6) 127–137www.elsevier.com/locate/yviro

Virology 355 (200

A novel in vitro system to generate and study latently HIV-infectedlong-lived normal CD4+ T-lymphocytes

Gautam K. Sahu, Kyeongeun Lee1, Jiaxiang Ji, Vivian Braciale, Samuel Baron, Miles W. Cloyd ⁎

Department of Microbiology and Immunology, The University of Texas Medical Branch, 301 University Blvd, BSB #3.132, Galveston, TX 77555, USA

Received 8 February 2006; returned to author for revision 13 March 2006; accepted 11 July 2006Available online 17 August 2006

Abstract

Studies of mechanisms of HIV-latency and its reactivation in long-lived resting CD4+ T-lymphocytes in patients have been limited due tothe very low frequency of these cells (∼1–10 cells per 106 CD4+ T-cells). To circumvent this obstacle, an in vitro culture system for post-activation long-term survival of normal CD4+ T-cells in a quiescent (non-cycling) state was developed and used to generate latently infected,long-lived quiescent CD4+ T-cells from HIV-infected, activated normal CD4+ T-lymphocytes. This yielded a frequency of ∼5×104 latentlyinfected cells per 106 cells in culture, which is ∼103- to 104-fold higher than that available from patients. Moreover, 5–10% of long-termsurviving non-cycling T-cells were found to make infectious HIV continuously at low levels, showing that HIV production from infected T-cellsdoes not require full cellular activation. This model system should facilitate studies of long-lived, latently infected and persistently HIV-producing quiescent normal CD4+ T-lymphocytes.© 2006 Elsevier Inc. All rights reserved.

Keywords: CD4+ T-lymphocytes; Feeder cells; Long-term survival; Quiescent cells; Latent HIV infection

Introduction

The establishment of latent HIV infection in resting memoryCD4 T-lymphocytes has been one of the major obstacles ineliminating HIV from patients by using highly active anti-retroviral therapy (HAART) (Siliciano et al., 2003).Although thefrequencyof latently infectedCD4T-lymphocytes is low (∼1–10cells per 106 CD4 cells) (Finzi et al., 1997; Siliciano et al., 2003;Wong et al., 1997), the very slow turnover of these cells (t1/2∼6–44 months) makes their elimination likely impossible by usingonlyHAART (Finzi et al., 1999; Ramratnam et al., 2000; Zhanget al., 1999a). Both wild-type and drug-resistant variants residein these cellular reservoirs (Persaud et al., 2000; Ruff et al.,2002). Therefore, the removal of latent reservoirs is required toobtain complete suppression of HIV replication during HAART.

⁎ Corresponding author. Fax: +1 409 747 6869.E-mail address: mcloyd@utmb.edu (M.W. Cloyd).

1 Current address: HIV Drug Resistance Program, National Cancer Institute,NIH, Frederick, MD 21702, USA.

0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2006.07.020

How HIV latency is established in resting CD4 T-lympho-cytes in vivo is still not fully clear. One postulation is thatHIV-producing activated CD4+ T-cells that survive fromactivation-induced cell death (AICD), viral cytopathicity orimmune-mediated elimination, exit the cell cycle after severaldivisions and become resting memory cells carrying silent(latent) integrated proviral DNA in their chromosomes(Pierson et al., 2000). Alternatively, resting CD4+ T-cells thatare refractory to productive HIV infection in vitro may allowHIV to complete reverse transcription and integration into theirchromosomes under more permissive conditions available inlymphoid tissue environments (Kinter et al., 2003; Oswald-Richter et al., 2004; Pope et al., 1994; Swingler et al., 2003;Unutmaz et al., 1999; Zhang et al., 1999b). Our laboratorypreviously showed that HIV replication is inherently bimodal:acutely increasing to a peak of replication within a few daysfollowing a 2-day eclipse period in activated CD4+ lympho-cytes, and then gradually reducing the rate of replication untilmany cells become latently infected (Li et al., 1996). This occursin both transformed lymphocytes that constitutively divide aswell as mitogen-stimulated normal CD4 lymphocytes grown inculture for a few weeks. However, because normal lymphocytes

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do not divide more than 7–9 times and then eventually die inculture, it was impossible to study HIV replication events long-term in these cells.

Although understanding the molecular mechanisms of howHIV latency is established is important, studies on how latentHIV can be reactivated without significantly affecting themetabolic state of host cells is required to gain knowledge fordesigning new therapeutic approaches to eliminate the latentHIV-reservoir. However, carrying out such studies has beendifficult due to the very low frequency of latently infected cellsin patients (Chun et al., 1997). Furthermore, there is not anavailable culture system that can generate latently infectedprimary CD4 T-cells in vitro. Although, HIV latency has beenstudied in transformed, actively proliferating cell line models,such as the ACH-2 or U1 clonal lines (Butera et al., 1994; Folkset al., 1988; Pomerantz et al., 1990), or in the CEM (Li et al.,1996) and Jurkat cell lines (Jordan et al., 2003), the resultsobtained from these studies may not represent what occursduring latent infection in primary CD4+ T-lymphocytes becauselatently infected CD4 T-cells in vivo are in a resting state (Chunet al., 1995). Moreover, the HIVs present in the clonal cell linemodels of latent infection have either a mutation in the TARregion or in the transcriptional transactivator, Tat (Emiliani et al.,1996, 1998), making these models of questionable significance.

