Suppression of interleukin-2 and interleukin-2 receptor expression in jurkat cells stably expressing the human immunodeficiency virus tat protein

CELLULAR

IMMUNOLOGY

Suppression

144,32-42

(1992)

of Interleukin-2 and Interleukin-2 Receptor Expression in Jurkat Cells Stably Expressing the Human lmmunodeficiency Virus Tat Protein’

SCOTT F. PURVIS,* DANIEL L. GEORGE&-~ THOMAS AND MICHAEL M. LEDERMAN*+~

M. WILLIAM&$

*Department of Environmental Health Sciences,and TDepartment of Medicine, Division of Infectious Diseases, Case WesternReserve University School of Medicine, University Hospitals of Cleveland, 2109 Adelbert Road, Cleveland, Ohio 44106; and *Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87106 Received March 24, 1992; acceptedMay 18, 1992 The Jurkat T cell line was stably transfectedwith an Epstein-Barr virus-basedepisomal replicon designed to express high levels of the HIV- 1 Tat protein. After selection in hygromycin B, highlevel Tat activity was detected in 3 of 18transfected cell lines. After stimulation with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), Tat transfectants with high Tat expression showed diminished expression of interleukin-2 (IL-2) and the interleukin-2 receptor a!chain (IL2R) when compared to untransfected Jurkat cells or Jurkat cell lines transfected with the parent control plasmid. Sublines derived from the high-level Tat transfectants with reduced Tat activity showed normalization of PHA/PMA-induced IL-2 expression. Northern analysis showed diminished expression of IL-2 and IL-2R mRNA in the stimulated Tat transfectants. Inhibition of IL2 and IL-2R expression by the HIV-1 Tat protein may contribute to the immune suppression that characterizes HIV-I infection. o 1992 Academic press. IILC.

INTRODUCTION Although the human immunodeficiency virus (HIV-l) is cytopathic to infected CD4 cells, the mechanisms of immune deficiency associatedwith HIV- 1 infection are incompletely understood. Virus-induced cytolysis (l), lysis of uninfected cells by cytotoxic lymphocytes (2, 3) or lymphocytotoxins (4), and heightened susceptibility to complement-mediated lysis (5) have been postulated as mechanisms of cell loss in HIV-l infection. These phenomena notwithstanding, the dramatic failure of antigenspecific responsiveness among CDCenriched T cell populations of HIV- 1-infected persons (6) combined with the infrequency of viral infection of these cells (7) indicates either that antigen-responsive cells are selectively depleted in HIV- 1 infection or that other factors, viral or virus-induced, contribute to the immune deficiency of HIV-l infection. ’ These studies were supported by a grant from the Alma M. and Harry R. Templeton Medical Research Foundation, by Awards AI25879 and CA54428 from the National Institutes of Health, and by an American Cancer Society Junior Research Award to T.M.W. ’ To whom correspondence should be addressed. 32 0008~8749192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved

