Ž .Journal of Immunological Methods 221 1998 107–117

Flow cytometric quantification of HIV-1 Tat protein intat-transfected Jurkat T cell lines

Davide Gibellini a,), Maria Carla Re a, Roberto Panaya a, Erika Venturi a,Daniela Milani b, Michele La Placa a, Giorgio Zauli b

a Microbiology Section, Department of Clinical and Experimental Medicine, UniÕersity of Bologna, Via Massarenti 9, 40138 Bologna, Italyb Human Anatomy Section, UniÕersity of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy

Received 9 July 1998; revised 29 July 1998; accepted 24 August 1998

Abstract

The transactivator Tat protein represents a pivotal factor for the replication of human immunodeficiency virus type 1Ž .HIV-1 . In this report, we describe a flow cytometry procedure designed to quantify the intracellular content of Tat proteinin Jurkat CD4q T lymphoblastoid cell lines, stably transfected with plasmids expressing full-length Tat protein. Variousexpression vectors were compared for their effectiveness to yield Tat protein in Jurkat cells, and several technical parameterswere analyzed to optimize the assay. This method offers a quick and efficient approach to select stably transfected cell linesexpressing different levels of specific protein. q 1998 Elsevier Science B.V. All rights reserved.

Keywords: HIV-1; Tat protein; Flow cytometry

1. Introduction

The construction of stably transfected eukaryoticcell lines represents a basic tool in cell biologyŽ .Kriegler, 1990; Glover, 1996 . Thanks to the im-provement of transfection methods and to the genera-tion of progressively more efficient expression vec-tors, this approach is now widely used. Stably trans-fected cell clones have several applications, allowingthe overexpression of proteins of biological interestand also being used as reporter gene carriers to

Ževaluate factor-induced transactivations Kriegler,.1990; Keller and Manak, 1995; Glover, 1996 .

) Corresponding author. Tel: q39-51-341652;Fax: q39-51-341632

Each new stably transfected cell line needs to betested to verify that the protein of interest is ex-pressed correctly. Classical methods, such as North-ern blotting and immunoblotting are usually appliedto ascertain the presence of mRNA and protein,

Žrespectively Kriegler, 1990; Keller and Manak,.1995; Glover, 1996 . Although specific, these tech-

niques do not permit precise quantitative evaluations.On the other hand, quantification of a given proteinrepresents a crucial parameter for the selection ofnew cell clones. For instance, it has been demon-strated that several proteins show biological effectsonly above a given threshold and within a well-de-fined range of concentrations. In fact, negative orparadoxical results can be observed when the amount

Žof synthesized protein is very high Corallini et al.,

0022-1759r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-1759 98 00169-0

( )D. Gibellini et al.rJournal of Immunological Methods 221 1998 107–117108

.1996 . An interesting example of this phenomenon isrepresented by the Tat protein of human immunode-

Ž .ficiency virus-type 1 HIV-1 , which plays a key roleŽin efficient HIV-1 replication Dingwall et al., 1989;

.Cullen, 1991; Jones and Peterlin, 1994 . Besidestransactivating the HIV-1 genome, Tat protein alsoinduces pleiotropic effects influencing the survival ofvarious cell types, mainly by transactivating specific

Žcellular genes Ensoli et al., 1990; Zauli et al., 1992;Ensoli et al., 1993; Zauli et al., 1993; Albini et al.,1994; Gibellini et al., 1994; Scala et al., 1994;Westendorp et al., 1994; Zauli et al., 1995a; Corallini

. Žet al., 1996 . In particular, opposite promotion vs..inhibition effects on cell survival have been un-

Ž .equivocally related to relatively small one log dif-ferences in the intracellular concentrations reached

Ž .by Tat protein in lymphoid cells Ensoli et al., 1993 .In this report, we describe a flow cytometric

procedure, which makes it possible both to determinethe presence and to quantify the amount of Tatprotein in Jurkat lymphoblastoid CD4q T cell lines.

2. Materials and methods

2.1. Cell lines

The parental Jurkat CD4q lymphoblastoid T cellline was obtained from American Type Culture Col-

Ž .lection Rockville, MD and kept in RPMI 1640Ž .GIBCO Laboratories, Grand Island, NY supple-

Ž .mented with 10% fetal calf serum FCS, GIBCO atan optimal cell density of 0.3–1.5=106.