A useful in vitro model for latent HIV infection in normalCD4 T-cells is still lacking primarily due to the unavailability ofa culture system that supports long-term survival of stimulatedCD4 T-cells. After stimulation with mitogen or anti-CD3, thesecells proliferate in the presence of IL-2, but they gradually dieafter 2–3 weeks of cultivation. This happens because most ofthese activated T-cells are destined to die and/or becauseoptimal culture conditions for their long-term survival are notavailable (Green et al., 2003; Sprent and Surh, 2003).

In attempts to maintain anti-CD3-stimulated normal CD4+T-lymphocytes from uninfected human donors in vitro, wefound that these activated cells could be rescued from deaththat typically ensues upon co-culturing on a brain tumor-derived attached cell line (H80). The rescued CD4+ T-cellssubsequently became quiescent and survived for many monthsin co-culture. We then tested our hypothesis that if cultures ofHIV-infected, activated primary CD4+ T-cells could bemaintained viably for a long-time, HIV present in many ofthese long-lived infected CD4 T-cells would eventually go intolatency. Here, we show that HIV-infected activated primaryCD4 T-cells when allowed to live for a long time, acquirednon-dividing, quiescent phenotypes and a portion of themcarried latent, but inducible HIV, and a similar portionchronically produced HIV at low levels.

Results

A co-culture system that allows long-term survival ofstimulated primary CD4+ T-lymphocytes

Purified normal CD4+ T-lymphocytes (∼95% pure and αβ-TCR+, Figs. 1a and b, respectively) from normal human donor'sblood were stimulated (see Materials and methods) for two days

with immobilized anti-CD3 and cultured in the presence of IL-2(40 U/ml). Cellular activation was confirmed by detecting theinduction of activation markers (such as CD69, CD25 and HLA-DR) on the cell surface (Figs. 1c and d). Cell proliferation wasevident from a rapid increase in cell numbers (Fig. 1f), as well asincreased DNA synthesis (Fig. 1g). By 2–3 weeks of culture,these cells had divided about 5–6 times and then cell divisionslowed down as expected. Although a low percentage (rangingfrom 10% to 20%) of dead cells could always be found inculture (assayed by Trypan blue dye exclusion) during thisperiod, dead cells gradually appeared at higher percentages after3 weeks of culture (Fig. 1h, see mock). This is due to the factthat cellular activation and proliferation are always followed bygradual cell death in long-term cultures. In attempts to keepthese activated CD4+ T-cells viable for a long time, we foundthat a brain tumor-derived adherent cell line, H80, rescued themfrom dying when used as a feeder line (Fig. 1h). About two-foldhigher live cells counts were achieved after 3 weeks of co-culture as compared to mock culture that was maintainedwithout H80 cells. The percentage of dead cells remained low aslong as the co-culture was continued (Fig. 1h). These datademonstrated that the feeder line, H80, could rescue primaryactivated CD4 T-cells from the death that normally occurs inlong-term cultures. We thus routinely maintained these cells formore than 5 months on H80 cells (Fig. 2a). The mechanism(s)behind the long-term survival of CD4 T-cells on this feeder cellline is currently under investigation.

Many of the long-term surviving T-cells were phenotypicallyquiescent

Activated CD4 T-cells were cultured for 3 weeks before theywere co-cultured on H80 cells for another 3 weeks, and thenvarious cell surface markers were analyzed by flow cytometry.About 98% of them were positive for CD3 (not shown),whereas about 93% cells were expressing CD4 (Fig. 2b). Theremaining 7% CD4-negative population was likely due to thepresence of low percentage (5%) of non-CD4 T-cells in theoriginal cell population (Fig. 1a) used for stimulation.Interestingly, the majority of this minor population of non-CD4 T-cells (also negative for CD8 marker) were γδ-TCRpositive (Fig. 1b), which also survived in long-term cultures onH80. This is not surprising because some tumor lines areknown to support growth of γδ T-cells (Fisch et al., 1992).Activation markers such as CD25 and HLADR were notexpressed by most of the T-cells rescued in co-culture (Fig. 2c);however, an early activation marker CD69, which is usuallyonly transiently expressed during activation, was expressedconstitutively by more than 50% of the total cells present inculture (Fig. 2). Propidium iodide staining followed by flowcytometry showed that 99% of the cells remained in the G0–G1 phase of the cell cycle (Fig. 2e). This is consistent with theminimal level of cellular DNA synthesis observed by 3H-thymidine incorporation (Fig. 2d). Therefore, it was apparentthat the T-cells were not cycling during their maintenance inco-culture with H80 cells. Although CD69−CD25−HLA-DR−

T-cells represent quiescent phenotype of cells, we termed the

Fig. 1. Flow cytometric analysis of cell surface markers: (a) the purity of primary CD4+ T-cells isolated from normal donor's blood using stem cell's CD4+ T-cellspurification kit and (b) the majority of purified cells were αβ-TCR positive. (c and d) The expression of activation markers (CD69, CD25, HLA-DR) 5 days afterstimulation with cross-linked anti-CD3 plus IL-2 (see Material and methods). (e) The percentage of αβ- and γδ-TCR expressing cells after activation. (f) Growth curvefor activated and proliferating CD4 T-cells in the presence of IL-2. Arrows indicate when cultures were split. Live cell counts were determined by Trypan blue dyeexclusion on a hemocytometer; and (g) the kinetics of DNA synthesis in freshly activated and proliferating CD4 T-cells as measured by 3H-thymidine incorporation.The radioactive counts per minute (c.p.m) for unstimulated cells were always <1000 (not shown). (h) The accumulation of dead CD4 T-cells in long-term cultures.Activated CD4 T-cells were grown for 21 days in the presence of IL-2 (40 U/ml) and then cultured with or without H80 cells. Live and dead cells were counted on daysindicated, using Trypan blue dye exclusion. Percentages of dead cells were calculated on the respective days and plotted. The data presented here are representative offive independent experiments.