HIV-I

Tat INHIBITS

IL-2 AND

IL-2R EXPRESSION

33

The Tat protein of HIV-I is an 86- to IOl-amino-acid nuclear protein that is a powerful transactivator of retroviral replication. The cis acting element required for Tat-mediated transactivation, the Tat-responsive element (TAR), is found at the 5’ end of all nascent HIV mRNAs. Tat binds to a pyrimidine bulge in the TAR stem loop (8), where interactions with host proteins involved in transcriptional regulation are likely (9). Whereassome reports suggestthat Tat augments transcriptional initiation (lo), most recent data indicate that the primary effect of Tat is on elongation of transcripts (11). Despite its nuclear localization, immunoprecipitable Tat can be found in the supernatants of HIV-infected cells and Tat transfected cells ( 12). Exogenous Tat is readily taken up by uninfected cells where nuclear localization is confirmed by transactivation of the HIV LTR (13). There is accumulating evidence that exogenous Tat also may have effects on host cell function. Male mice made transgenic for the HIV- 1 tut gene develop dermal tumors resembling Kaposi’s sarcoma (14) although Tat expression cannot be detected within the tumor. In vitro, purified Tat protein can enhance the growth of tumor cells derived from Kaposi’s sarcoma tumors ( 12). Tat protein may also be immunosuppressive. Recombinant Tat protein can inhibit the lymphocyte blastogenic response to soluble antigen (15), and preliminary data suggestthat this may be effected through Tat-mediated transactivation of TGF-P transcription ( 16). To examine further the effects of Tat on immunocompetent cell function we established high-level expression of HIV-l Tat in the human CD6positive tumor line, Jurkat. We found that high-level Tat expression in these cells was associated with diminished expression of IL-2 and IL-2R after activation of the transfectants with phytohemagglutinin and phorbol myristate acetate. METHODS Plasmids, probes. A full-length reconstructed tat gene derived from the HIV- 1 SF1 isolate was excised from pSVTat (kindly provided by Dr. Paul Luciw, Davis, CA) and cloned into the episomal replicon Rep5P (17). The resulting construct, RepTatP (Fig. 1), encodes the HIV-l tat gene under control of the Rous sarcoma virus (RSV) LTR and is flanked by the SV40 polyadenylation signal. A hygromycin phosphotransferasegene within the Rep-based plasmids permits selection of stable transfectants in the presence of the toxic aminoglycoside hygromycin B, and an Epstein-Barr virus EBNA- 1 gene and origin of replication permit high-level extrachromosomal replication of the plasmid in transfected cells (17). Rep4 was also used in control transfections. Rep4 and Rep5/3differ only in the orientation of their expression cassettes.Tat activity in stable transfectants was assessedby cotransfection with the plasmid pLTR-CAT (provided by Dr. Paul Luciw) which contains the bacterial chloramphenicol acetyltransferasegene linked to the HIV- 1 LTR. Plasmid pBC 12/CMV/T2 was kindly provided by Dr. B. Cullen (Durham, NC). For Northern analysis, probes for actin (18) IL-2 (19) IL-2R (20) TGF-P (G. Bell, Chicago, IL), and c:jun (P. Leder, Cambridge, MA) were used. Cell lines, transfectants. Jurkat clone E6- 1 was provided by Dr. A. Weiss (San Francisco, CA), obtained from the AIDS Research and Reference Reagent Program (Bethesda, MD), and maintained in RPM1 1640 medium (MA Bioproducts, Walkersville, MD) supplemented with 10% fetal bovine serum (Hyclone Logan, Utah), 2 mA4 Lglutamine, 10 mA4 Hepes buffer, 100 U/ml penicillin, 100 pg/ml streptomycin at

34

PURVIS

ET AL.

RepTat p ( 10.6 Kb)

FIG. 1. RepTatP exprpssion vector. Construction of the Rep plasmid has been described (17). A fully reconstructed tat gene wasexcised from the plasmid SVTat and inserted into the episomal expression vector RepSo.