Stably tat-transfected Jurkat cell clones were ob-Ž .tained, as previously described Caputo et al., 1990 ,

by transfecting Jurkat cells with the following plas-Ž .mid constructs: i pRPneoSL3rtat containing HIV-

Ž .1tat cDNA cloned in sense under the control of theSL3 murine leukemia virus long terminal repeat

Ž .region, ii pSVneortat containing HIV-1tat cDNAŽ .cloned in sense under the control of SV40 pro-

Ž . Ž .moter region, iii pRPneoSL3 and iv pSVneo rep-resenting backbone control plasmids. The pLTRCATplasmid represents a pCAT vector, where the longterminal repeat region of HIV-1 was cloned in frontof the bacterial chloramphenicol-acetyltransferaseŽ .CAT gene. All stably transfected cell clones weregrown in RPMI plus 10% FCS and kept under

continuous selective pressure by adding 800 mgrmlŽ .of the G418 antibiotic Sigma, St. Louis, MO .

2.2. Dot–blot assay

2=106 cells were washed in PBS, centrifuged,Žand treated with lysis solution 2.3% SDS, 5% b-

.mercaptoethanol, 0.015M Tris–HCl pH 6.8 , as pre-Ž .viously described Musiani et al., 1989 . The pro-

teins were boiled for 5 min and chilled in ice. ProteinŽextracts and recombinant Tat protein Intracell, Cam-

.bridge, MA were filtered onto nylon membranesŽ .Amersham, Airlington, IL . The filters were blocked

Žusing 2% blocking buffer Boehringer Mannheim,.Germany and then treated with a rabbit polyclonal

Ž .anti-Tat serum Intracell diluted 1r100 in 2% bovineŽ .serum albumin BSA rPBS for 3 h at room tempera-

Ž .ture RT . After three washes with 0.5% Tween20rPBS for 15 min at RT, the samples were pro-cessed with anti-rabbit IgG peroxidase-conjugatedantibody diluted 1r200 in 2% BSArPBS for 1 h atRT. The cells were washed three times with 0.5%Tween 20rPBS for 15 min at RT. After these washes,nylon filters were developed with 4-chloro-1-naph-

Ž .thol, as previously described Musiani et al., 1989 .

2.3. CAT assay

Transient transfection experiments were carriedout using the DEAE-dextran method, as previously

Ž . 6described Zauli et al., 1995b . Briefly, 10=10 ofpRPneoSL3rtat, pSVneortat, pRPneoSL3, pSVneoand Jurkat cell clones were transfected with 5 mg ofLTR-CAT plasmid and 500 mg DEAE-dextran for60 min in 2 ml of medium. After transfection, cellswere seeded again in 10 ml of RPMI plus 10% FCSfor 24 h. Cells were then lysed and the clarifiedlysates were assayed for CAT activity using volumesof extracts corresponding to equal amounts of pro-tein as determined by the Bio-Rad protein assay

Ž .system Bio-Rad Laboratories, Richmond, CA .

2.4. Fixation, permeabilization and staining of thecells

Ž 6Actively growing Jurkat cells 2=10 cells for.each sample were washed twice in PBS and then

( )D. Gibellini et al.rJournal of Immunological Methods 221 1998 107–117 109

fixed and permeabilized using one of the two proce-dures described below.

In the first approach, cells were fixed in 0.5 mlŽ .70% ethanol EtOH for 30 min at 48C. Samples

were centrifuged, washed twice in PBS, treated withŽ200 ml of Guinea pig normal serum 10% vrv in

.PBS, DAKO, Copenhagen, Denmark for 15 min atRT to saturate nonspecific binding sites, and thenincubated for 2 h at 378C in 200 ml containing eitheranti-Tat polyclonal serum or anti-Tat monoclonalantibody directed against the first 14 aminoacids of

Ž . Ž .Tat protein Intracell , both diluted 1:100 in 2%BSArPBS. After two washes in PBS, samples weretreated with a fluorescein-conjugated swine anti-rab-

Ž .bit FITC-SAR, Dako or fluorescein-conjugated rab-Ž .bit anti-mouse FITC-RAM, Dako IgG diluted

Ž .1r200 in PBSr2% BSA. After several washes inPBS, samples were resuspended in 300 ml PBS andanalyzed by flow cytometry. Negative controls con-sisted of normal rabbit or mouse serum or anti-HIV-1

Ž .p24 polyclonal or monoclonal antibody Intracellfollowed by identical second layer labeling as above.The cells were processed for flow cytometric analy-sis. This procedure represents the method of choiceunless stated otherwise.

