EXPERIMENTAL CELL RESEARCH 225, 102–111 (1996)ARTICLE NO. 0161

Specific Binding of Adenosine Deaminase but Not HIV-1Transactivator Protein Tat to Human CD26

JULIA BLANCO, ISABELLE MARIE, CHRISTIAN CALLEBAUT, ETIENNE JACOTOT,BERNARD KRUST, AND ARA G. HOVANESSIAN1

Unite de Virologie et d’Immunologie Cellulaire, UA CNRS 1157, Institut Pasteur, 28 rue Dr. Roux, Paris Cedex 15, France

INTRODUCTIONAdenosine deaminase (ADA) and the HIV-1 transacti-

vator protein Tat have been reported to bind to human CD26 is a 110-kDa type II membrane glycoprotein,CD26, also known as dipeptidyl peptidase IV (DPP IV). constitutively expressed in different cell types, [1], butIn order to demonstrate the specificity of such binding highly regulated in lymphocytes, in which it was firstunder native conditions of CD26, i.e., when expressed on defined as an activation antigen [2]. Several biologicalthe cell surface, we established murine cell lines express- activities have been associated to CD26. It was founding transfected human CD26, either wild-type or mu- to be identical to the ectopeptidase dipeptidyl peptidasetated at its serine-630, which inactivates the DPP IV ac- IV (DPP IV) (EC 3.4.14.5), which can cleave N-terminaltivity. This experimental system is advantageous since dipeptides from a variety of potential substrates pro-murine ADA does not bind human CD26, whereas human

vided that the penultimate residue is proline. CD26and bovine ADA bind. Consequently, murine cell clonesappears to be implicated in antigen stimulation or inexpressing either the wild-type or mutated form of hu-anti-CD3/anti-CD26 costimulation of T cells [3, 4]. Weman CD26 were found to bind specifically bovine 125I-have previously reported that CD26 may play an im-labeled ADA with a high affinity (KD Å 12 { 2 nM and 11portant role in HIV infection, both for viral entry and{ 4 nM, respectively). No specific binding of 125I-labeledits cytopathic effect [5, 6]. This controversial role, alsoADA was observed to murine clones not expressing hu-reported recently by others [7], is most probably medi-man CD26. The binding of 125I-labeled ADA to CD26 wasated by the V3 loop of the HIV envelope glycoproteinfurther characterized by the use of monoclonal antibod-gp120. In accord with this, a synthetic V3 loop peptideies specific to human CD26. The results obtained werehas been shown to bind the catalytic domain of CD26in accord with those reported previously using other

experimental models. These observations indicated that [8]. Independent of its role in HIV entry and cytopathicthe murine cells expressing human CD26 provide a effect, CD26 has been reported to interact with HIV-1highly suitable model to investigate the potential bind- Tat protein, and this interaction has been suggested toing of HIV-1 Tat to CD26. In contrast to previously pub- be responsible for the immunosuppressive activity oflished results, however, we could not demonstrate a spe- Tat on memory T cells [9]. In this subset of T cells,cific interaction between Tat and human CD26. The 125I- CD26 could be found physically associated with thelabeled ADA-specific binding to human CD26 was not membrane-linked phosphatase CD45 [10]. Finally, sev-affected by Tat, even at concentrations which induced eral groups have identified CD26 or DPP IV as thecell death. Similarly, the binding of several monoclonal adenosine deaminase (ADA, EC 3.5.4.4) complexingantibodies to human CD26 was not modified by the addi- protein [1, 11, 12].tion of Tat. More significantly, Tat binding to different Adenosine deaminase (ADA) is a 43-kDa protein [13,murine cell clones (human CD26 negative or positive) 14] that can be found in a monomeric form or in awas found not to be correlated with the expression of

280-kDa molecular mass complex, corresponding to twohuman CD26. Finally, the toxic effect of Tat on themolecules of ADA bound to a CD26 dimer [15]. ADAgrowth of different murine cell clones was independentcatalyzes the deamination of adenosine or 2-deoxy-of human CD26 expression. Taken together, these obser-adenosine to inosine or 2-deoxyinosine and ammonia.vations further confirm the specific binding of ADA toIts localization is mainly cytoplasmic, but it may behuman CD26 and point out that CD26 is not the targetfound on the cell surface in virtue of its binding to CD26of HIV-1 Tat protein. q 1996 Academic Press, Inc.[16]. This existence on the cell surface allows ADA totake part in the general purine ectometabolism withthe inositol-anchored enzyme 5*-nucleotidase or differ-1 To whom correspondence and reprint requests should be ad-ent ectophosphatases [17] and in the costimulatory ef-dressed at Institut Pasteur, Departement Retrovirus/SIDA, 28 rue

du Dr. Roux, 75725 Paris 15, France. Fax: (33.1) 4061 3012. fect during the activation events mediated by the T-cell

1020014-4827/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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103CHARACTERIZATION OF ADA AND HIV-1 TAT BINDING TO CD26