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physiological state of CD69+CD25−HLA-DR− T-cells as a“semi-quiescent” state. The constitutive expression of CD69ranging from detectable to undetectable levels may be anindication of the physiological states of low-level activation ofnon-dividing T-cells as well in vivo (Dorfman et al., 2000;Stefanova et al., 2002).

HIV-infected activated primary CD4+ T-lymphocytes becomequiescent when co-cultured on the feeder cell line, H80

Freshly purified and activated primary CD4 T-cells wereinfected with a T-cell line (VB)-adapted CCR5-dependent (non-cytopathic) HIV strain, JRCSF, at moi∼1–10. Virus replicationand spreading occurred with the characteristic kinetics shown inFigs. 3a and b, as expected. After 3–4 weeks,∼10% of total livecells in culture remained HIV-producing. Because it is knownthat the uninfected cells (mock) normally start to die at this timepoint, the cells from infected cultures were overlaid on ∼50%confluent H80 cells to rescue them from death. After∼2 monthsof co-culture, T-cells from infected cultures were phenotyped byanalyzing cellular activation markers, cell cycle status andcellular DNA synthesis. It was found that∼98% of them did notexpress activation markers CD25 and HLA-DR (Fig. 3d). However,

the majority of them constitutively expressed CD69, an earlyactivation marker (∼76% in Fig. 3c). Most of these cells did notexpress CD4 on their surface (∼73% in Fig. 3c), but about half ofthem was positive for αβ-TCR and the remaining was γδ-TCR-positive (Fig. 3e). About 98% of T-cells in HIV-infected culturesremained in G0/G1 phase of the cell cycle, similar to uninfectedcultures (Fig. 2e), and the majority of them looked much smaller(Fig. 3f, left) than activated cells (Fig. 3f, right). Furthermore, the rateof cellular DNA synthesis was very minimal as detected by 3H-thymidine incorporation assays (Fig. 3g). These data suggested thatHIV-infected, activated CD4 T-lymphocytes when rescued from celldeath and maintained long-term on the feeder line becamephenotypically quiescent or “semi-quiescent” represented byCD69−CD25−HLA-DR− or CD69+CD25−HLA-DR− status,respectively.

Persistent low-level HIV production occurred in some of thelong-lived, quiescent memory T-lymphocytes

The levels of HIV production from CD4 T-lymphocytesdepend on cellular activation status because transcription of theHIV-LTR requires several cellular transcription factors, liketranscriptionally active NF-kB, which are only present at higher

Fig. 3. Kinetics of HIV replication in activated primary CD4 T-cells and theirphenotypic analyses after long-term culture on H80 cells. (a) Cells were infectedwith HIVat different multiplicity of infection (m.o.i.) after 5 days of stimulation(see Materials and methods) and cultured in the presence of IL-2. Thepercentages of HIV-expressing cells were detected by fixed-cell IFA at daysindicated. (b) Detection of HIV-p24 levels in culture fluid by Coulter's p24antigen-capture ELISA. Cells harvested at different time points were cultured for48 h at density of 1.5×106 per ml, and then supernatants were tested for p24-levels by ELISA. Flow cytometric analyses of various surface markers areshown in panels c, d and e. The percentages of various activation marker-positive HIV-infected CD4 T-cells that were cultured on H80 cells for∼2 months are shown in panels c and d. CD69, an early activation markerwas expressed by ∼76% of T-cells (c), whereas almost none expressed CD25 orHLA-DR (d). (e) Indicates the expression of αβ- and γδ-TCR by long-termcultured CD4 T-cells. Forward scatterings of long-term cultured infected cells(d, left) and freshly stimulated CD4 T-cells (d, right) are shown. (g) Representscellular proliferation measured by 3H-thymidine incorporations in various cells.Activated CD4 T-cells were prepared by stimulating freshly isolated resting CD4T-cells with immobilized anti-CD3 antibodies (see Materials and methods) andculturing in the presence of IL-2 for several days as indicated.

Fig. 2. Long-term survival of primary CD4 T-cells and their phenotypes. (a)Representative long-term survival curve for activated CD4+ T-cells with orwithout H80 feeder cells. Activated CD4+ T-cells were grown in the presence ofIL-2 (see Material and methods). Four weeks later, equal numbers of cells werecultured with or without H80 cells. Live cell counts were measured by Trypanblue dye exclusion. Although primary CD4+ T-cells die increasingly in vitroafter 3–4 weeks of culture, H80 cells rescued them from dying and maintainedthem viably for more than five months. A representative figure from more thaneight independent repeat experiments giving similar data is shown here. Cellularphenotypic analyses of activated CD4+ T-cells co-cultured on H80 cells for amonth are shown in panels b–e. (b and c) Flow cytometric analyses of the cellsurface activation markers (CD69, CD25 and HLA-DR); 3H-thymidineincorporations are shown in (d). CD4 T-cells were labeled in triplicate with3H-thymidine for 20 h and counts were measured. Freshly purified CD4 cellsand stimulated CD4 cells that were co-cultured long-term on H80 cells did notshow significant incorporation of 3H-thymidine. Cell cycle status was analyzedby propidium iodide staining/flow cytometry and is shown in panel e.

