Eur. J. Immunol. 1992. 22: 3155-3160

Ana G. Santis, Miguel R. Campanero, Jose L. Alonso and Francisco Sanchez-Madrid Secci6n de Inmunologia, Hospital de la Princesa. Universidad Autonoma de Madrid, Madrid

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Regulation of TNF-a/TNFreceptor through CD2

Regulation of tumor necrosis factor (TNF)-a synthesis and TNF receptors expression in T lymphocytes through the CD2 activation pathway* The involvement of the CD2 (T11) molecule, an alternative activation pathway for T lymphocytes, in the regulation of tumor necrosis factor (TNF)-a/TNF receptor system in humanT lymphocytes has been investigated. It has been found that bothTNF-a synthesis and secretion were induced after incubation of purified T lymphocytes with the appropriate mitogenic combination of antibodies specific for two different epitopes on the CD2 molecule. Moreover,TNF-a secretion was also observed by activation of Tlymphocytes either through CD3 or CD69 molecular pathways, or with other stimulating agents such as Ca2+ionophore in combination with phorbol esters. The expression of TNF receptors has been studied in both nonactivated and CD2-activated T lymphocytes. Unstimulated T cells weakly expressed a functional 75-kDa receptor form, whereas they lacked detectable levels of the 55-kDa receptor form. Triggering of T cell activation through the CD2 molecule also markedly increased the expression of the p75-kDa TNF receptor form, but did not exert any inductive effect on the expression of the p55-kDa TNF receptor. In addition, we have found that TNF-a enhanced the proliferative response triggered by the mixture of anti-CD2 monoclonal antibodies. Taken together, these results support a role for the CD2 activation pathway in the functional regulation of TNF-a/TNF receptor system in T lymphocytes, and reinforce the view of CD2 as an alternative pathway for regulation of the cytokine network that modulates the function of T lymphocytes.

1 Introduction CD2 (T11) is a 50-kDa transmembrane glycoprotein that is expressed on the surface of the majority of thymocytes and mature peripheral blood lymphocytes [l]. CD2 acts as an adhesion molecule that participates in interactions with other cells, and hence contributes to antigen-specific responses of both cytotoxic and helper T lymphocytes. The physiologic ligand for CD2 is CD58 (LFA-3), which is a 55to 70-kDa protein that is expressed on a variety of cell types, including endothelial, epithelial, connective tissue and most blood cells [2]. Three functionally important epitopes have been defined on the human CD2 molecule: T1l1, T112. and T113. Both T l l l and T112 epitopes are present on resting and activated T lymphocytes, whereas T113 is restricted to activated T lymphocytes. The combined action of mAb directed against the T l l l or T112 and T113 CD2 epitopes activates human T cells [ 11.

be similar to that triggered through the TcK/CD3 complex, and thus both pathways stimulate the tyrosine phosphorylation of indistinguishable patterns of polypeptides [5]. TNF-a, also termed cachectin, is a pleiotropic cytokine produced mainly by macrophages [6].TNF-a appears to be necessary for normal immune response, but in excessive quantities produce dramatic pathogenic effects [7]. TNF-a production by T lymphocytes can be induced through different molecular pathways involving CD3, CD69 or CD28 antigens. Thus, the combination of phorbol esters with mAb against these antigens [S-lo], or with a calcium ionophore induces a strong TNF-a production by T lymphocytes [11].

Protein purification and cDNA cloning studies have led to the recent identification of two distinct TNF receptors, the 55-kDa and the 75-kDa molecular forms [ 12-14]. Each of the two receptors can bind TNF-a or TNF-p with a similar Signal transduction through CD2 has been reported to high affinity [14, 151. There is strong evidence indicating involve stimulation of phospholipase C, elevation in [Ca2+]i that each of the receptors themselves can transduce the [l],increase in cAMPi 131, and PKC activation 141. CD2- signal needed for biological activity 1161. The expression of mediated signal transduction in T lymphocytes appears to the 55- and 75-kDaTNF receptors varies among all cell lines studied. Certain cell lines, such as the epidermoid carcinoma HEp2 were found to express only the 55-kDa receptor whereas most other cell lines examined have both receptors, expressed at different proportions [6, 121. [I 107481 In this report, we have studied the regulation of TNF-a -I This research w a supported ~ by a grant from CDTI-Laboratorios Andrcimaco S.A. (n. 880051), FIS (n. 91/0259) and a fellowship synthesis and TNF receptors cell surface expression in T lymphocytes upon activation through the CD2 antigen. from Comunidad Autonoma de Madrid We have found an induction of TNF-a secretion and a Correspondence: Francisco Sinchez-Madrid, Seccihn de Inmuno- differentially regulated expression of p75- and p55-kDa logia, Hoyxtal de la Princesa. Diego de Leon, 62, E-28006 Madrid, TNF receptors in Tcells activated with a mitogenic combiSpain nation of anti-CD2 mAb. 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992

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0014-2980/92/1212-3155$3.50 .25/0

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A . G . Santis, M. R. Campanero, J. L. Alonso and F. Sanchez-Madrid

Eur. J. Immunol. 1992. 22: 3155-3160

2 Materials and methods

2.4 Immunofluorescence flow cytometry

2.1 Reagents and monoclonal antibodies (mAb)

Flow cytometry analysis were performed using a FACScan flow cytometer (Becton Dickinson) .Binding of anti-TNF receptor antibodies was done by mixing aliquots of 106cells with biotinylated mAb (10 pg/ml) and streptavidin PE essentially as described [ 161. Blocking analysis were carried out by addition of 100ng/ml rTNF-a. Specific linear fluorescence was obtained by subtracting background fluorescence produced by streptavidin PE in the absence of biotinylated mAb.