1503 specific for HIV-1 Tat protein was generously provided by thereceptor–CD3 complex [18]. The inherited deficiency ofAIDS Research and Reference Reagent Program (NIAID, NIH: mAbADA causes a severe combined immunodeficiency into HIV-1 Tat from American Bio-Tecnologies, Inc.).

man [19]. In mice however, the lack of ADA activityCells. The murine hybridoma T-cell line T54S1, stably expressing

does not disturb the immune system but causes perina- human CD4, was a gift of U. Blank (Institut Pasteur, Paris, Francetal death due to liver cell degeneration [20]. Significant [32]). These cells were cultured in DMEM (ICN) supplemented with

10% FCS (Dominique Dustcher), 2 mM glutamine (Gibco), and 10species specific differences have also been reported formg/ml gentamicin (Panfarma). Transfections were performed in PBSthe capacity of ADA to bind to CD26. For example,at 1 1 107 cells/ml by addition of 6 mg of pKG5 plasmid DNA (con-bovine and human ADA bind to human but not murinetaining the neomycin resistance gene) with a 10-fold molar excess of

CD26, whereas murine ADA does not appear to bind either pSRa (for the establishment of human CD260 clones) or pSRato murine nor human CD26 [21, 22, and unpublished carrying the human CD26 either the wild-type or mutated at the

serine-630 to alanine (a generous gift of Dr. C. Morimoto; Dana-results]. In these studies, the characterization of ADAFarber Cancer Institute, Boston, MA). Cells were electroporated atbinding to CD26 has been performed by using purified250 V, 250 mF, and at 48 h after transfection were selected by cultur-preparations of both proteins or by binding studies of ing in medium containing 2 mg/ml G418 (Gibco). Jurkat cell lines,

purified ADA to cell membrane preparations. parental expressing low levels of endogenous CD26 (clone p32), orThe other soluble protein which has been reported transfected to express high levels of human CD26 (clone 11) were

also obtained from Dr. C. Morimoto [3]. CD260 C8166 cells (fromto bind human CD26 is the HIV-1 Tat protein [9]. TatDr. G. Farrar) were generously provided by MRC AIDS Directedis a 14-kDa regulatory protein which trans-activatesProgramme Reagent Project (UK). Jurkat and C8166 cells were cul-HIV genes and thus it is essential for viral replication tured in suspension medium RPMI 1640 (Bio-Whitaker, Verviers,

[23]. This protein, mainly localized in the nucleus, can Belgium) containing 10% (v/v) fetal calf serum.be released by Tat expressing cells to be taken up by Analysis of human and murine CD26 and human CD4 expression.

Human CD26 and human CD4 expression in different murine clonessurrounding cells [24] and might effect the expressionwas determined by FACS analysis using the anti human CD26 PE-of different cellular genes such as interleukin 2 andlabeled mAb Ta1 and the anti-human CD4 FITC-labeled mAb OKT4,its receptor [25], Mn-dependent superoxide dismutaserespectively. In some experiments human CD26 expression was de-

[26], or cyclin-dependent kinases [27]. In addition to termined by indirect immunofluorescence using mAb 1F7 [2], re-its ability to enter cells, extracellular Tat has been vealed with FITC-labeled rabbit anti-mouse (Immuno Quality Prod-

ucts). Incubations were performed in FACS buffer (1% bovine serumshown to have two major activities, acting as a growthalbumin and 0.01% sodium azide in PBS) at 47C for 30 min. Afterfactor for AIDS-associated Kaposi sarcoma cells [24]each incubation cells were washed twice, fixed in FACS buffer con-and supressing T-cell antigen-induced stimulation [28]. taining 1% formaldehyde, and analyzed in a FACScan flow cytometer

Finally, Tat has been shown to be toxic to cells and (Beckton–Dickinson, Mountain View, CA). In all experiments, PE-may induce apoptosis in several cell lines and periph- labeled mAB B4 (specific for CD19; Coulter) or FITC-labeled mouse

isotype control antibody MCG1 (Immuno Quality Products) wereeral blood mononuclear cells [27, 29–31].used as controls.In order to study the binding of ADA to human CD26

Cell surface expression of CD26 was also monitored by determiningwhen it is expressed on the cell surface, we established the cleavage of GP-pNA (glycylprolyl-p-nitroanilide, Sigma) as de-murine cell lines transfected to express human CD26. scribed previously [5].In this heterologous model, the recombinant human Bovine ADA and HIV-1 Tat iodination. Bovine ADA (Sigma TypeCD26 is ADA free, allowing the specific characteriza- VIII) and HIV-1 Tat 1-86 protein (AIDS Research and Reference

Reagent Program, NIAID, NIH: Recombinant HIV-1 Tat from Ameri-tion of the ADA/CD26 interaction. This model was thencan Bio-Tecnologies, Inc.) were iodinated by using the Bolton-Hunterused to test the ability of HIV-1 Tat to bind humanreagent (DuPont, NEN). Briefly 1 nmol of each protein was sus-CD26. The results pointed out that there is no specific pended in 10 ml 0.1 M borate buffer, pH 8.3, and allowed to react

binding of Tat to human CD26, under experimental overnight at 47C with 250 mCi of 125I-labeled Bolton-Hunter reagent.Reaction was stopped by the addition of 500 ml ice-cold 0.2 M glycine,conditions in which Tat-induced apoptosis was ob-0.1 M borate buffer, pH 8.3. Reaction mixtures were applied ontoserved in all tested cell lines regardless of the level ofEcono-Pac P6 cartridge columns (Bio-Rad) and eluted in 0.5% bovineCD26 expression. Our results confirm the specificity ofserum albumin (BSA) in PBS buffer in order to remove the unincor-