37°C in a humidified 5% COz-enriched incubator. The murine cytotoxic T cell line CTLL-N (kindly provided by Dr. J. Finke, Cleveland Clinic, Cleveland OH) was maintained as described (2 1). Stable transfection was established by electroporation of 10’ log phase Jurkat cells with 10 pg purified RepTat& Rep4, or Rep50 plasmid and selection in culture medium supplemented with 400 r.lg/ml hygromycin B (Sigma, St. Louis, MO). Transient transfections were performed by incubating 10’ log phase cells with 5- 15 pg plasmid DNA for 1 hr in STBS buffer (25 mM Tris, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HP04, 0.7 rnY4 CaC& , and 0.5 mM MgCl*) supplemented with 200 pug/mlDEAEdextran (Pharmacia, Piscataway, NJ). Cells were then washed, suspended in culture medium, and incubated as above. Assay of chloramphenicol acetyltransferase activity. Forty-eight to 72 hr after transfection with pLTR-CAT, cells were washed, counted, and assessedfor viability by trypan blue exclusion. Equivalent numbers of cells were resuspendedin 0.125 M Tris, pH 7.4, and lysed by repeated freezing and thawing in a dry ice/ethanol bath. Lysate or chloramphenicol acetyltransferase standard (Boehringer Mannheim, Indianapolis, IN) was incubated with 0.5 &i [3H]acetyl-coenzyme A (DuPont NEN, Boston, MA) and gently overlayered with organic scintillant (Econofluor, DuPont NEN, Boston, MA). CAT activity was measured by scintillation counting after l-2 hr incubation at room temperature. Counts per minute were linear in the rangesof CAT activity assayed. Production and assay of interleukin-2. Jurkat cell lines were washed, resuspended in fresh hygromycin-free media, and cultured overnight in medium alone-or with medium supplemented with phytohemagglutinin (PHA, Burroughs Wellcome, Research Triangle Park, NC) and with phorbol 12-myristate 13-acetate(PMA, Sigma). Supernatant IL-2 activity was assayedby incubating supernatant or IL-2 standard in

HIV-I Tat INHIBITS IL-2 AND IL-2R EXPRESSION

35

duplicate wells with 1O4freshly washed CTLL-N cells. The cells were cultured at 37“C in a 5% COz-enriched incubator. After 18 hr of culture, 0.1 PCi of [3H]thymidine (ICN, Irvine, CA) was added to each well. Cells were harvested 6 hr later and [3H]thymidine incorporation was measured using scintillation spectroscopy. Data are expressedin cpm and units of IL-2/ml as derived from a standard curve using human rIL-2 (Roche, Nutley, NJ). Surface antigen detection. The murine monoclonal antibody anti-Tat (Becton Dickinson, Mountain View, CA) was used to monitor surface expression of the 55kDa IL-2 receptor (Ychain. Expression of T cell activation-associated antigens was detected using the monoclonal antibodies 4D12 and 4F2 (22) (kindly provided by Dr. Barton Haynes, Durham, NC). After incubation with these monoclonal antibodies or an irrelevant monoclonal antibody as a control for nonspecific fluorescence, cells were washed and incubated with fluorescein-conjugated goat anti-mouse IgG (Tago, Burlingame, CA) and then fixed in PBS containing 1%paraformaldehyde. Ten thousand cells were counted using an Ortho Diagnostics Cytofluorograph II-3 flow cytometer. Fluorescence intensity of reactive cells was expressedon flow histograms. Detection qfmessenger RNA. Cells were lysed in 5 M guanidine isothiocyanate, 50 mM Tris, pH 7.5, 10 mM EDTA, 5% /3-mercaptoethanol and enriched preparations of Poly(A)+ RNA were obtained using oligo(dT) cellulose chromatography (FastTrack, Invitrogen, San Diego, CA). RNA was quantified by uv absorption at 260 nm. RNA preparations were electrophoresed in 1% agarose gels containing formaldehyde and transferred to nylon membranes.Purified DNA probeswere denatured and radiolabeled using Klenow enzyme and random hexanucleotide primers (Boehringer Mannheim). After overnight hybridization, filters were washed and hybridizing probes were detected by autoradiography. For some experiments, filters were stripped by incubation in 50% formamide 3X SSPE(540 mM NaCl, 30 mM NaHPO,, pH 7.7, and 3 mM EDTA) for 1 hr at 65°C autoradiographed to confirm stripping, and rehybridized as above using a different probe. RESULTS Establishment of stable cell lines. After electroporation of Jurkat cells and selection in medium containing hygromycin B, stable transfectant cell lines were established. The stable transfectants were examined for expression of the HIV-l Tat protein by transient transfection of pLTR-CAT and assayed for CAT activity in cell lysates. Jurkat cells and cell lines stably transfected with the parent Rep4 plasmid expressed no CAT activity. Three of eighteen lines stably transfected with the RepTatP construct (JRTat A,B,C) showed extremely high CAT activity indicative of high-level Tat expression (Table 1). Two lines stably transfected with Rep4 (JR4 3 and A) and four lines stably transfected with Rep5P (JR5 1,2 A, and C) consistently showed no CAT activity (data not shown). DNA was prepared from several of these lines, was examined by Southern blotting, and was found to contain between 1 and 30 plasmid copies per cell (data not shown). These cell lines are tested periodically for mycoplasma and for Tat activity. They are mycoplasma free and have maintained a high level of expression of Tat for over 10 months in continuous culture. Interleukin-2 production by stably transfected Jurkat cell lines. Cell lines stably transfected with RepTatP or Rep4 plasmids were incubated with concentrations of PHA and PMA optimal for expression and detection of IL-2. After overnight culture,