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levels in activated cells (Malek et al., 2001; Zhong et al., 2002).To check if HIV is produced from quiescent T-lymphocytespresent in long-term cultures, these cells were stained intracellu-larly with antibodies to HIV core antigen and simultaneouslysurface-stained with antibodies to various cell surface markers(Fig. 4). Culture supernatants were also tested for the presence ofHIV-p24, which is a structural component of extracellularlyreleased virus particles, by HIV-p24 enzyme-linked immuno-sorbent assay (ELISA). The data presented in Fig. 4 showed that∼8%of total T-cells present in the feeder cell co-culturewere stillmaking HIV-p24 intracellularly (see Figs. 4a–d). HIV-p24 wasalso detected in the culture supernatants by ELISA (see Fig. 5c),indicating that virus particles were released into themedium. The

majority of virus producing cells wereαβ-TCR+CD4− (compareFigs. 4a and c) and CD45RA− (Fig. 4e). However, only about55% of total virus producing cells were positive for CD45ROmarker (Fig. 4f). Approximately 58% of total virus producingcells were expressing the early activation marker, CD69 (Fig.4b). We further purified CD69−CD25−HLADR− cells by FACSor by using a stem cell column and cultured them for several days.HIV-p24 was found to be accumulating in culture supernatantsharvested at different time points (Fig. 4h), demonstrating that

Fig. 4. Detection of chronically HIV-infected cells by intracellular staining and flow cytometry. A HIV-core antibody was used in intracellular staining to detect coreantigen-expressing cells and simultaneously, the expression of various cell surface markers [(a) CD4; (b) CD69; (c) αβ-TCR; (d) γδ-TCR; (e) CD45RA; (f) CD45RO;and (g) CD62L] on HIV-expressing cells was assessed by surface staining. (h) Persistent HIV production from quiescent T-cells generated after long-term co-culture ofHIV-infected, activated CD4 T-cells on H80 cells. T-cells were stained with CD69-, CD25- and HLA-DR-antibodies conjugated to FITC and quiescent cells (i.e.,CD69−CD25−HLA-DR− cells) were isolated by FACS. The sorted cells were cultured for several days. The accumulation of HIV-p24 in culture supernatants wasmeasured by ELISA at several time points as indicated and presented as fold-increase over the p24-levels accumulated on day 1. (i) Shows that HIV present in freshlyharvested supernatants of quiescent T-cell cultures could spread in a CD4 and CCR5 expressing T-cell line, VB (solid square). The percentages of HIV-antigen (Ag)expressing cells were determined by fixed-cell IFA. Solid triangle indicates the background staining on uninfected VB cells.

Fig. 5. Reactivation of latent HIV by a protein kinase C activator, prostratin, in infected primary CD4 T-cells. (a) Cells that were co-cultured on H80 for ∼2 monthswere treated with 500 nM prostratin for 24 h (right) and stained with core antibody conjugated to PE (intracellularly) and CD4 antibody-conjugated to FITC (surface).Prostratin increased the percentage of HIV-p24+ cells in culture from 6.3% (in mock) to 13.1%. (b) The reproducibility of generation of latently infected cells in long-term cultures from three different experiments. Student's t test was used to calculate p value. (c) Cells were treated with different concentrations of prostratin for4 days. Phytohemagglutinin (PHA) or anti-CD3/CD28 antibodies were also used at 2 μg/ml and 3 μg/ml concentration, respectively, to stimulate these cells.Supernatants were harvested on day 1 and day 4 for p24 measurements by antigen-capture ELISA. The levels of p24-production are shown as bars. In parallel, 2 daysafter treatment, cells were labeled with 3H-thymidine for 20 h. The radioactive counts incorporated in DNA as an indicator of cellular DNA synthesis and cellproliferation [shown as bars in panel d] were measured.

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phenotypically quiescent T-cells were also making HIV, inaddition to the HIV production from “semi-quiescent” (CD69+

CD25−HLADR−) T-cells.To test further that the released virus particles were actually

infectious and could spread in target cells, supernatants wereadded to a T-cell line (VB) expressing CD4 and CCR5receptors. The appearance of infected cells was determinedfrom time to time by fixed-cell indirect immunofluorescenceassay as previously reported (Li et al., 1996). As daysprogressed, the percentage of infected cells increased in culture(Fig. 4i), demonstrating that the culture supernatant possessedinfectious virus that were released by quiescent T-cells in long-term cultures. These data suggested that neither fully activatedCD4 T-cells, nor being in cell cycle, are required for HIVproduction from normal CD4 T-cells; rather quiescent T-cellssupport HIV production, albeit, at low levels. However, fullyactivated status of infected CD4 T-cells is likely required for theoptimal HIV-replication.

Latent HIV infection is evident in long-lived quiescent primaryT-lymphocytes maintained in long-term cultures

Although it is not clear how latent infections in restingmemory CD4 lymphocytes are established in vivo, we sought totest whether HIV-infected activated CD4 T-cells that survive andbecome quiescent harbor HIV in a latent state. Therefore, tocheck the possibility that quiescent T-cells that did not stainintracellularly with the antibodies to HIV core antigens,possessed latent HIV, a portion of total T-cells in culture wastreated with a phorbol ester, prostratin (protein kinase Cactivator), which would not induce cells to full activation andcell division, but it would induce latent HIV if present in thesecells (Gulakowski et al., 1997; Korin et al., 2002). As shown inFig. 5a, the percentage of p24-positive cells increased by ∼2-fold (from 6.3% to 13.1%), when prostratin was used at aconcentration of 500 nM or higher, with a concomitant increaseof HIV-p24 levels in the culture supernatants (Fig. 5c), and theyield was highly reproducible (p<0.05 in Fig. 5b). Thisdemonstrated the presence of latently infected primary T-cellsin these cultures at >5% of total T-cells. Note that, as reported byothers, no significant incorporation of 3H-thymidine were seenin these cells at the concentrations of prostratin used (Fig. 5d).The expression of the CD4 molecule was, however, down-modulated with prostratin (Gulakowski et al., 1997). These datademonstrated that latently infected primary T-cells weregenerated in our culture system and the latent HIV could bereactivated with a protein kinase C-activator, as has been shownfor latently infected CD4 T-cells isolated from patients(Kulkosky et al., 2004).