ADA binding to human CD26 and discard the implica- porated Bolton-Hunter reagent.tion of CD26 as a high-affinity receptor of Tat. 125I-Labeled ADA binding. Cells were washed twice in serum-free

culture medium and resuspended at 1 1 107 cells/ml. Aliquots of 0.51 106 cells were preincubated for 15 min at 377C in the presence of

MATERIALS AND METHODS antibodies or binding inhibitors (such as unlabeled bovine ADA orTat) and then incubated with the desired amounts of radiolabeledproteins in a final volume of 100 ml. After incubation (usually 1 h),Antibodies. Five different monoclonal antibodies (mAb) specific

for human CD26 were used: BA5 (Immunotech, Marseille, France), cells were washed twice in cold PBS and lysed in SDS–PAGE samplebuffer (125 mM Tris–HCl, 2% SDS, 20% glycerol, 2% 2-mercaptoeth-Ta1 (Coulter, Miami, FL), 4H12 (Endogene, Cambridge, MA), 1F7

(kindly provided by Dr. C. Morimoto, Dana-Farber Cancer Institute, anol, 0.002% bromophenol blue). Following SDS–PAGE analysis,gels were fixed and dried and radioactivity associated with the 43-Boston, MA; [2]), TA5.9-CC1-4C8 (kindly provided by Dr. E. Boss-

man, Eurogenetics, Tessenderlo; [12]). The mAb OKT4 specific for kDa 125I-labeled ADA band was quantified in a Phosphorimager (Mo-lecular Dynamics, Sunnyvale, CA).human CD4 was purchased from Ortho Diagnostics Systems. The

polyclonal antibody raised against human ADA was kindly provided ADA binding was also monitored by measuring the ADA activityassociated to the cell membrane of whole cells. Briefly, cells wereby Dr. R. Franco (University of Barcelona, Spain; [16]). The mAb

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104 BLANCO ET AL.

incubated with 200 nM cold ADA and washed as described. Ecto-ADA clones) or expressing human CD26, wild-type CD26, oractivity was assayed in control and ADA-treated cells by incubating 2 serine-630 to alanine-mutated CD26 (S/A630) [3]. G418-1 105 cells in buffer containing 10 mM Hepes, pH 7.4, 138 mM NaCl,

resistant clones were tested for the expression of hu-5 mM KCl, 1.2 mM MgSO4, 8 mM glucose, 50 mM adenosine, 1 mMman CD26 and human CD4 by FACS analysis, usingnitrobenzylthioinosine (NBTI, an inhibitor of adenosine uptake). In-

cubations were performed at 377C for 15 or 30 min, and deamination mAb Ta1 specific for human CD26 and mAb OKT4 spe-of adenosine was determined by measuring the absorbance at 265 cific for human CD4 (Fig. 1). Cell clones were alsonm of cell-free incubation media. FACS analysis of membrane-bound tested for ecto-DPP IV activity (Table 1). This method,ADA was performed as described [33].

which does not differenciate between human and mu-Tat binding. 125I-Labeled Tat binding was determined as de-

rine CD26, was used here to evaluate the expressionscribed for [125I] ADA binding. Tat binding was also determined byof endogenous murine CD26.FACS analysis, using unlabeled Tat as ligand. Incubations and wash-

ings were performed as described above. Cells were incubated in the Table 1 gives the characteristics of the selectedFACS buffer at 47C for 30 min with anti-HIV-1 Tat (mAb 1503) which clones. As expected, the clones transfected with therecognizes a region near the N-terminus of Tat, amino acids 6–14. pSRa control plasmid are human CD260; however, aCells were then washed twice with the same buffer and revealed

low expression of murine cell surface CD26 was found,with FITC-coupled rabbit anti-mouse. After washing twice, cells wereas monitored by the ecto-DPP IV activity (Table 1 andfixed in FACS buffer containing 1% formaldehyde and analyzed in

a FACScan flow cytometer. In some binding experiments Tat was Fig. 1). The FACS analysis revealed a high expressionused in a reduced state. Reduction of Tat was performed by incubat- of human CD26 in the clones transfected with pSRaing the protein (25 mM) in nonsupplemented culture medium con- plasmid containing wild-type or mutated CD26 (clonestaining 200 mM 2-mercaptoethanol at 377C for 2 h. When reduced

W and M, respectively; Fig. 1). Determination of theTat was used, 1 mM 2-mercaptoethanol was added in all incubationbuffers. DPP IV activity confirmed the phenotype of the human

