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Journal of Virology, February 2002, p. 1673-1681, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1673-1681.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Effect of Latent Human Immunodeficiency Virus Infection on Cell Surface Phenotype

David G. Brooks1 and Jerome A. Zack1,2,3*

Department of Microbiology, Immunology and Molecular Genetics,1 Department of Medicine,2 AIDS Institute, School of Medicine, University of California at Los Angeles, Los Angeles, California 900953

Received 18 September 2001/ Accepted 13 November 2001


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Highly active antiretroviral therapy has succeeded in many cases in suppressing virus production in patients infected with human immunodeficiency virus (HIV); however, once treatment is discontinued, virus replication is rekindled. One reservoir capable of harboring HIV in a latent state and igniting renewed infection once therapy is terminated is a resting T cell. Due to the sparsity of T cells latently infected with HIV in vivo, it has been difficult to study viral and cellular interactions during latency. The SCID-hu (Thy/Liv) mouse model of HIV latency, however, provides high percentages of latently infected cells, allowing a detailed analysis of phenotype. Herein we show that latently infected cells appear phenotypically normal. Following cellular stimulation, the virus completes its life cycle and induces phenotypic changes, such as CD4 and major histocompatibility complex class I down-regulation, in the infected cell. In addition, HIV expression following activation did not correlate with expression of the cellular activation marker CD25. The apparently normal phenotype and lack of HIV expression in latently infected cells could prevent recognition by the immune response and contribute to the long-lived nature of this reservoir.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infection with human immunodeficiency virus (HIV) is characterized by high plasma viral loads, CD4+ T-cell depletion, and ultimately immune system failure (18). The administration of highly active antiretroviral therapy has succeeded in reducing viral loads in many patients to undetectable levels (19, 28, 35). However, termination of therapy results in a rebound in viral load, even in patients with previously undetectable viral levels, due to the presence of a viral reservoir not depleted by the present drug regimens (9). Previous studies have indicated that one such reservoir able to rekindle productive infection following cessation of highly active antiretroviral therapy is the latently infected T cell (10, 40). Based on the lack of sequence evolution in the latent population compared to virus isolated from the periphery, it appears that HIV in the latent state remains hidden from the immune response for the duration of therapy (16, 17, 38, 40). Unlike other suspected cellular reservoirs, such as productively infected T cells and persistently infected macrophages, which have a relatively short half-life (19, 27, 35), the population of latently infected T cells is estimated to remain stable up to 60 years on therapy (15, 30).

Much of the latent infection in the periphery likely occurs during the differentiation of infected, transcriptionally active effector T cells into quiescent memory cells. It is thought that infection of the transcriptionally active T cell allows for reverse transcription and integration of the viral genome; however, prior to either viral or immunologic cytopathic effects, cellular and viral transcription ceases and the virus becomes dormant. In this form HIV is resistant to the effects of antiviral drugs and is maintained until the cell is stimulated to increase RNA production. Environmental stimuli which trigger T cell transcription are then capable of reactivating the virus from latency (5, 12, 13, 25, 33). Formation of the latent reservoir in vivo, although very rare (~1 per million resting CD4+ T cells), occurs early during the acute phase of infection and does not seem to decrease substantially with duration of therapy, making it a formidable barrier to viral eradication (8, 11, 15, 30).

There is an emerging body of evidence demonstrating HIV infection of naïve T cells in vivo (3, 24, 26, 29), but exactly where and how infection of this cell type occurs is not clear. The presence of CCR5-tropic strains of HIV in the naive T-cell population, which is resistant to direct infection by HIV in vitro, and the recently demonstrated presence of HIV proviral DNA in naïve, CD45RA+ CD8+ T cells suggest that infection may occur during differentiation in the thymus (21, 24, 26, 29). We have previously established using the SCID-hu (Thy/Liv) mouse that some latent HIV infection can be generated during thymopoiesis and that the loss of cellular gene transcription during the later stages of thymopoiesis is associated with the transition into viral latency (5). These latently infected thymocytes are stable and can be exported into the periphery where the virus remains latent until the cell becomes transcriptionally activated. These studies strengthen the notion that latently infected naïve T cells identified in HIV-positive patients may have arisen in the thymus during thymocyte differentiation.