We checked the kinetics of reactivation of latent HIV in thesenon-cycling T-cells by stimulating with prostratin (500 nM). Aprotease inhibitor, indinavir (200 nM), or a reverse transcriptaseinhibitor, AZT (0.13 μg/ml), was used to inhibit new infection,if any, with HIV during prostratin-induced cell activation. Weobserved the increase in the percentages of HIV-antigenproducing cells within 12 h of treatment with prostratin (datanot shown), further substantiating the reactivation of latent HIV.

This is in agreement with the recent report showing the rapidactivation of latent HIV in primary CD4 T-lymphocytes and alsoin the clonal cell line, ACH-2 (Arlen et al., 2006).

Latent HIV infection in vitro was not due to mutations inTat/TAR-axis

The cell line models of HIV-latency, such as ACH-2 and U1cells, possess TAR and Tat mutations, respectively, that causefunctional defects in these elements, leading to the apparent shut-down ofHIV transcription (Emiliani et al., 1996, 1998). To checkwhether Tat/TARmutations that correspond to the inactivation ofLTR-transcription are present in the presumed latently infectedprimary T-cells generated in our culture system, chromosomalDNAs were isolated from total T-cells containing bothchronically and latently infected cells. The HIV-tat (exon-1)and TAR-regions were PCR-amplified, cloned and sequenced.The derived amino acid sequences from each clone were aligned.The Tat mutations that could potentially inactivate or attenuateits' function were not observed (Reza et al., 2003). Similarly, nomutation was also found to occur in the TAR region (data notshown). Therefore, the occurrence of latent infection in quiescentCD4 T-cells in vitro was not due to the “pseudo latent infection”that could be caused by Tat/TAR-defects.

Discussion

Latent HIV resides in long-lived, resting memory CD4+T-cells in infected subjects (Blankson et al., 2002). To test thehypothesis that such latently infected cells are generated in vivowhen HIV-infected activated CD4+ T-cells become quiescent,the development of a culture system was required which couldrescue activated CD4 T-cells from death in culture and maintainthese cells long-term in a quiescent state. Using the feeder cellline, H80, we found that after a month of co-culture on H80cells, ∼99% of previously activated and dividing T-cellsbecame non-cycling (Figs. 2d and e). Activation markers likeCD25 and HLA-DR were not expressed by the majority(98.7%) of these cells (Fig. 2c); however, an early activationmarker, CD69, was constitutively expressed on more than half(∼63%) of total cells in culture (Fig. 2b). Approximately 34%of co-cultured CD4 T-cells were of CD69−CD25−HLA-DR−

phenotype representing their quiescent state, and ∼58% CD4T-cells showed a partial activation state as identified byCD69+CD25−HLA-DR− phenotype, which we termed as a“semi-quiescent” state. In general, all CD4 T-cells on H80 cellsin long-term cultures looked much smaller than fully activated,proliferating CD4 cells, but they are approximately 25% largerthan freshly isolated resting T-cells from human blood.Phenotypic analyses of long-term, healthy CD4 T-cells suggestthat these cells remained in higher metabolic states rather thanin the ‘truly’ resting state that is difficult to obtain in vitro. Invivo, the presence of such semi-quiescent cells with detectableto undetectable levels of activation markers cannot be ruled out(Stefanova et al., 2002). Most of the T-cells in co-culture werealso phenotypically CD45RA−CD45RO+ (not shown), repre-senting memory cells. Impressively, these normal CD4 T-cells

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could survive for more than 5 months with a slow decrease intotal number cells in co-culture, yielding about 2- to 3-fold lessin number of live CD4 cells than their initial numbers taken forco-culture (Fig. 2a). These quiescent CD4 T-cells could furthertake up 3H-thymidine upon re-stimulation with mitogen orimmobilized anti-CD3 (see in Fig. 5d), indicating that they stillhad proliferation potential. The mechanisms behind this long-term survival of primary CD4 T-cells on H80 cells are notknown.