Tat cytotoxicity. The effect of Tat on cell proliferation was deter- wild-type compared to the mutated CD26 expressed inmined in 96-well plates by incubating of 2 1 105 cells at different these W and M clones (Table 1). In accord with theconcentrations of Tat in a final volume of 200 ml. Cells were counted expression of the wild-type CD26 on the surface of Wat 24 and 48 h, and Tat-induced cell death by apoptosis was deter-

clones, the DPP IV activity was high. On the othermined by fluorescence microscopy, using a TUNEL-based fluoresceinhand, the endogenous murine DPP IV activity was notin situ cell death detection kit (Boehringer-Mannheim), according to

the instructions of the manufacturer. significantly different between the M and T clones, thusconfirming that the mutated CD26 was devoid of DPPIV activity.RESULTS

In order to corroborate the lack of murine ADA bindingto human CD26, the different murine clones were moni-The characterization of the binding of exogenoustored for the presence of ecto-ADA by FACS analysis andbovine ADA to human cells expressing human CD26by enzyme activity determination. By FACS analysis, nois handicapped by the presence of endogenous humanecto-ADA labeling was observed in control (T) clones,ADA. In preliminary experiments using human Jur-while a weak labeling was found in CD26-transfected (Wkat cell lines expressing different levels of CD26 andand M) clones. The presence of ADA in these clones isby assaying for ecto-ADA activity, we estimated thatmost probably due to contamination from the fetal calf35–70% of ADA binding sites on the cell surfaceserum used in cell culture media and was evaluated toCD26 were occupied by endogenous ADA (not shown).occupy less than 5% of the potential ADA binding sites.The presence of ecto-ADA may affect the binding ofBy ecto-ADA activity determination, low levels of activityexogenous ADA, as ADA associated to CD26 repre-were found in all clones tested; however, no significantsents a steady-state equilibrium between bound anddifferences were observed between control (T) clones andfree ADA. In order to investigate the specific bindingCD26-transfected (W and M) clones (data not shown).of ADA to CD26 without competition interference ex-

erted by the endogenous ADA, we studied the binding125I-Labeled ADA Binding to Murine Cell Clonesof bovine ADA to human CD26 expressed on the sur-

Expressing Human CD26face of murine cells. In this heterologous system, bo-vine ADA can specifically bind to human CD26, which

In order to determine the specificity and the kineticis ADA free since endogenous murine ADA fails toparameters of ADA binding to human CD26, saturationbind to human CD26.isotherms of 125I-labeled ADA and competition with un-labeled ADA were performed using the different mu-Characterization of Murine Cell Clones Expressingrine clones. Control (clone T6) and wild-type CD26 ex-Human CD26pressing (clone W12) cells were incubated with increas-ing concentrations of bovine 125I-labeled ADA rangingA murine hybridoma T-cell line, stably expressing

human CD4, was cotransfected with the plasmid pKG5 from 1 to 100 nM. The binding of 125I-labeled ADA tothe W12 clone reached a saturation at 10–25 nM,carrying the neomycin resistance and the plasmid

pSRa, either as such (for the generation of control whereas no binding was observed for the control T6

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105CHARACTERIZATION OF ADA AND HIV-1 TAT BINDING TO CD26

FIG. 1. Characterization of human CD26 expressing clones. FACS analysis of human CD26 (Hu CD26) and human CD4 (Hu CD4)expression in the control clone T6 and in clones expressing recombinant forms of human CD26; either the wild-type (clone W12) or theS630A mutated form (clone M9). PE-labeled anti-Ta1 mAb specific for human CD26 and FITC-labeled mAb OKT4 specific for human CD4were used. Peaks C-PE and C-FITC give the fluorescence of cells incubated with control PE- or FITC-labeled antibodies.

clone (Fig. 2). Similar results were observed using other saturation experiment and correlates with the valueobtained in the displacement experiments (15 { 4 nM).control (clones T1, T2, and T3) and human CD26 ex-

pressing (W6, W10, and W5) murine cells (data not The lack of specific binding of bovine 125I-labeled ADAto the control clone T6, even at the highest concentra-shown). These results illustrated that the binding of

125I-labeled ADA to the murine clones is dependent on tion used (100 nM), confirmed the strict specificity ofbovine ADA binding to the human CD26. Interestingly,the expression of cell surface human CD26. The bind-

ing of 125I-labeled ADA was specific, since it was com- the dissociation constant values for the binding of 125I-labeled ADA to the clones expressing the S630A mu-pletely prevented by excess unlabeled ADA (Fig. 3).