Experimental data have illustrated that HIV infection perturbs many normal cellular events. In vitro evidence indicates that the HIV gene products nef, vpu, and env are capable of down-regulating expression of CD4 (1, 6, 7) and that nef can also induce down-regulation of major histocompatibility complex I (MHC-I) (14, 31) and up-regulation of FasL (39) from the cellular surface. HIV has further been shown to alter the expression of the homing and chemotactic receptors CD54, CD62L, and CCR5 on the surface of productively infected cells (23). In vitro microarray analysis has illustrated that nef alone is capable of triggering a cell activation program similar to anti-CD3 stimulation and can alter gene expression of proteins known to be crucial to HIV replication (32). Similarly, other studies have demonstrated that the viral protein tat, in addition to increasing HIV transcription, also transactivates cellular interleukin 2 gene expression (34, 36). While it is not known if HIV causes changes in cellular phenotype prior to the transition into latency, it is possible that infection induces a change in cellular physiology that is not reversed when the infected cell enters a quiescent state. Alternatively, the coincident loss of viral gene transcription during cellular quiescence may cause these phenotypic changes to revert and the cell would then appear similar to an uninfected T cell. Due to the extremely low levels of latent virus in vivo, characterization of the latent reservoir at the cellular level has been difficult. In the SCID-hu system, latent HIV infection is generated at high frequency and the use of reporter viruses allows identification of productively infected cells (5, 20). This system thus provides a powerful model to assess the properties of latently infected T cells and the effects of virus reactivation on the infected cell. Herein we demonstrate that latent infection does not significantly alter the normal phenotype of human thymocytes or peripheral blood lymphocytes. However, upon stimulation of the latently infected cell, viral proteins are produced and the characteristic, virally mediated manifestations of CD4 and MHC-I down-regulation are observed. These data suggest that the latently infected cell will appear normal to the immune system and thus escape immune clearance.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Infection of SCID-hu mice. SCID-hu mice were prepared by implantation of human fetal liver and thymus (Advanced Bioscience Resource, Alameda, Calif.) as previously described (2). Thy/Liv implants were mock infected with medium or directly injected with viral stocks such that a total of 20 ng of HIVNL-r-HSAs was introduced. In general 1 ng of p24 contains approximately 100 infectious units. Viral stocks were prepared by electroporation of cloned proviral DNA into CEM cells. p24 gag expression was assessed by enzyme-linked immunosorbent assay (Coulter, Hialeah, Fla.) and was used to quantitate viral titers and reactivation from latency. Tissue donor numbers are provided in the figure legends to differentiate separate experiments.

Cell isolation and culture. Thy/Liv implants were harvested 5 weeks postinfection, and single-cell suspensions of thymocytes were immediately pooled in the presence of 100 ng of indinavir (Merck, West Point, Pa.)/ml and, where indicated, also in 10 µM zidovudine (AZT) (Sigma, St. Louis, Mo.). Thymocytes from mock-infected implants were isolated and cultured in parallel as controls in all experiments. Where indicated, thymocytes were stained with monoclonal antibodies specific for human CD4, CD45, murine CD24 (muCD24) directly conjugated to phycoerythrin (PE), allophycocyanin (APC), and fluorescein isothiocyanate (FITC) (Coulter) and with an antibody to human CD8 conjugated to peridinin chlorophyll protein (PerCP; Becton-Dickinson) and sorted on a FACStarPlus flow cytometer. Postsort analysis showed that more than 98% of cells were CD4 positive, CD8 negative, and muCD24 negative.