Using this co-culture system, we found HIV-infectedactivated primary CD4+ T-cells would also become quiescentin long-term cultures (Fig. 3). However, cells making HIV coreantigens were readily detected, despite their quiescent char-acteristics (Fig. 4b). Interestingly, an early activation marker,CD69 was constitutively expressed by more than half of thetotal T-cells in H80 co-cultures (Figs. 3c and 4b). This stableexpression of CD69 was not dependent of HIV infection inculture because its expression was also constitutive onuninfected CD4 T-cells in long-term co-cultures (Fig. 2b),indicating that the co-culture conditions could provide astimulatory micro-environment to the surviving CD4 T-cells.Nevertheless, T-cells that were making HIV core antigens inlong-term cultures were of both CD69+ (“semi-quiescent”) andCD69− (quiescent) phenotypes. Moreover, HIV productionwas sustained in the presence of a protease inhibitor, indinavir(not shown here, unpublished results), indicating that contin-uous HIV production was occurring at the single cell levelrather than due to on-going spreading of the virus in culture.This continuous low-level HIV production from infected T-cells might be reminiscent of what is actually being observed inpatients during HAART. For example, virus productioncontinues at very low levels (<50 copies per ml) despiteextensive HAART (Dornadula et al., 1999). In addition,numerous studies have shown that HIV transcripts arefrequently detected in lymphoid tissues and peripheral bloodmononuclear cells (PBMCs) of many HAART-treated patientsby sensitive assays like in-situ hybridization or real time PCR(Cavert et al., 1997; Furtado et al., 1999; Gunthard et al., 2001;Hockett et al., 1999; Stellbrink et al., 2002; Zhang andCrumpacker, 2001). Haase and colleagues have shown thatreplication of HIV and SIV in phenotypically resting T-cellsoccurs in vivo (Li et al., 2005; Zhang et al., 1999b). Non-proliferating quiescent CD4+ T-cells isolated from HIV-infected PBMC cultures in vitro were also shown to produceHIV (Gondois-Rey et al., 2001; Scales et al., 2001). It is notclear whether these HIV-producing cells in vivo continuouslyproduce HIV during HAART. Although multi-drug-resistantmutants can reproduce themselves by infecting new target cellsin the presence of anti-retroviral drugs, stable viral loads of lessthan 50 copies per ml in plasma during effective HAARTwould presumably require the “chronic” low-level productionof viral copies from a stable source, one of which could beHIV-infected, long-lived quiescent or semi-quiescent CD4 T-cells. Interestingly, the majority of low-level virus circulating inblood during HAART are of wild-type character (Persaud et al.,2004), indicating that constant production of these viruses arenot solely due to drug-resistant mutants. Therefore, the chronically

infected cells that are generated in our culture system may berepresentative of such cells in vivo (Li et al., 2005; Zhang et al.,1999b), and if so, our culture system would provide a means tostudy these cells in greater detail.

Although low percentages (5–10%; in some experiments upto 20%) of T-cells making HIV chronically were present in long-term H80 co-cultures (Figs. 4a and 5a, left), the presence oflatently infected cells was evident because a protein kinase Cactivator (prostratin) increased the percentage of HIV-producingcells in these cultures by ∼2-fold (Figs. 5a, right, and 5b).Prostratin is known to reactivate latent HIV (Korin et al., 2002;Kulkosky et al., 2004). The frequencies of latently infected cellspresent in our culture system is therefore ∼10,000-fold higherthan the low frequency of these cells present in patients' blood,i.e., ∼1–10 cells per 106 resting CD4 T-cells. The question canbe raised asking whether the latently infected quiescent primaryT-cells formed in our culture system are truly latent. It is possiblethat the ‘presumed’ latently infected cells were actually making avery low-level of HIV, which is below the detection limit ofintracellular staining/flow cytometry, and therefore these cellscould not be detected by these techniques. However, treatmentwith prostratin would result in the increased production(detectable levels) of HIV core proteins. This explanation,which cannot be ruled out at present, requires the presence of, atleast, a minimal level of viral unspliced mRNA in stably infectedquiescent T-cells generated in long-term cultures, but the levelcould be insufficient for the production of detectable levels ofHIV core proteins by intracellular staining. It is also not entirelyclear if latently infected resting memory CD4 T-cells present invivo are truly latent or if they express a very low level ofunspliced HIV mRNA. The low frequency of latently infectedcells in patients, and therefore any low level of viral transcripts,if present in these cell populations, would not be readily detectedby even sensitive RT-PCR assays. Nevertheless, occasionally,unspliced viral mRNA can be detected in purified restingmemory CD4 T-cells from patients (Hermankova et al., 2003;Zhu et al., 2002) and numerous studies have shown that viralgene expression can be detected in phenotypically resting CD4T-cells residing in various tissue locations in vivo (Eckstein etal., 2001; Li et al., 2005; Zhang et al., 1999b). This may reflectthe existence of a spectrum of T-cells' activation status in vivo.Our in vitro generated latently infected cells may fall into thislow-level activation spectrum as opposed to the truly restingCD4 T-cells in vivo.

Recently, attempts have been made by others to generatelatently infected cells in vitro. One study has shown formation oflatently infected cells at low percentages by the spinoculationtechnique (Swiggard et al., 2005). Others have infectedstimulated PBMCs with HIV and then isolated latently infectedquiescent CD4 T-cells (Gondois-Rey et al., 2001). However, inboth studies, these latently infected cells did not possess theability to survive long-term, as they likely do in vivo, whereasthe latently infected cells that we generate in our system are long-lived. Moreover, our data provide evidence that HIV-infectedactivated CD4 T-lymphocytes can carry HIV during their exitfrom the cell cycle, and they become either chronic low-levelHIV-producing or latently infected cells. Using our culture system,

134 G.K. Sahu et al. / Virology 355 (2006) 127–137

the mechanisms of latency establishment and the reactivation oflatent HIV can be studied to possibly yield useful therapeuticintervention in the era of HAART. Moreover, chronically infectedcells generated in our culture system may be similar to thosefrequently observed in patients, even duringHAART, and these canalso be studied in greater detail to understand the biology of thesecells and to design any drugs that can be used to kill them orsuppress HIV production from them.