The KD value calculated after quantitation of the radio- tant form of human CD26 (clones M) was found not tobe significantly different compared to those observedactivity associated to each band was 12 { 2 nM in thewith the wild-type CD26 expressing clones (clones W).For example the KD values for 125I-labeled ADA bindingin M9 and W12 clones were 11 { 4 and 12 { 2 nM,TABLE 1respectively (Fig. 4). Previously, it has been reported

Expression of Human CD26 in Different Murine Cells

DPP IV activity onFACS analysis the cell surface

Cell clone (human CD26) (nmol pNA cleaved)

T1 0 16.4T2 0 10.2T5 0 12.4T6 0 8.7W6 HuCD26; wt /// 55.5W10 HuCD26; wt /// 50.5W11 HuCD26; wt /// 51.7W12 HuCD26; wt /// 102.6M2 HuCD26; S630A /// 23.8M8 HuCD26; S630A /// 16.2M9 HuCD26; S630A /// 10.4M14 HuCD26; S630A /// 15.0

Note. Cell surface expression of human CD26 was determined indifferent murine clones by FACS analysis and also by assaying for FIG. 2. 125I-Labeled ADA binding to human CD26. Saturation

analysis of 125-labeled ADA binding to human CD26 negative murinecell surface DPP IV activity as described under Materials and Meth-ods. Clones T1, T2, T5, and T6 represent control cells; clones W6, clone T6 (A) and the human CD26 expressing clone W12 (B). Cells

were assayed for 125I-labeled ADA binding as described under Mate-W10, W11, and W12 represent cells expressing the wild-type (wt)recombinant form of human CD26; clones M2, M8, M9, and M14 rial and Methods. The concentrations of labeled bovine ADA used

were 2, 10, 25, 50, and 100 nM (lanes 1 to 5, respectively). Therepresent cells expressing a mutant form of recombinant humanCD26 with a point mutation in serine-630 to alanine (S630A). Note figure shows a typical autoradiogram of one of three independent

experiments. The position of molecular weight protein markers (kDa)that DPP IV activity values are the addition of human and murineenzyme activities in clones expressing the wild-type human CD26. is given in the middle of the gel (lane M).

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106 BLANCO ET AL.

FIG. 3. Specific binding of 125I-labeled ADA to human CD26.Binding of 10 nM 125I-labeled ADA to human CD26 expressing W12clone was determined in the absence or in the presence of increasingamounts of unlabeled ADA. Experimental conditions are those de-scribed under Materials and Methods. Cells were preincubated for15 min at 377C with unlabeled bovine ADA before addition of 125I-labeled ADA. The concentrations of unlabeled ADA used were 40,100, and 500 nM and 1 and 2 mM (lanes 2 to 5). Lane 1 shows 125I-labeled ADA binding in the absence of unlabeled ADA. The positionof molecular weight markers (kDa) is given (lane M).

that ADA binding to CD26 does not affect the DPP IVactivity [11, 12]. Here we clearly demonstrate that the FIG. 5. Characterization of 125I-labeled ADA binding to humanS630A point mutation which completely inactivates the CD26. The effect of different anti human CD26 mAbs on the binding

of 125I-labeled ADA (10 nM) was determined using the W12 clone ascatalytic activity of CD26 [3] does not modify 125I-la-described under Materials and Methods. Anti-human CD26 mAbsbeled ADA binding.used were Ta1, 1F7, BA5, 4H12, and TA5.9 (clone 4C8). The mAbOKT4A specific for CD4 was used as a control monoclonal antibody.The different mAbs were used at 1 and 10 mg/ml as indicated. Apolyclonal antibody anti-human ADA was used to demonstrate theblockage of 125I-labeled ADA binding to human CD26; an irrelevantrabbit serum was used as control. The rabbit sera were used at1/100th and 1/10th dilution. 100% binding represents the controlvalue, i.e., the binding of 125I-labeled ADA to the W12 clone in theabsence of antibodies (None). The values are given as the percentageof binding in the presence of the different antibodies compared tothe control value.

Other saturation binding experiments were carriedout using a Jurkat cell line that expresses increasedlevels of transfected wild-type human CD26 (clone 11)[3]. The binding of 125I-labeled ADA was specific onthese Jurkat cells with an apparent KD value of 14 {2 nM (not shown), similar to the value found in themurine clones expressing human CD26.

Characterization of 125I-Labeled ADA Binding toFIG. 4. Binding of 125I-labeled ADA to human CD26 devoid of Human CD26

DPP IV activity. Saturation isotherms of 125I-labeled ADA bindingto wild-type human CD26 (clone W12; closed symbols) and to the The efficiency of our heterologous murine system wasS630A mutant form of human CD26 (clone M9; open symbols). Each further demonstrated by determining the inhibitory ef-point represents total 125I-labeled ADA binding determined by quan- fect of different monoclonal antibodies specific for hu-titation of the 43-kDa band-associated radioactivity as described

man CD26 on the binding of 125I-labeled ADA (Fig. 5).(Materials and Methods). Nonspecific binding was negligible, asshown in Fig. 2. The epitope recognized by mAb TA5.9 has been sug-

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107CHARACTERIZATION OF ADA AND HIV-1 TAT BINDING TO CD26

gested to be at the ADA binding site in the humanCD26 [12]. Consequently, this mAb at 1 mg/ml blockedalmost completely the 125I-labeled ADA binding toCD26. Other anti-CD26 mAbs, except mAb Ta1, inhib-ited to different extents the binding of 125I-labeled ADAto CD26. However, it should be noted that their inhibi-tory effect was less than 75% even at high concentra-tions (10 mg/ml) of the antibody. This latter might bethe consequence of steric hinderance or it might bepossible that certain anti-CD26 mAbs modify the con-formation of CD26, thus affecting its affinity to ADA.These results are in agreement with those previouslyreported by others, who found no effect of mAb Ta1 andonly a partial effect of mAb 1F7 on ADA binding [12].As expected, polyclonal anti-ADA antibody blocked 125I-labeled ADA binding to CD26, while control rabbit anti-bodies had no effect. MAb OKT4A directed against hu-man CD4 (expressed stably in these murine cells) hadno effect on 125I-labeled ADA binding.

