and i) and plus anti-CD8 mAb (b, d, f, h, andj), as described expression, IL-2 production, and 23-kDa protein phosphorylation for Fig. 3, was carried out for 48 hr. Duplicate cultures stimulated during activation of purified T cells stimulated by a low density (0.05 with anti-CD3/CD8 in addition to 24 tuM H7 were carried out and ,ug/ml during coating, o) and a high density (1.5 jig/ml, *) of analyzed in parallel (solid bars, b, d,f, h, andj). CD3 was determined immobilized anti-CD3 mAb alone (Left) and by low- and high-density by flow cytofluorometry and expressed as mean percent of channel anti-CD3/CD8 (Right). Anti-CD8 mAb was coated at 1.5 ,g/ml. fluorescence of nonstimulated control cells as in Fig. 2. IL-2R was Cultures without anti-CD3 stimulation (x) were included. T cells measured by flow cytofluorometry (Fig. 2), and IL-2 activity of were obtained from one blood donation and cultured under identical culture supernatants diluted 1:4 was measured in the CTLL prolif- conditions except that [32P]orthophosphate was present at 30 uCi/ml eration assay; maximum [3H]thymidine incorporation in the refer- in some cultures for the determination of protein phosphorylation. ence curve with recombinant IL-2 was 31,400 cpm. [NB: the H7 CD3 was determined by anti-CD3 mAb measurement in lysates of concentration in the IL-2 assay due to carryover from H7-treated anti-CD3 mAb-coated cells by using an ELISA. IL-2R was also T-cell cultures (6 ,uM) gave only 15% inhibition of [3H]thymidine quantified by ELISA as described for CD3. Phosphorylation of incorporation in a control assay with recombinant IL-2-stimulated proteins with 32p was done only under stimulation with a high density CTLL.] (1.5 ,ug/ml) of immobilized anti-CD3 mAb (for comparison, see Figs. la and 4 i and j). IL-2 activities in culture supernatants diluted 1:4 CD3 alone with the mAb BMA030 did not activate these cells. determined by the CTLL proliferation assay (cpm of [3Hlthymidine incorporated) were all within the linear range of the IL-2 standard By using this mode of stimulation with immobilized antibod- curve generated with recombinant human IL-2, which had a maxi- ies but with a high cell density, we showed the existence of mum incorporation of 79,400 cpm. two signals. The first, generated by TCR-CD3 crosslinking alone and affecting both CD4+ and CD8+ cells, induced those T-cell surface structures involved in MHC-restricted IFN-y production and IL-2R expression. The second signal, antigen recognition. These structures are the TCR-CD3 generated by crosslinking CD8 with TCR-CD3, was required complex and either CD8 or CD4 for MHC class I- and class for substantial IL-2 production and a strong proliferative TI-restricted recognition, respectively. Although stimulation response that did not require exogenous IL-2. This result as of the TCR-CD3 complex by crosslinked (immobilized) well as the IL-2R expression induced by anti-CD3 stimulation anti-CD3 or anti-ar TCR antibodies is sufficient to trigger a alone differ from the earlier findings of Emmrich et al. (6, 7). proliferative response in T-cell clones (23-25), resting T cells They used, however, an additional purification step for require a second signal provided by accessory cells that have monocyte depletion and a 20 times lower cell density during to be in direct contact with the T cells and cannot be replaced stimulation. This could explain the dependency on exoge- by soluble factors like interleukin 1 (26). Earlier experiments nous IL-2 for proliferation of anti-CD3/CD8-stimulated cells (6, 9) had shown a pronounced synergism between TCR-CD3 due to dilution of endogenous IL-2 (6). IL-2R expression was and CD8 crosslinking by the respective immobilized antibod- stimulated by anti-CD3 alone at high cell density (our present ies for the induction of polyclonal proliferation in resting results) but not at low cell density (7). As suggested by human T cells at low cell density while crosslinking of TCR- Heumann and Vischer (27), this might be due to small Downloaded by guest on September 25, 2021 Immunology: Samstag et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9693 Table 1. Proliferative response of T cells in the absence and Furthermore, we discovered the selective strong phospho- presence of exogenous recombinant IL-2 rylation ofa 22- to 24-kDa protein as a phenomenon ofhuman [3H]Thymidine incorporation, cpm T-cell activation. This phosphoprotein appeared not to be associated with the TCR-CD3 complex. The kinetics of its IL-2 IL-2 phosphorylation was quite similar to those of IL-2R expres- Addition(s) None (60 units/ml) (120 units/ml) sion and IFN-y and IL-2 production, indicating that the None 254 15,494 15,754 23-kDa protein phosphorylation is not an initial event of Anti-CD3 63,757 64,774 88,118 activation. Anti-CD3/CD8 160,900 