Materials and methods

Media, reagents and cell lines

All cells were grown in RPMI 1640 media supplementedwith 2 mM L-glutamine (Cellgrow, USA), 10% fetal bovineserum (FBS) and 5% penicillin-streptomycin, and incubated at37 °C, 5% CO2 humidified incubator. All antibodies (e.g.,antibodies to CD3, CD25, CD69 and HLA-DR eitherconjugated to FITC or PE), FITC-conjugated antibody toCD4 domain 2 region and propidium iodide (PI) werepurchased from BD Bioscience (USA), Ancell Corporation(USA) and Sigma (USA), respectively. HIV-core antibody(RD1; mouse IgG1 isotype) conjugated to phycoerythrin (PE)and prostratin were purchased from Beckman Coulter and LClaboratories, respectively. H80 cell line was originally derivedfrom a human glioma and obtained from Dr. Darell Bigner,Duke University, USA. H80 cells grow as an adherentmonolayer. The following reagents were obtained through theAIDS Research and Reference Reagent Program, Division ofAIDS, NIAID, NIH: VB cells (Lifson et al., 1986; Stein et al.,1987); MAGI-CCR5 and TZM-bl, indicator cell lines suscep-tible for both macrophage (M)-tropic and T-cell line (T) adaptedHIV-1 strains (Chackerian et al., 1997; Wei et al., 2002).

Isolation, stimulation and culture of CD4+ T-lymphocytes

Buffy coats (each equivalent to 500 ml of blood drawn fromhealthy donors) were purchased from the Blood Center(Houston, TX) and were subjected to isolation of peripheralmononuclear cells (PBMCs) by Ficoll–Hypaque method asdescribed previously (Wang et al., 1999). CD4+ T-cells werepurified by negative selection method using stem cell's CD4+T-cells purification kit in which non-CD4 cells are removed by amagnetic field. Briefly, PBMCs were resuspended in phosphatebuffer saline (PBS), 2% FBS. A cocktail of CD8, CD16, CD19,CD56, CD36, glycophorin A antibodies in bispecific tetramericantibody complexes was added to PBMCs, incubated for 30 minon ice. Then colloidal magnetic dextran iron particles wereadded and incubated in ice for further 30 min. These particlescould bind to the cells already bound to antibody complexes.The whole mixture was passed through a column placed in amagnetic field. Cells with antibody complexes and thereforedextran iron on their surfaces were retained within the column.Unbound CD4+ T-cells as flow-through were collected.

Purified CD4+ T-cells (routinely >95% pure) were stimu-lated for 2 days in RPMI, 10% FBS with anti-CD3 antibody(3 μg/ml), which was cross-linked by plate bound goat anti-

mouse IgG antibody (5 μg/ml). The stimulated cells werecollected on day 2 from plastic dish and cultured in thepresence of IL-2 (40 U/ml) in flasks. Cell numbers graduallyincreased up to 2–3 weeks. Every 2–3 days, half of culturemedia was replaced by fresh media containing IL-2 (40 U/ml).The cultures were maintained at a density of ∼1.5–2.0 millionlymphocytes per milliliter of media. Whenever required, cellviability was monitored by Trypan blue dye exclusion.

HIV infection and detection of HIV-producing cells

Laboratory-adapted non-cytopathic R5-tropic HIV stain,JRCSF, was propagated in a T-cell line (VB) which is susceptibleto R5-tropic strains. The kinetics of virus infection in cultureswere monitored by determining the percentages of HIV-infectedcells using fixed-cell indirect immunofluorescence assay (IFA)described elsewhere (Li et al., 1996). At early time points when∼100% of the cells became positive in fixed-cell IFA, culturesupernatants were harvested after overnight culture of infectedcells in fresh media and stored at −70 °C as stocks. Infectivity ofviral stocks (i.e., number of infectious particles per ml) wasdetermined by MAGI assay (Chackerian et al., 1997; Wei et al.,2002). Briefly, MAGI-CCR5 or TZM-bl cells were plated(∼50,000 cells per well) in 96-well plate and cultured. Next day,culture supernatants were removed and 30 μl of 10-fold serialdilutions prepared from virus stocks were added to each well induplicate in the presence of 40 μg/ml concentration of DEAE-dextran (Sigma, USA). After 2 h of incubation, 70 μl of freshmedia was added to each well and incubated for 2 days at 37 °C,CO2 incubator. Cells were washed with PBS and fixed with0.5% glutaraldehyde in PBS for 10 min at room temperature.After washing with PBS twice, cells were stained for 2 h with50 μl of a staining solution containing 4 mM each of potassiumferrocyanide and potassium ferricyanide, 2 mM MgCl2 and0.5 mg X-gal per ml. The numbers of blue colonies, eachcorresponding to an infectious unit, were counted in wellsincubated with highest dilutions of virus stock and the virus titerper ml of stock was calculated using dilution factor. Wheneverrequired, HIV-producing cells were also detected by intracellularp24 staining and flow cytometry (see below).

Co-culture of uninfected and infected activated CD4+ T-cellson H80 cells

Uninfected or infected CD4+ T-cells were harvested after 3–4 weeks of cultivation and overlaid on ∼30% confluent H80cells in flasks. Every 2–3 days, half of culture media wasreplaced by fresh IL-2-containing media. However, every week,T-lymphocytes were transferred to fresh H80 cells. AlthoughH80 cells grew to full confluence by 3–4 days after seeding,they still remained attached to flasks for another 3–4 days.