Rabbit anti-ADA antibodies were raised against hu-FIG. 6. The specific binding of 125I-labeled ADA is not affectedman ADA [16]. This and the fact that this antibody com-

by HIV-1 Tat protein. Binding of 125I-labeled ADA (10 nM) was deter-pletely inhibited the binding of bovine ADA to human mined in the absence (No addition) and in the presence of differentCD26 (Fig. 5) indicate that bovine ADA behaves like hu- concentrations of HIV-1 Tat protein (as indicated) or unlabeled bo-

vine ADA (4 mM). HIV-1 Tat protein and unlabeled ADA were addedman ADA with respect to its binding to human CD26.15 min before starting incubation. Values are the means { SD oftwo independent experiments.Effect of Tat on 125I-Labeled ADA Binding

Gutheil et al. [9] have reported that HIV-1 Tat pro-tein directly interacts with human CD26 in a region of CD26 expression (data not shown). To enhance theclosely related or identical to the Ta1 epitope (identified sensitivity of the binding assay, a FACS-based analysisby mAb Ta1) and thus might be responsible for an in- was used to evaluate Tat binding (Fig. 7). Cells werehibitory effect on antigen-stimulated lymphocyte pro- first incubated with Tat before washing extensively toliferation. In order to assess the effect of such a poten- remove unbound Tat. The cell surface bound Tat wastial interaction on the 125I-labeled ADA binding to then revealed by FACS analysis using an anti-Tat mAbCD26, binding experiments were carried out in the (1503). The efficiency of this method was demonstratedpresence of different concentrations of purified Tat (10 by the lack of any Tat signal on cells which have notnM to 1 mM). As shown in Fig. 6, Tat protein had no been preincubated with Tat (Fig. 7, sections T5 0 Tateffect on 125I-labeled ADA-specific binding, even at ele- and W12 0 Tat; the peak C for the control mAb andvated concentrations. Consequently, in order to demon- peak 1503 for the anti-Tat mAb were superimposable).strate any potential interaction between Tat and hu- Incubation of murine cell clones with 200 nM of Tatman CD26, we investigated the direct binding of Tat at 377C for times ranging from 2 to 12 h resulted into human CD26. the association of Tat on the surface of both human

CD260 (clones T) and CD26/ (clones W) murine cells.Interaction of Tat with Murine Cell Is Independent ofA typical result is shown in Fig. 7 for the clones T5 andHuman CD26 ExpressionW12. Clearly, Tat binding in these cell clones was notcorrelated to human CD26 expression; the bound TatThe 125I-labeled ADA binding studies discussed

above demonstrated that the heterologous murine sys- average mean fluorescence (a. m. f.) values in the T5and W12 clones were 29 { 5 and 16 { 4, respectively.tem expressing human CD26 provide an efficient ex-

perimental model to investigate the interaction of a As Gutheil et al. [9] have reported that Tat binds theTa1 epitope in human CD26, we studied the effect ofpotential ligand with human CD26. Therefore, we used

such murine cell clones (T5 and W12) to confirm the Tat on the binding of Ta1 mAb to the murine W12 cloneexpressing human CD26. The anti-human CD26 mAbbinding of Tat to human CD26. In a preliminary experi-

mental approach, 125I-labeled Tat was used to study 1F7 was used in order to verify that cell surface expres-sion of CD26 was not modified by Tat during the experi-both the binding and internalization in the murine

clones T5 and W12. However, the binding of 125I-labeled ment (Fig. 7). The results show that the expression ofboth epitopes (Ta1 and 1F7) in human CD26 was notTat to these cells was found to be very low, irrespective

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108 BLANCO ET AL.

Similar experiments were carried out using the hu-man Jurkat cells expressing either low levels of endoge-nous CD26 or increased levels (100-fold) of transfectedCD26 [34]. No correlation was observed between thebinding of Tat to such Jurkat cells and the level ofCD26 expression (data not shown).

Tat Is Cytotoxic to Murine Cells Independent of theExpression of Human CD26

Human cells treated with Tat undergo cell death byapoptosis [27]. In view of this, we investigated the effectof different concentrations (0.1 to 5 mM) of Tat on cellproliferation and apoptosis, using the murine cellclones negative (clones T) or positive (clones W and M)for the expression of human CD26 and also the humanJurkat cell lines expressing low endogenous or en-hanced transfected CD26. In all the different types ofcells, more than 50% inhibition of cell proliferation(monitored by the cell number) was observed following2 days of treatment with 5 mM of Tat (data not shown).

