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).