In the second approach, samples were fixed inŽ . Ž .300 ml PBSr4% paraformaldehyde PF Sigma for

30 min at RT. Cells were washed twice in 300 ml

Ž . Ž . Ž . Ž .Fig. 1. A Dot–blot analysis of protein extracts obtained from pRPneoSL3rtat lane 2 , pSVneortat lane 3 , pSVneortat lane 4 ,Ž . Ž . Ž .pRPneoSL3 lane 5 , wild-type lane 6 Jurkat cells. The positive control is represented by recombinant Tat protein lane 1 whereas the

Ž . Ž .negative control is represented by BSA lane 7 . B CAT assay after transient transfection of pLTRCAT in the same cell lines employed forthe dot–blot assay. Reported values correspond to the ratio between the acetylation values achieved in the different transfected cell lines andthose obtained in wild-type Jurkat cells.

( )D. Gibellini et al.rJournal of Immunological Methods 221 1998 107–117110

PBS and permeabilized with 200 ml 0.5% TritonŽ .X100 Sigma or alternatively with 200 ml 0.5%

Ž .Tween 20 Sigma in PBS for 5 min in ice. After twoadditional washes in PBS, samples were stained andtreated as above.

Triplicate samples were next analyzed using aŽFACScan flow cytometer Becton-Dickinson, Palo

.Alto, CA , equipped with an Argon laser tuned at488 nm. Gates were carried out on FSC and SSCparameters, and analysis was performed using the

Ž .Lysis II software Becton Dickinson . In some exper-

iments, samples were spun on coverslips, then fixedand stained with anti-Tat monoclonal antibody asdescribed above. The cells were then analyzed by

Žmeans of an MCR-1000 confocal microscope Bio-.Rad Microscience Ltd, Hemel, Hempstead, UK

equipped with a kryptonrargon ion laser emitting at488 nm. The signal was measured after passage

Žthrough an Epidetector filter passing band 522r35.nm , analyzed by CoMOS software and printed on a

Ektachrome 64T-Kodak film by a Focus Im-Ž .agecorder Plus Focus Graphics, Foster City, CA .

Fig. 2. Flow cytometric detection of Tat protein after indirect immunofluorescence performed using a monoclonal anti-Tat monoclonalantibody in: pSVneortat, pRPneoSL3rtat, pSVneo, pRPneoSL3, and wild-type Jurkat cells. Shaded histograms represent cells stained withanti-Tat antibody, while unshaded histograms represent cells stained with anti-p24 antibody. X axis, mean fluorescence intensityŽ .logarithmic scale . Y axis, relative cell number. A representative of three separate experiments is shown.

( )D. Gibellini et al.rJournal of Immunological Methods 221 1998 107–117 111

2.5. Quantification of Tat-specific epitope bindingsites

In order to quantify the Tat-specific epitope bind-ing sites, cells were treated as above and stained witha monoclonal anti-Tat antibody directly conjugated

Ž .to FITC, diluted 1r100 in PBSr2%BSA for 2 h at378C. After two washes in PBS, samples were ana-lyzed by flow cytometry. The negative control con-sisted of FITC-conjugated anti-CD41 monoclonal an-tibody. Before assaying the samples, the FACScan

was calibrated by quantitative fluorescein mi-crobeads standards labelled with increasing numbers

Ž 4 4of FITC molecules 0, 1.8=10 , 8.7=10 , 1.6=

105 and 2.4=105; Flow Cytometry Standards, Re-.search Triangle Park, NC . A standard curve was

constructed by plotting the mean fluorescence inten-sity against the log of FITC molecules per bead. Themean fluorescence intensity values achieved by sam-ples were compared to the reference curve in orderto obtain the absolute number of FITC molecules ineach sample. Relative Tat protein binding sites per