TS2/18 [ 171 antLCD2 mAb was used as purified immunoglobulin to a final concentration of 10 pg/ml; D66 [18] anti-CD2 mAb, a gift of Dr. A. Bernard (Nice, France),was used as ascites fluid diluted 1/500;and TP1/8 anti-AIM [19] was used at 10 pg/ml. TS2/16 anti-VLA-Bl (CD29) [20], included in every experiment as control mAb, was employed at 10 pg/ml. SPV-T3b anti-CD3 mAb [21] was kindly provided by Dr. De Vries (Unicet Laboratories, Dardilly, France) and used soluble at 1 pg/ml. Utr-1 and Htr-9 anti-p75- and p55-kDa TNF receptors, respectively [16] were kindly provided by Dr. M. Brockhaus (F. Hoffman-La Roche, Basel, Switzerland). Anti-TNF-a mAb, 9/17 and 10/18 used for the radioimmunoassay, have been previously described [9]. P3-X63 IgGl myeloma culture supernatant was used as negative control for immunoprecipitation and flow cytometry analysis.

PMA and Ca2+ionophore (A23187) were purchased from Sigma (St. Louis, MO). Human recombinant TNF-a (rTNF-a) was purchased from Genentech, Inc. (San Francisco, CA). It was >99% pure with a specific activity of 4.5 x 107 U/mg.

2.5 Radiolabeling, immunoprecipitation, and electrophoresis For metabolic labeling, resting and activated T lymphocytes were washed with PBS and resuspended at 5 x lo6 cells/ml in methionine-free RPMI medium.Then, cells were pulsed with 100 pCi/ml = 3.7 MBq/ml of [35S]methionine (Amersham Int., Amersham, GB) at 37°C during 4 h. After labeling, cells were lysed and immunoprecipitation was carried out as previously described [9]. Precipitates were analyzed by SDS-15% PAGE and autoradiography.

2.6 Proliferation assays 2.2 Cells

Highly purified Tcells (2 x lo5 cells/well) in 200 pl were incubated by triplicate in flat-bottom 96-well plates at 37 "C PBMC were obtained from heparinized venous blood of normal volunteers by Ficoll Hypaque (Pharmacia Fine for 3 days in a 5% C02 atmosphere. Tcells were cultured Chemicals, Uppsala, Sweden) centrifugation. T cells were with different stimuli either in the presence or in the purified from PBMC by removal of adherent cells on plastic absence of TNF-a (100 ng/ml). Cell proliferation was petri dishes followed by passage through a nylon wool estimated by [3H]dThd (New England Nuclear, Boston, column. Phenotypic analysis of purified Tcells by flow MA) incorporation during the last 16 h of culture. Cells cytometry analysis showed that T cell population contained were harvested and the radioactivity was measured in a > 90% CD3+, > 90% CD2+, < 1% CD14+, and < 1% liquid scintillation counter. CD20+ cells.

3 Results 2.3 TNF-a production assays and quantitative estimation of TNF-a concentrations

It has been documented that a combination of two anti-CD2 mAb can stimulate peripheral blood T lymphoPurified T cells were cultured (4 X lo5 cells/well) in RPMI cytes to produce IL-2 [1].To evaluate the possible role of the 1640 (Flow, Irvine, Scotland) supplemented with 1% CD2 activation pathway in the induction of TNF-a secrepenicillin-streptomycin (Flow), 2 mM L-glutamine, and 5% tion, peripheral blood T lymphocytes were cultured in the FCS (Flow), which is referred to as complete medium, in presence of different anti-CD2 mAb either alone or in round-bottom microtiter plates (Costar, Cambridge, MA) combination. The combination of two different antLCD2 in the presence of different combinations of the indicated mAb, TS2/18, directed to the T l l , epitope, and D66, stimuli. After the indicated times, culture supernatants specific for a distinct epitope and previously described to be were harvested and TNF-a concentrations were deter- comitogenic with anti-Tll1 mAb [MI, was able to induce mined as previously described by a quantitative radioim- TNF-a secretion (Table 1). The CDZmediated TNF-a munoassay [9]. Briefly, anti-TNF-a mAb specific for two secretion in T lymphocytes was compared to that triggered different epitopes were employed, one of them was through other T cell molecular and signalling pathways. As adsorbed onto 96-well vinyl plates. Then, plates were shown inTable 1,the highest TNF-a response was observed washed andsaturated with 1%BSA PBS,washed again, and with the mitogenic combination of anti-CD2 mAb, in the then culture supernatant samples were incubated in tripli- absence of PMA. A synergistic TNF-a response to CD3, cate wells. After washing, 1251-labeledsecond anti-TNF-a CD69, and the Ca2+ionophore A23187 was observed in the mAb was added, and incubated. Then, plates were washed presence of PMA (Table 1), that correlates with the fully and dried. Bound radioactivity was measured in a gamma proliferative T cell responses triggered under the same counter. TNF-a concentration in the test samples was experimental conditions (data not shown). determined on the basis of a standard curve, defined in each experiment by using rTNF-a. The semilogarithmic plot of Next, we studied the kinetics of CD2-induced TNF-a cpm vs. TNF-a concentration was linear from 0.1 ng/ml to secretion in the absence of PMA.The production of TNF-a 30 ng/ml. was maintained along 5 days of culture, whereas PMA or