176,256 188,646 Anti-CD3/CD8 + H7 21,904 61,979 70,098 We thank Georges Kohler for valuable discussions, Ms. C. Anti-CD3/CD8 + Riesterer for excellent technical assistance, Mr. U. Brugger for flow anti-IL-2R 685 ND ND cytofluorometric analyses, Ms. S. Kleinhans for graphics work, and Mrs. B. Carter and Ms. P. Meneghinello for typing ofthe manuscript. Approximately 2.7 x 105 T cells per 0.1 ml were cultured for 62 hr Y.S. was the recipient of a postdoctoral fellowship from the in triplicate flat-bottom microtiter wells with 1 ,uCi of [3H]thymidine Deutsche Forschungsgemeinschaft. present for the final 16 hr. Stimulating anti-CD3 (BMA030) and anti-CD8 (T811) mAbs were coated on the microtiter wells at 1.5 1. Swain, S. L. (1981) Proc. Nat!. Acad. Sci. USA 78, 7101-7105. ug/ml. Anti-IL-2R (anti-Tac) mAb at 6 ,ug/ml or 24 ,tM H7 was 2. Meuer, S. C., Schlossman, S. F. & Reinherz, E. L. (1982) Proc. present as indicated throughout the culture period. Cells were Nat!. Acad. Sci. USA 79, 4395-4399. harvested onto glass fiber filters and radioactivity was measured in 3. Gay, D., Coeshott, C., Golde, C., Kappler, J. & Marrack, P. (1986) a liquid scintillation spectrometer. Deviation of individual measure- J. Immunol. 136, 2026-2032. ments was <11% of the mean. ND, not determined. 4. Dembic, Z., Haas, W., Zamoyska, R., Parnes, J., Steinmetz, M. & von Boehmer, H. (1987) Nature (London) 326, 510-511. numbers of accessory cells because IL-2 responsiveness to 5. Doyle, C. & Strominger, J. L. (1987) Nature (London) 330, 256-259. pure T cells stimulated at low cell density with immobilized 6. Emmrich, F., Strittmatter, U. & Eichmann, K. (1986) Proc. Nat!. Acad. Sci. USA 83, 8298-8302. anti-CD3 was restored by adding 0.5% accessory cells. Pure 7. Emmrich, F., Kanz, L. & Eichmann, K. (1987) Eur. J. Immunol. 17, interleukin 1 could not replace accessory cells. Similar results 529-534. were obtained by Halvorsen et al. (26). Alternatively, the 8. Eichmann, K., Jomsson, J. I., Falk, J. & Emmrich, F. (1987) Eur. direct cell-cell contact at high cell density might provide an J. Immunol. 17, 643-650. mediated the inter- 9. Walker, C., Bettens, F. & Pichler, W. J. (1987) Eur. J. Immunol. 17, additional signal(s) by specific reciprocal 873-880. action of other T-cell surface structures, such as CD2 and 10. Weiss, A., Imboden, J. B., Shoback, D. & Stobo, J. D. (1984) Proc. lymphocyte function-associated antigen 3 (LFA3), known to Natl. Acad. Sci. USA 81, 4169-4173. trigger the so-called alternative pathway of T-cell activation 11. Imboden, J. B. & Stobo, J. D. (1985) J. Exp. Med. 161, 446-456. (28). Our results showing the importance ofCD8 crosslinking 12. Samelson, L. E., Harford, J., Schwartz, R. H. & Klausner, R. D. with for IL-2 corroborate findings from (1985) Proc. Nat!. Acad. Sci. USA 82, 1%9-1973. CD3 production 13. Cantrell, D. A., Davies, A. A. & Crumpton, M. J. (1985) Proc. transfection experiments with murine T-cell lines. Transfec- Nat!. Acad. Sci. USA 82, 8158-8562. tion of either human CD4 (29) or CD8 (30) into murine T-cell 14. Krangel, M. S. (1987) J. Exp. Med. 165, 1141-1159. hybridomas resulted in a 6- to 20-fold increase of IL-2 15. Cantrell, D., Davies, A. A., Londei, M., Feldman, M. & Crumpton, production dependent on the presence of human class II or M. J. (1987) Nature (London) 325, 540-542. 16. Isakov, N., Scholz, W. & Altman, A. (1986) Immunol. Today 7,271- class I MHC, respectively, on the target cells. On the other 277. hand, blocking of CD8-CD3 or CD4-CD3 crosslinking with 17. Garotta, G. & Neri, T. M. (1985) Immunol. Methods 2, 163-185. soluble antibodies against CD8 or CD4 on T cells or against 18. Laemmli, U. K. (1970) Nature (London) 227, 680-685. MHC class I or II structures on antigen-presenting cells 19. Gallati, H., Pracht, J., Schmidt, J., Haring, P. & Garotta, G. (1987) prevented the second signal required for proliferation (IL-2 J. Biol. Regul. Hemostat. Agents 1, 109-118. 20. Gillis, S., Ferm, M., Ou, W. & Smith, K. A. (1978)J. Immunol. 120, production) of CD8+ or CD4+ cells, respectively (3, 31-34). 2027-2032. The finding by Walker et al. (9) that crosslinking of many 21. Hidaka, H., Inhagaki, M., Kawamoto, S. & Sasaki, Y. (1984) other T-cell surface structures to CD3 can synergize recep- Biochemistry 23, 5036-5041. tor-mediated activation may suggest that yet unknown nat- 22. Zerial, M., Suomalainen, M., Zanetti-Schneider, M., Schneider, C. ural ligands to these molecules might elicit signals similar to & Garoff, H. (1987) EMBO J. 6, 2661-2667. those of class I MHC-CD8 interaction. 23. Meuer, S. C., Hussey, R. E., Cantrell, D. A., Hodgdon, J. C., Schlossman, S. F., Schmith, K. A. & Reinherz, E. L. (1984) Proc. TCR-CD3 modulation has been reported to occur in Nat!. Acad. Sci. 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