Fig. 3. Flow cytometric detection of Tat protein after indirect immunofluorescence performed using a polyclonal anti-Tat serum in:pRPneoSL3rtat, pSVneortat, pRPneoSL3, and pSVneo Jurkat cells. Shaded histograms represent cells stained with anti-Tat antibody,

Ž .while unshaded histograms represent cells stained with anti-p24 antibody. X axis: mean fluorescence intensity logarithmic scale . Y axis:relative cell number. A representative of three separate experiments is shown.

( )D. Gibellini et al.rJournal of Immunological Methods 221 1998 107–117112

Ž .cell N were obtained by applying the following0

equation:

N yNŽ .1 2N s0 3

where N is the number of FITC molecules of each1

sample and N is the number of FITC molecules2

obtained in the background control represented bycells treated with a FITC-conjugated anti-CD41monoclonal antibody. The denominator is the inversemean number of FITC molecules conjugated to anti-

body. Once the epitope binding site number specificfor anti-Tat monoclonal antibody was derived, theTat cellular content was readily extrapolated. Allexperiments were performed under antibody satura-tion conditions.

2.6. Statistical analysis

Statistical analysis was carried out by using Stu-dent’s t-test for paired data.

Fig. 4. Confocal microscopy analysis of Tat protein after indirect immunofluorescence performed using an anti-Tat monoclonal antibody inpRPneoSL3rtat, pSVneo tat, pRPneoSL3 Jurkat cells. Tat-positive cells were identified by their intense yellow-green staining.

( )D. Gibellini et al.rJournal of Immunological Methods 221 1998 107–117 113

3. Results

3.1. Characterization of Tat protein expression intat-transfected CD4q Jurkat T cell clones

In the first set of experiments, the presence of Tatprotein was evaluated in homogenates obtained fromstably tat-transfected Jurkat cell clones by means ofa dot–blot assay developed with a polyclonal anti-Tat

Ž .serum Fig. 1A . Tat protein expression was ob-served in Jurkat cells stably transfected with pRP-neoSL3rtat or pSVneortat plasmids, but not inJurkat cells transfected with pRPneoSL3 or pSVneocontrol vectors, nor in untransfected controls.

The biological activity of Tat protein, producedby either pRPneoSL3rtat or pSVneortat Jurkatcells, was next determined in transient transfectionassays using the pLTRCAT reporter plasmid. TheCAT assay unequivocally demonstrated that Jurkatcells stably transfected with pRPneoSL3rtat orpSVneortat yielded a Tat protein with a full trans-

Žactivating activity on the HIV-1 LTR promoter Fig..1B . On the other hand, when pRPneoSL3, pSVneo

Jurkat cell clones were co-transfected with pLTR-CAT plasmid, no transactivating activity was ob-served.

In order to analyze whether Tat protein was ho-mogeneously expressed by the whole tat-transfectedJurkat cell population or only by a subset of cells,samples were subjected to indirect immunofluo-rescence staining and flow cytometric analysis. Afterstaining with either a monoclonal anti-Tat antibody

Ž .or an anti-Tat polyclonal serum Figs. 2 and 3 , aclear positive signal corresponding to intracellularTat protein was detected in virtually allpRPneoSL3rtat, and pSVneortat Jurkat cells. Onthe other hand, the mean fluorescence intensity ofpRPneoSL3 and pSVneo Jurkat cells stained withanti-Tat monoclonal or polyclonal serum did notshow any significant difference with respect to thebackground levels, represented by cells stained withanti-p24 monoclonal antibody.

Indirect immunofluorescence followed by confo-cal microscopy analysis confirmed the presence of aprevalent nuclear signal corresponding to Tat proteinin pRPneoSL3rtat and pSVneortat cells, while thesignal was completely absent in cells transfected

Ž .with the empty vector Fig. 4 .