36

PURVIS ET AL. TABLE 1 Tat Activity in Jurkat Cells Stably Transfected with RepTatP and Rep5B Plasmids Cell line JRTat JRTat JRTat JR4 JRTat Jurkat

A B C A

Plasmid used for transient transfection

cpm per 1O6cells

Fold increase in Tat activity’

pLTR-CAT pLTR-CAT pLTR-CAT pLTR-CAT -

173,713 248,858 166,221 970 1,079 10,719

179 257 171 II

pBC12/CMV/T2’ pLTR-CAT

Note. Tat activity was assessedafter transient transfection of cell lines with 5 pg of pLTR-CAT using DEAE-dextran. Forty-eight hours after transfection, cells were washed, counted, and lysed. CAT activity in the cell lysateswas detected by measurement of incorporation of [‘Hlacetyl-CoA into chloramphenicol using scintillation counting. acpm in Tat transfectantstransfectedwith pLTR-CAT divided by the cpm in control cells (JR4) transfected with pLTR-CAT. bTat expressionplasmid containing the first Tat exon under the control of the cytomegalovirus IE promotor.

supernatants were harvested and IL-2 activity was assessedby incubating the supernatants with the murine cytotoxic T cell line CTLL-N and measuring [3H]thymidine incorporation. Supernatants prepared from unstimulated cell lines failed to produce IL-2 (Table 2). After activation with PHA and PMA, cell lines stably expressing high levels of HIV-l Tat protein showed diminished IL-2 production when compared to the Jurkat cell lines stably transfected with the parent Rep4 plasmid. Interleukin-2 receptorexpression by stably transfectedJurkat cell lines. Unstimulated Jurkat cells and Jurkat cell lines stably transfected with the Rep50 or RepTatP plasmids expressedno detectable surface IL-2R. To induce expression of IL-2R, these cells were stimulated for 18 hr with PHA and PMA. Jurkat cell lines stably expressing high levels

TABLE 2 Interleukin-2 Production by PMA/PHA-Stimulated Jurkat Cells Stably Transfected with RepTat and Rep5B IL-2 activity Cell line

PHA/PMA

cpm

Units/ml”

JRTat A JRTat A JR4 JR4 Media

+ + +

1,804 41,889 1,136 279,135 4,179

<0.2 2.5 <0.2 >20.0 <0.2

Note. Supernatants of resting cells or cells stimulated with 0.5 &ml PHA and 10 rig/ml PMA were collected after 18 hr culture and tested for their ability to support [‘Hlthymidine incorporation by the murine cytotoxic T cell line CTLL-N. Data are presented as mean cpm of duplicate samples. SD < 10%of mean. a Units/ml derived from standard curve using rIL-2.