This inhibition of cell growth was highly associatedwith the occurrence of apoptosis monitored by the TU-FIG. 7. HIV-1 Tat does not bind to human CD26. The binding

of Tat to human CD26 was investigated by using murine cells ex- NEL method. Figure 9 shows typical results in murinepressing (clone W12) or not expressing (clone T5) human CD26. Fluo- cell clones negative (clone T6) or positive (clone W12)rescence labeling was determined in cells incubated for 12 h in the for the expression of human CD26 and in two humanabsence (0Tat) or in the presence (/Tat) of 200 nM Tat as described

Jurkat cells lines [3] expressing endogenous low levelsunder Materials and Methods. Peak C in each section gives the fluo-of CD26 (parental cells P32) or high levels of CD26rescence obtained using a control antibody. mAb 1503 gives the fluo-

rescence associated to Tat protein bound to the cell surface. Anti- (obtained by transfection; clone 11). In both human andhuman CD26 mAb Ta1 was used to verify whether Tat binding blocks murine cell lines, a significant degree of apoptosis wasthe Ta1 epitope (as it has been reported previously by Gutheil et al., observed in the presence of 5 mM Tat, independent of1994), whereas mAb 1F7 anti-human CD26 was used to demonstrate

human CD26 expression (Fig. 9). No apparent effectthat Tat does not modulate human CD26 expression on the cell sur-face. Average mean fluorescence was determined for each peak (see was observed in the murine and human cell lines incu-text). bated with 1 mM Tat. These results, therefore, indicate

that the cytotoxic effect of Tat is not dependent on thepresence of CD26, and moreover this effect is not spe-cies specific since murine cells were as sensitive to Tataltered by preincubation of cells with Tat; the mAb Ta1as human cells.a. m. f. values in the control and Tat-preincubated cells

Further evidence that the Tat-induced cytotoxic ef-were 360 { 30 and 390 { 50, respectively.fect is independent of CD26 expression was providedTaking into account the importance of the redox stateby the use of human C8166 cells which are CD26 nega-for Tat activity [34], further binding experiments weretive [35]. Tat at 5 mM concentration resulted in a com-carried out using reduced Tat (Materials and Methods).plete arrest of cell proliferation (data not shown).In this latter case 1 mM 2-mercaptoethanol was main-

tained in all the buffers during the experiment. TheDISCUSSIONpresence of 2-mercaptoethanol did not affect the capac-

ity of anti-human CD26 mAbs 1F7 and Ta1 to bindtheir respective epitopes. In contrast, the binding of The activation antigen CD26 has been identified as

the ectopeptidase DPP IV and also as the ADA com-reduced Tat to the T5 and W12 clones was significantlyhigher compared to that of unreduced Tat (Fig. 8). In plexing protein (for a review see [36]). However, al-

though the precise function of extracellular peptidasespite of this, the enhanced binding of reduced Tat wasnot correlated with the expression of human CD26, and deaminase activities, and their relationship, if any,

are not yet defined, both CD26 and ADA have beenthus indicating once again that Tat does not bind hu-man CD26. Consistent with this, the binding of mAbs implicated in T-cell activation events [18, 37]. It should

be noted that human adenosine deaminase has beenTa1 and 1F7 to human CD26 (expressed on the surfaceof W12 cell clone) was not affected by the preincubation shown to exist as different isoforms designated as

ADA1 (ADA as referred to herein), ADA2, and a double-of cells with the reduced Tat (Fig. 8).

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109CHARACTERIZATION OF ADA AND HIV-1 TAT BINDING TO CD26

FIG. 8. Interaction of reduced Tat with the different murine clones. Tat protein was reduced as described under Materials and Methods.The control clone T5 (not expressing human CD26) and the human CD26 expressing clone W12 were assayed for their ability to bind bothnontreated Tat (/Tat) and reduced Tat (/red Tat). FACS analysis with anti-Tat (mAb 1503) and anti-CD26 (mAbs Ta1 and 1F7) was performedas indicated in Fig. 7. Both cell types were also assayed by FACS analysis in the absence of Tat. Incubations of reduced Tat were performed inthe presence of 1 mM 2-mercaptoethanol; as observed, this addition did not modify CD26 labeling with mAbs Ta1 or 1F7.

stranded RNA-specific ADA [15, 38]. However, in con- in the range of 10 nM [22]. Other studies have comeup with a similar KD value for bovine ADA bindingtrast to ADA1 or ADA little is known about the two

other isoforms. to purified and agarose-conjugated human CD26 [11].Further qualitative experiments have provided evi-Binding of ADA to CD26 has been characterized pre-

viously in various species by using different experimen- dence for binding of bovine ADA to intact human pe-ripheral blood lymphocytes [12] and for the lack of bo-tal models. Equilibrium constant studies for the bind-

ing of rabbit, monkey, bovine, and human ADA to glu- vine ADA binding to murine CD26 [21].Our heterologous experimental model describedtaraldehyde-fixed rabbit kidney membranes have

indicated that the binding was specific, with KD values herein—the binding of bovine ADA to human CD26 ex-

FIG. 9. Tat-induced apoptosis is not correlated to the CD26 expression. Two human Jurkat cell lines (A), expressing either low levelsof CD26 (parental Jurkat cells; P32) or enhanced levels of CD26 (obtained by transfection; clone 11), and the murine clones (B) expressing(W12) or not expressing (T6) human CD26 were incubated in their respective culture medium in the absence (0) or in the presence ofindicated amounts of the Tat protein. After 48 h incubation cells were assayed for terminal transferase labeling (TUNEL) by fluorescencemicroscopy. Three different fields were examined for the proportion of apoptotic cells; the mean values are given for the % apoptotic cells.