3.2. Optimization of parameters

In order to optimize the protocol for the stainingof intracellular Tat protein, two distinct procedures

Ž .of fixation 70% EtOH or 4% PF and permeabiliza-Ž .tion 0.5% Tween 20 or 0.5% Triton X100 were

used.On flow cytometry analysis, the mean fluores-

cence values were lower in PF-fixed cells comparedŽ .to cells fixed with 70% EtOH Fig. 5 . In fact, it was

observed that the fluorescent cellular content was notimproved when PF fixation was followed either by aTween 20 permeabilization step or by Triton X100

Ž .treatment Fig. 5 . Analysis of various fixation timesŽ . Ž5, 15, 45, 60 min , fixative concentrations 50%,

.80%, 100% EtOH, and 1%, 2%, 3% PF , and TritonŽ .X100 or Tween 20 amounts 0.25%, 0.75%, 1%

showed that EtOH 70% for 30 min at 48C repre-sented the optimal fixation conditions, at least when

Ž .using our experimental conditions data not shown .

3.3. Quantification of anti-tat epitope binding sites

In order to compare the mean fluorescence valuesrelative to Tat protein observed in various tat-trans-

Fig. 5. Analysis of Tat protein expression in pRPneoSL3r tatJurkat cells by flow cytometry after different cellular fixation

Ž .conditions. In 1 , cells stained with anti-p24 control serum; inŽ . Ž .2 – 4 , cells stained with anti-Tat antibody, after fixation with

Ž .70% EtOH 2 , 4% PF followed by permeabilization with 0.5%Ž .Triton X100 3 , 4% PF followed by permeabilization with 0.5%

Ž . ŽTween 20 4 . X axis: mean fluorescence intensity logarithmic.scale . Y axis: relative cell number. A representative of three

separate experiments is shown.

( )D. Gibellini et al.rJournal of Immunological Methods 221 1998 107–117114

Fig. 6. Quantitative analysis of the number of Tat molecules in pRPneoSL3rtat Jurkat cells. Cells were stained with either aFITC-conjugated anti-CD41 control monoclonal antibody or a FITC-conjugated anti-Tat monoclonal antibody. Peaks 1, 2, 3, 4, 5correspond to quantitative fluorescent microbeads labelled with increasing numbers of FITC molecules. X axis: mean fluorescence intensityŽ .logarithmic scale . Y axis: relative cell number. A representative of four separate experiments is shown.

fected Jurkat cell clones, a reference curve wasobtained using calibrating beads conjugated with in-

Ž .creasing amounts of FITC molecules Fig. 6 . Takinginto account the fact that for an accurate quantifica-tion of intracellular Tat molecules the recognition ofa single epitope is required, we chose to employ ananti-Tat monoclonal antibody, directly conjugated toFITC. In fact, polyclonal anti-Tat serum could not beused due to the unknown number of recognizedtarget epitopes. By comparing the mean fluorescencevalues obtained using flow cytometric analysis of thereference beads and the mean fluorescence values ofsamples treated with FITC-conjugated anti-Tat mon-oclonal antibody, we could extrapolate the number ofTat specific-epitope binding sites. Assuming satura-tion conditions, a single FITC-conjugated mono-clonal antibody appears associated with a singleepitope, while two to four molecules of FITC areconjugated with each molecule of anti-Tat mono-

clonal antibody. Thus, the data relative to the epitopebinding sites could be easily transformed into therelative number of Tat molecules present in each

Ž .single cell Table 1 . It is particularly noteworthythat, among tat-transfected Jurkat cell lines, the

ŽpRPneoSL3rtat cells produced a significantly p-

Table 1Flow cytometric quantification of Tat protein in stably tat-trans-fected cell lines

Cell line Tat moleculesŽ .average numbers per cell

Jurkat 0pRPneoSL3r tat Jurkat 2300"350pSVneor tat Jurkat 1450"210pRPneoSL3 Jurkat 0pSVneo Jurkat 0

Data are expressed as means"SD of four separate experimentsperformed in duplicate.

( )D. Gibellini et al.rJournal of Immunological Methods 221 1998 107–117 115

.0.01 larger amount of Tat protein with respect toŽpSVneortat cells 2300"350 vs. 1450"210 Tat

.molecules, respectively . This was probably due tothe higher transcriptional efficiency of the SL3murine leukemia virus promoter with respect to theSV40 promoter region in lymphoid cells.