37

HIV-l Tat INHIBITS IL-2 AND IL-2R EXPRESSION

of the HIV-l Tat protein failed to express IL-2 receptors after activation (Fig. 2). Addition of recombinant human IL-2 (1, 10, and 100 U/ml) to PHA/PMA-stimulated cultures of Tat transfected cells did not restore IL-2R expression (data not shown). Thus the failure of IL-2R expression was not attributable to diminished production of IL-2. Failure of IL-2 receptor expression by the Tat transfectants did not represent a generalized inhibition of surface antigen expression since expression of CD4 and CD3 in the Tat transfectants was unaffected (data not shown). PMA/PHA activation of the Tat-expressing cell lines resulted in heightened expression of other surface acRestirg

PHA I PMA

Jurkat

.. 200

JR5C

400

6w

ace

Fluorescent

Intensity

Fluorescent

Intensity

two

Fluorescent Intensity

.

v

200

400

Fluwescent

0

mo

lwo

800

low

Intensity

JRTat B

1

200

‘loo

Fluorescent

wo Intensity

800

loco

1

200

400

wo

Fluoreecent Irdeneity

FIG. 2. Expression of interleukin-2 receptors in Jurkat transfectants.Cells were washedand then cultivated at IO6cells/ml in medium without hygromycin B in the presence or absence of 1 pg/ml PHA and 0.1 ng/ ml PMA for 18 hr. IL-2 receptor expression was detected by indirect immunofluorescence using anti-Tat monoclonal antibody, FITC-conjugated goat anti-mouse Fab’*, and flow cytometry. The solid lines are the flow cytograms obtained using control murine monoclonal antibody, and the shaded areas represent the patterns obtained with anti-Tat.

38

PURVIS

ET AL.

Resting

PHA I PMA

Fluorescent Intensity

FIG. 3. Expression of the activation antigen 4D12 in Jurkat transfectants. Cells were washed and then stimulated with 1 rg/ml PHA and 0.5 rig/ml PMA for 18 hr. 4D12 expression was detected by indirect immunofluorescence using anti-4D12 monoclonal antibody, FITC-conjugated goat anti-mouse Fab2, and flow cytometry as described in the legend to Fig. 2.

tivation antigens including those recognized by the monoclonal antibody 4D12 (Fig. 3) and 4F2 (data not shown), indicating that the diminished expression of IL-2 and IL-2R in these cells did not represent a global failure of activation. Northern analysis for IL-2 and IL-2R mRNA expression. Poly(A) mRNA prepared from lysates of resting and PHA/PMA-activated cells was electrophoresed on formaldehyde agarose gels, transferred to nylon, and hybridized to purified radiolabeled probes. Expression of IL-2 mRNA (Fig. 4) and IL-2R mRNA (Fig. 5) was markedly decreasedin the Tat transfectants. To examine the specificity of the impaired IL-2 and IL-2R mRNA expression in the Tat transfectants, and to ascertain if Tat-mediated inhibition of IL-2 expression might be mediated through transactivation of TGF-0, filters were probed for c-jun and TGF-P mRNA. Activation of both cell lines resulted in increased expression of both c-jztn and TGF-P mRNA (Fig. 6). Restoration of IL-2 expression with loss of Tat activity. From each high-level Tatexpressing cell line (JRTat A,B,C), sublines with diminished Tat activity were derived (JRTat A-2,B-2,C-2). Simultaneous assay of PHA/PMA-induced IL-2 expression revealed inhibition of IL-2 expression in the high Tat expressors and a normalization

JR43 75

STIM

JRTAT A 5

STIM

IL-2

ACTIN FIG. 4. Expression of IL-2 mRNA in Jurkat transfectants. Poly(A)+ RNA was prepared from control Rep4 (JR4 3) and RepTat (JRTat A) transfected Jurkat cells 4 hr after stimulation with 1 fig/ml PHA and 10 rig/ml PMA. RNA was electrophoresed on a 1%agaroseformaldehyde gel, transferred to nylon, hybridized with IL-2 and actin probes, and autoradiographed.