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110 BLANCO ET AL.

pressed by murine cells—provided an efficient model to onstrated that Tat could interact with murine, rat, andhuman cells in a nonspecific manner [29, 40]. In thefurther characterize the specific binding of bovine ADA

to human CD26 expressed in its native state on the cell case of growth stimulatory effect of Tat on the Kaposisarcoma tumor cells in which Tat is functional at 0.1–1membrane. Calculations based on ecto-ADA activity in

ADA-saturated cells and from 125I-labeled ADA saturation ng/ml, it has been proposed that Tat’s action is throughspecific interaction with the integrin receptors [39]. Onbinding experiments suggested that the expression of hu-

man CD26 in transfected murine cell clones was of high- the other hand, no specific receptors have been desig-nated for the other Tat-induced effects which requiredensity type (1–1.51 105 ADA binding sites/cell). Interest-

ingly, human CD26 expressed by such clones was ADA concentrations higher than 100 ng/ml, such as effectson cellular gene expression, immunosuppression, cellfree because of the failure of murine ADA to bind CD26.

Indeed, less than 5% of CD26 was found to be occupied cytotoxicity, and induction of apoptosis [27, 29–31]. AsTat has a high content of basic amino acids, it is possi-by ADA in these murine cell clones. Equilibrium binding

parameters for bovine 125I-labeled ADA binding to human ble to suggest that in these latter situations Tat maybind cells through nonspecific interactions with differ-CD26 showed a high-affinity binding site, with a KD value

in the low nM range, and a behavior similar to that de- ent cell surface antigens.scribed in human cells expressing CD26. In view of theseresults, therefore, we believe that the experimental condi- This work was supported by grants from Institut Pasteur and

Agence Nationale de la Recherche sur le SIDA, ANRS. J.B. is recipi-tions were very well adapted for the study of the potentialent of a postdoctoral fellowship from Spanish Ministerio de Educa-interaction of HIV-1 Tat with human CD26.cion y Ciencia.Previously, Gutheil and collaborators [9], by flow cy-

tometry analysis using anti-Tat mAb and by the inhibi-REFERENCEStory effect of Tat on the DPP IV activity of purified CD26,

have suggested that Tat binds human CD26. It should1. Morrison, M. E., Vijayasaradhi, S., Engelstein, D., Albino,be noted that in these latter experiments, the specificity

A. P., and Houghton, A. N. (1993) J. Exp. Med. 177, 1135–1143.of Tat/CD26 binding was not demonstrated by these au-

2. Dang, N. H., Torimoto, Y., Schlossman, S. F., and Morimoto, C.thors in the flow cytometry assay. Furthermore, the in- (1990) J. Exp. Med. 172, 649–652.hibitory effect of Tat on DPP IV activity was observed 3. Tanaka, T., Kameoka, J., Yaron, A., Schlossman, S. F., andonly at salt concentrations less than 60 mM, and no Morimoto, C. (1993) Proc. Natl. Acad. Sci. USA 90, 4586–4590.inhibitory effect was observed at physiological salt con- 4. Tanaka, T., Duke-Cohan, J. S., Kameoka, J., Yaron, A., Lee, I.,

Schlossman, S. F., and Moromoto, C. (1994) Proc. Natl. Acad.centrations. In order to confirm and further characterizeSci. USA 91, 3082–3086.the potential interaction of Tat with CD26, we investi-

5. Callebaut, C., Krust, B., Jacotot, E., and Hovanessian, A. G.gated the specificity of Tat/CD26 binding by using our(1993) Science 262, 2045–2052.murine cell clones expressing or not expressing human

6. Callebaut, C., Jacotot, E., Marie, I., Blank, U., Robert, N.,CD26. This experimental model therefore included anKrust, B., and Hovanessian, A. G. (1994) The role of CD26 inunquestionable control, provided by the murine cell HIV infection: Viral entry and its cytopathic effect., in Retrovi-

clones not expressing human CD26, to assess the speci- rus of human AIDS and related animal diseases, pp. 141–149,Ixe Colloque des Cent Gardes.ficity of Tat/CD26 binding. The results obtained indi-

cated that Tat binds to murine cells but this binding is 7. Oravecz, T., Roderiquez, G., Koffi, J., Wang, J., Ditto, M., Bou-Habib, D. C., Lusso, P., and Norcross, M. A. (1995) Nature Med.not correlated with the expression of human CD26, thus1, 919–926.ruling out a specific interaction with human CD26.

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Received January 23, 1996Revised version received March 7, 1996

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