Flow cytometry

Analyses of cell surface markersCD4+ T-cells were stained with antibodies against various

surface markers e.g., CD4, CD3, αβ-TCR, CD69, CD25, HLA-

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DR. Briefly, ∼5×105 cells were stained with two antibodies(10 μl each, one conjugated to FITC and other conjugated toPE) in 50 μl volume for 45 min at 4 °C, washed twice in Hanksbalanced salt solution (HBSS), 2% fetal bovine serum (FBS)buffer and analyzed by Fluorescence activated cell sorter(FACS) available in a core facility at UTMB. Appropriateisotype controls were included in each experiment. The flowdata were analyzed using cell Quest program (BectonDickinson).

Intracellular staining with HIV-core antibodyApproximately 0.5×106 cells were permeabilized in

100 μl of cytoperm/cytofix solution (BD Biosciences) for30 min at 4 °C, washed twice with perm wash buffer andresuspended in 200 μl of perm wash buffer. Five microliter ofHIV-core antibody conjugated to PE was added and incubatedat 4 °C for 30 min. Then unbound antibodies were washed offtwice by incubating stained cells with perm wash buffer for30 min at room temperature and finally cells were fixed in0.5% paraformaldehyde in PBS. For dual staining such assurface and intracellular staining simultaneously, first surfacestaining was done as above and then intracellular staining wasdone. The percentages of HIV-expressing cells were deter-mined by FACS.

Analyses of cell cycleThis was done by propidium iodide (PI) staining following

(Yu et al., 1993) with minor modifications. Briefly, 1.5×106

cells were suspended in 1 ml of a low salt solution containing30 mg polyethylene glycol 8000 (Sigma), 50 μg PI, 0.1%sodium citrate, 0.01% Triton X-100 (Sigma) and 180 U ofRNAse A (Worthington Biochemicals) and incubated for20 min at 37 °C water bath. The resulting permeabilized cellswere kept in the dark at 4 °C overnight before analyzing by flowcytometry, mixing with 1 ml of a high salt solution derived fromlow salt solution by replacing RNAse and sodium citrate withNaCl (400 mM final concentration). At least 10,000 cells wereanalyzed for each sample. Cells with subdiploid amounts ofDNA were considered apoptotic, and viable cells showed thefollowing distribution of typical DNA content based on theircell cycle phases: G0–G1 (diploid), S (DNA content betweendiploid and tetraploid) and G2+M (tetraploid).

Cell proliferation assay

Cellular proliferation was assayed by measuring DNAsynthesis. About 1×105 cells were labeled in triplicate with3H-thymidine (10 μCi/ml) for 16 h at 37 °C, 5% CO2 incubatorand then harvested onto glass fiber filters with a Cell Harvester.The incorporated radioactive thymidine was measured as countsper min (c.p.m.) in a liquid scintillation counter (Beckman).

HIV-p24 assay

The level of HIV-p24 in culture supernatant was determinedby Coulter's p24 antigen capture ELISA kit. The assay wascarried out by following the manufacturer's instructions.

Analyses of HIV-tat and TAR sequences isolated fromlong-lived HIV-infected cells

HIV-infected CD4 cells' DNAs were isolated by usingWizard genomic DNA purification kit (Promega). HIV tat andTAR-amplification was done by nested PCR as follows: a 50-μlPCR mixture containing ∼1 μg of DNA as a template, 1×expand high fidelity buffer with 1.5 mM MgCl2 (Roche),200 μM of each dNTP, 300 nM of each primer and 2.6 U ofexpand high fidelity enzyme (Roche) was set up and denatured at94 °C for 2 min followed by 30 cycles' amplification at 94 °C for20 s, 56 °C for 20 s and 72 °C for 30 s and one final cycle ofextension at 72 °C for 5 min. Five microliter of first round PCRproducts was used as template for next 30 cycles of PCR and theother components of reaction mixture were same except primers.PCR was done using a program; one cycle of denaturation at94 °C for 2 min, 30 cycles of amplification at 94 °C for 20 s,56 °C for 20 s, 72 °C for 30 s, followed by an extension step at72 °C for 5 min. PCR was carried out in an Gene Amp PCRSystem 2400 thermal cycler (Applied Biosystem). Outer primersused for tat-exon 1 region: 5′-GGATACTTGGGCAGGAGTG-GAAGC-3′(forward) and 5′-GTTTGTCTTAATATTTTCCTA-TATTCTATG-3′ (reverse); inner primers used for tat exon 1region: 5′-GCAGAATTGGGTGTCGACATAGCAG-3′(for-ward) and 5′-GATGGACCACACAACTATTGCT ATTATT-3′(reverse). Outer primer pair used for TAR-amplification: 5′-GAAGTGTTAGAGTGGAG GTTTGACAGC-3′ (forward)and 5 ′-GCTTCAGCAAGCCGAGTCCTGCGTCG-3 ′(reverse); inner primer pair used for TAR-amplification: 5′-GCCCGAGAGCTGCATCCGGAGTAC-3′ (forward) and 5′-GATTTTCCACACTGACTAAAAGGG-3′ (reverse). PCR pro-ducts were electrophoresed in 1% agarose gel and DNA bandswere cut and subjected to purification using Gene clean Kit(Bio101, Inc.). Purified DNA products were ligated into a TA-cloning vector (pGEM-T) and cloned in JM109. Ten clones weresequenced by automatic DNA sequencer in the UTMB-corefacility using M13 forward primer. DNA sequences wereanalyzed by Vector NTI program (Invitrogen) in whichCLUSTALWalgorithm was used to generate multiple sequencealignment.

Acknowledgments

This study was supported by the Clayton Foundation forResearch. We are thankful to Mark C. Griffin for excellentassistance with flow cytometry and cell sorting in UTMB-core.We are grateful to Dr. Rolf Konig for helpful discussion.

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