39

HIV-l Tat INHIBITS IL-2 AND IL-2R EXPRESSION JR5 A

JRTAT

t

0

6

24

6

24

0

PMA

0

.l

.l

.5

.5

0

6

A

24

6

24

.l

.5

.5

.l

IL-2 R

ACTIN

FIG. 5. Expression of IL-2R mRNA in Jurkat Transfectants. Poly(A)+ RNA was prepared from control RepSP (JR5 A) and RepTat@(JRTat A) transfected Jurkat cells 0, 6, or 24 hr after stimulation with 1 fig/ ml PHA and 0.1 or 0.5 rig/ml PMA as indicated. RNA was electrophoresed on a 1%agaroseformaldehyde gel, transferred to nylon, hybridized with IL-2R and actin probes, and autoradiographed.

of IL-2 expression in the derived sublines expressing lower Tat activity (Table 3). These experiments indicate that in the three high-level Tat-expressing cell lines with impaired IL-2 production, inducibility of IL-2 expression can be restored as Tat activity is lost. DISCUSSION A EBV-based episomal vector was used to achieve high-level stable expression of the HIV- I Tat protein in Jurkat tumor lines. High-level Tat expression in Jurkat cells was associatedwith inhibition of IL-2 and IL-2R expression after stimulation of these cells with PHA and PMA. PMA/PHA stimulation of control Rep transfectants and Tat transfectants increases surface expression of the activation-associated antigens recognized by the monoclonal antibodies. 4D 12 and 4F2 and increased expression of mRNA for the protooncogene c:jzln. Thus, these data suggestthat inhibition of expression of IL-2 and IL-2R in the Tat transfected cells is not associated with a generalized impairment in activation of JRTat

0

B

STIM

JR4 A

0

STIM

FIG. 6. Expression of TGF-6 and c-jun mRNA in Jurkat transfectants. Poly(A)+ RNA was prepared from control Rep4 (JR4 A) and RepTat (JRTat B) transfected Jurkat cells 4 hr after stimulation with 0.5 pg/ml PHA and 10rig/ml PMA as indicated. RNA waselectrophoresedon a 1%agaroseformaldehyde gel. transferred to nylon, hybridized with IL-2 and actin probes, and autoradiographed.

40

PURVIS ET AL. TABLE 3 Normalization of IL-2 Expression with Loss of Tat Activity IL-2 activity’ Cell line

Tat activityb

cpm

Units/ml

JRTat A JRTat B JRTat C JRTat A-2 JRTat B-2 JRTat C-2 JR5 I JR5 2 JR5 C Jurkat Media

478 260 198 60 45 31 -

8,896 7,575 31,072 126,827 192,246 186,830 45,587 183,996 157,563 176,723 12,633

<0.2 <0.2 0.8 14.1 18.0 17.8 4.5 17.6 16.3 17.4 -

Note. High-level Tat-expressing cell lines (JRTat A, B, C), low-level Tat expressors derived from these lines (JRTat A-2, B-2, C-2), control cell lines transfected with the parent RepSP vector (JR5 1, 2, 3), and Jurkat cells were examined for Tat activity after transient transfection of pLTR-CAT and assay for chloramphenicol acetyltransferaseactivity. ’ Jurkat and Jurkat transfectants were stimulated with 1 pg/ml PHA and 0.5 rig/ml PMA, and cell supernatants were assayedfor IL-2 activity by measuring [3H]thymidine incorporation by CTLL-N cells. Data are expressedas cpm and units/ml calculated as in Table 2. b As described in Table 1.