Regulation of TNF-a/TNF receptor through CD2

Eur. J. Immunol. 1992. 22: 3155-3160

Table 1. TNF-a production induced through different activation pathways in T lymphocytes Activation pathway

TNF-a (ng/ml)a)

Treatmentb)

Without PMA With PMAC) -

Medium TS2/18 + D66 T3b TP1/8 A23187

CD2 CD3 CD69/AIM Ca'+ influx

0.1 k 0.0 4.1 -I 0.1 1.1 k 0.1 0.3 k 0.1 0.4 k 0.1

6.3 k 0.0 6.6 f 0.3 15.8 k 0.4 18.6 k 0.4 12.0 k 0.4

a) Culture supcrnatants were harvested at 16 h after activation and the concentration of TNF-a was quantitatively measured as described in Sect. 2.3. Data are from a representative experiment out of fivc different arid independent ones. b) Purified T lymphocytes were cultured with the indicated stimuli: Medium. absence of any reagent or mAb; TS2/18 anti-CD2 mAb (10 pg/ml); D66 anti-CD2 mAb (ascites fluid diluted MOO); SPV-T3b anti-CD3 mAb (1 pg/ml); TP1/8 antiCD69/AIM mAb (10 pg/ml); and, Ca2+ ionophorc A23187 (1 p ~ ) All . of mAb were added as soluble protein. c) In this set of experiments PMA was added at the beginning of the culture at a final concentration of 10 ng/ml.

anti-CD2 TS2/18 mAb alone induced a weak and transient TNF-a secretion within the first 24 h of culture (Fig. 1A). No TNF-a synthesis was observed by addition of other control mAb such as an anti-CD29 mAb (data not shown). Immunoprecipitation analyses with anti-TNF-a mAb from ["S] methionine-labeled CD2-activated cell lysates further confirmed the induction of TNF-a biosynthesis through the

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CD2 molecule. As shown in Fig. 1B, TNF-a was precipitated by the 10/18 anti-TNF-a mAb as the 26-kDa TNF-a transmembrane form from CD2-activated (lane 4) but not from restingT lymphocytes (lane 2). Altogether these data indicate that mAb engagement of CD2 induces TNF-a synthesis. To study the regulation of TNF receptor expression through the CD2 activation pathway, we first analyzed the expression of p55- and p75-kDa TNF receptor polypeptides in unstimulated purified peripheral blood T lymphocytes. Two specific mAb, the Htr-9 anti-55-kDa TNF receptor, and the Utr-1 anti-75-kDa TNF receptor, raised against purified TNF-binding proteins were used [12]. As observed in Fig. 2 A , flow cytometry analysis showed that resting T cells weakly expressed the 75-kDa receptor form, whereas no detectable staining with the anti-55-kDa receptor was found. The binding of biotinylated anti-75-kDaTNF receptor mAb to resting T lymphocytes was virtually abolished by simultaneous treatment with rTNF-a, indicating that the p75-kDaTNF receptor is able to bind TNF-a on these cells (Fig. 2 A). Immunoprecipitation analysis from [35S]methionine-labeled purified resting T lymphocytes further confirmed the active biosynthesis of the p75, but not that of the p55-kDa TNF receptor (Fig. 2 B). The expression of TNF receptors was also studied in activated T cells. Flow cytometry analysis revealed that upon activation with the appropriate combination of anti-CD2 mAb, T lymphocytes showed a markedly increased expression of the p75-kDaTNF receptor, whereas minor changes in the expression of the p55-kDa receptor were detected (Table 2). Treatment of Tcells with PMA alone or in combination with anti-AIMED69 also induced

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Regulation of tumor necrosis factor (TNF)-alpha synthesis and TNF receptors expression in T lymphocytes through the CD2 activation pathway.

The involvement of the CD2 (T11) molecule, an alternative activation pathway for T lymphocytes, in the regulation of tumor necrosis factor (TNF)-alpha...
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