4. Discussion

In this report, we have described a flow cytomet-ric procedure which permits an accurate quantifica-tion of intracellular Tat protein in stable tat-trans-fected Jurkat cell clones. In order to check either theeffective transcription of specific genes or the ex-pression of full-length proteins, different techniquessuch as Northern blotting and immunoblotting arecurrently carried out. However, these assays provide,at best, semiquantitative information, whereas theprecise quantification of specific protein in trans-fected cells represents an important parameter that

Žneeds to be assessed Kriegler, 1990; Keller and.Manak, 1995; Glover, 1996 . In this respect, flow

cytometry offers a multiparametric quantitative anal-ysis of viral and cellular targets. This technique, hasbeen applied successfully to analyze simultaneouslythe presence of specific proteins expressed on thecell membrane and within intracellular compart-ments. Such an assay also represents a simple andeffective method of quantifying either the levels of agiven protein within the cells or the number of the

Ž .cells carrying a specific antigen Rigg et al., 1995 .Using calibration with fluorescence beads, it is

possible to correlate the mean fluorescence values ofdifferent samples with the epitope binding sites andthen to determine the relative protein levels in eachcell. An important step of this technique is clearly

Žrepresented by the fixation procedures Rigg et al.,.1995 . Several reports emphasize the importance of

this technical step in order to study intracellularŽproteins Jacobberger et al., 1986; Kastan et al.,

.1989 . In fact, it has been well documented that aprotein epitope can be lost after incorrect use offixatives. For the optimal determination of intra-cellular Tat protein, 70% EtOH was the best fixativewhereas the aldehyde fixatives were less effective.Moreover, we demonstrated that the intracellular

amounts of Tat protein are strictly dependent uponthe expression vector used in Jurkat cells. Indeed,our data demonstrate that the pRPneoSL3rtat Jurkatcells showed higher levels of intracellular Tat proteinthan pSVneortat Jurkat cells. This was due to dif-ferential activity of the plasmid promoters employedto regulate Tat expression in Jurkat cells. In fact, tattranscription is managed by a retroviral promoter inthe pRPneoSL3rtat plasmid and by the SV40 pro-moter in the pSVneortat plasmid. It is well knownthat the basal transcriptional efficiency of promotersdepends on the amount and the pattern of cellular

Žtranscription factors present in each cell type Krie-.gler, 1990 .

Of note, Jurkat T cell clones stably transfected bypRPneo SL3rtat are more resistant to serum with-drawal-induced apoptosis than pSVneortat Jurkat

Ž .cell clones Gibellini et al., 1995 . Hence, the deter-mination of epitope binding sites in stably trans-fected cells represents important information forcloned selection in order to evaluate dose-dependentbiological effects. This is particularly crucial fortat-expressing cell clones, since Tat protein showsstrikingly different biological effects in relation tothe concentrations employed. In fact, differentamounts of Tat can lead to dramatic changes in cellsurvival and proliferation, as well as in viral and

Žcellular gene activation Viscidi et al., 1989; Ensoliet al., 1990; Helland et al., 1991; Zauli et al., 1992;Ensoli et al., 1993; Subramanyam et al., 1993; Zauliet al., 1993; Albini et al., 1994; Buonaguro et al.,1994; Gibellini et al., 1994; Scala et al., 1994;Westendorp et al., 1994; Chirmule et al., 1995;Gibellini et al., 1995; Li et al., 1995; Vellutini et al.,1995; Westendorp et al., 1995; Zauli et al., 1995a;Zauli et al., 1995b; Corallini et al., 1996; McCloskey

.et al., 1997 . In conclusion, we have developed areliable and effective technique based on flow cy-tometry. This method represents a quantitative assaywhich can be used for specific determination of theaverage protein content in each single cell in order toselect specific protein-expressing clones.

Acknowledgements

This work was supported by ‘AIDS project’ ofthe Italian Ministry of the Health, Fund for Selected

( )D. Gibellini et al.rJournal of Immunological Methods 221 1998 107–117116

Research Topics of the University of Bologna, andCNR Strategic Project on Apoptosis.

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