these cells but rather that the defect is selective for signals involved in regulation of IL-2 and IL-2R expression. Moreover, the failure to correct the impaired IL-2R expression of the Tat transfectants with exogenous IL-2 is also compatible with the hypothesis that Tat may interfere with signals common to activation and expression of IL-2 and IL-2R. Viscidi et al. have found that recombinant Tat protein inhibited the lymphocyte blastogenic response to soluble antigen but not to mitogens (15). Lotz et al., in a preliminary report, also found that Tat inhibited antigen-induced T lymphocyte blastogenesis and that antibody to TGF-/I reversed this inhibition (16). Using a reporter gene linked to the TGF-P promotor, this group demonstrated that Tat expression was associatedwith increased transcription from the TGF-P promotor. Using a tumor cell line, we have found that high-level Tat expression is associatedwith a failure of IL-2 and IL-2R expression after activation of these cells through the T cell antigen receptor. We were however, unable to demonstrate an effect of Tat on TGF-0 mRNA expression (Fig. 6) or on TGF-P production as detected by immunoblot (data not shown). Thus in Jurkat cells, Tat-mediated inhibition of IL-2 and IL-2R expression is not related to TGF-0 activity. Bielinska et al. were unable to show alterations in IL-2 expression in Jurkat cells stably expressing the HIV-l Tat protein (23). This apparent discrepancy can be explained by the differences in the levels of Tat expression in the two systems.Bielinska’s transfectants expressed a modest, 42-fold increase in CAT activity over background after transient transfection with an HIV LTR-CAT plasmid. Our data indicate that higher Tat activity is required to inhibit IL-2 expression after PMA/PHA stimulation.

HIV-l Tat INHIBITS IL-2 AND IL-2R EXPRESSION

41

We have examined eight other Jurkat cell lines stably expressing HIV Tat in which Tat activity is regularly lessthan 50-fold greater than background and failed to observe significant inhibition of IL-2 or IL-2R expression in these lines. Moreover, sublines derived from high-level Tat expressorswith diminished Tat expression recovered their ability to express IL-2 as Tat activity was lost. The biological significance of these high levels of intracellular levels of Tat is unclear. There are no data reporting the concentrations of intracellular Tat achieved during HIV infection. Likewise, there are no reports of measurements of “serum Tat” among HIV-infected persons, although the rapid uptake of Tat by cells expressing a large number of Tat binding sites (24) would suggestthat the “extracellular half-life” of Tat would be short. In this regard, extracellular recombinant Tat protein can achieve intracellular levels (as measured by biological activity) exceeding those achieved in our stable transfectants (24). Conceivably, Tat may concentrate at sites such as lymph nodes where HIV replication is thought to be brisk (25) and may accumulate in surrounding uninfected lymphocytes. Uptake of exogenous Tat by uninfected cells could account for the apparent immunologic dysfunction (6) of cell populations with frequencies of HIV infection less than 1 in 100 (7). The mechanism of Tat-mediated inhibition of IL-2 expression is not clear. Whereas IL-2R expression is regulated transcriptionally, IL-2 expression is likely regulated at both transcriptional and post-transcriptional levels (26,27). The specific and coordinate impairment of IL-2R and IL-2 expression and the failure to enhance IL-2R expression by exogenous IL-2 suggestthat Tat interferes with signals required for expression of both of these proteins. Studies are underway to examine the transcriptional regulation of IL-2 and IL-2R expression by Tat. Inhibition of IL-2 and IL-2R expression by HIV Tat may contribute to the immune deficiency seenin HIV infection. Therapy of HIV infection with Tat inhibitors therefore may not only halt replication of HIV but also may be associated with restoration of immunologic responsiveness.Conversely, Tat agonists might provide a novel approach to immunosuppressive therapy. ACKNOWLEDGMENTS The authors thank Dr. Zahra Toosi for performing the TGF-0 assays.Dr. Mark Tykocinski. Dr. HsingJien Kung. and Dr. Jerry Elmer for their advice. and Ms. Marla Manning for her assistancein preparing the manuscript.

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Lane, H. C., Depper, J. M., Greene, W. C.. Whalen G., Waldmann, T. A., and Fauci, A. S., ,%‘.f+tg/.

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42

PURVIS ET AL.

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