In other experiments, total thymocytes were stained with murine antibodies to human CD8 (Becton-Dickinson, Mountain View, Calif.) and CD4 single-positive (SP) cells isolated by negative selection via panning with goat anti-mouse antibody (Coulter)-coated flasks. These cells were then stained with rat antibody to muCD24 (Pharmingen, San Diego, Calif.) and were panned in flasks coated with rabbit anti-rat antibody (Sigma). Purity was assessed with different antibody clones to prevent epitope masking (Coulter).

To obtain human peripheral blood lymphocytes from SCID-hu mice, blood from multiple mice was pooled and peripheral blood mononuclear cells were isolated over a Ficoll-Paque gradient. To deplete murine monocytes/macrophages, the remaining cells were adhered for 2 h at 37°C in medium containing 100 ng of indinavir/ml and 10 µM AZT. Peripheral blood lymphocytes were stained as described above.

All cells were cultured in RPMI 1640 supplemented with 10% human AB serum, 100 U of penicillin/ml, 100 µg of streptomycin/ml, 2 mM glutamine, and 100 nM indinavir. Where indicated cells were cultured in the presence of 10 µM AZT (Sigma). Cells were costimulated with {alpha}CD3 monoclonal antibody bound to goat anti-mouse antibody-coated plates and 0.1 µg of soluble {alpha}CD28 (5)/ml.

Flow cytometry. Cells from cultures were stained with CD4-PE, CD8-PerCP, CD45-APC, and muCD24-FITC. For phenotypic analysis, cells were stained with the indicated antibody conjugated to FITC, PE, or APC. Thymocytes for Fig. 4 were stained with an antibody to human CD25 conjugated to APC. HLA-A2 expression was assessed with an antibody specific to HLA-A2 (and HLA-B17) conjugated to biotin and counterstained with streptavidin-APC (Pharmingen). Isolation of this antibody has been previously described (37). Samples were analyzed on a FACSCalibur flow cytometer using the Cell Quest Program (Becton-Dickinson). Forward- versus side-scatter profiles were used to define the live population. All cells were further gated on the human CD45+ population to exclude murine cells. Quadrants were set based on isotype controls from uninfected cells for each condition.



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  		FIG. 4. CD25 expression on thymocytes before and after HIV reactivation from latency. Thymocytes from pooled HIVNL-r-HSAs-infected Thy/Liv implants were negatively selected as done for Fig. 2a and were analyzed for CD45, CD25, and muCD24 expression by flow cytometry (day 0). Thymocytes were then costimulated for 3 days in the presence of a viral protease inhibitor, and the percentage of CD45-positive thymocytes expressing muCD24 was assessed by flow cytometry (day 3, left panel). Backgating on muCD24-negative (top right), muCD24-dim-positive (middle right), and muCD24-bright-positive (bottom right) thymocytes illustrated the percentage of cells expressing CD25 in each population. The mean fluorescence intensity (MFI) of CD25 expression for each panel is indicated above each histogram.

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Latently infected cells are phenotypically similar to uninfected cells. In order to determine if latently infected thymocytes and peripheral blood lymphocytes are phenotypically different from uninfected cells, we infected SCID-hu (Thy/Liv) mice with the pathogenic, CXCR4-tropic, reporter virus HIVNL-r-HSAs (20) and 5 weeks postinfection harvested thymocytes and peripheral blood lymphocytes. This virus contains the coding sequence for muCD24 inserted in place of the vpr gene, and productive infection directs the expression of muCD24 on the surface of infected cells, allowing quantitation by flow cytometry. Infection resulted in depletion of CD4/CD8 double-positive thymocytes and loss of CD4+ T cells in the periphery (Fig. 1). We next isolated mature, CD4 SP, muCD24-negative thymocytes by negative selection, resulting in a population of cells that expressed less than 1% CD8 or muCD24 (Fig. 2a). These cells were then subjected to costimulatory signals to induce latent virus expression. Uninfected thymocytes were obtained in the same manner from normal Thy/Liv implants and were similarly cultured for comparison (data not shown). Cells in all experiments were cultured in the presence of a viral protease inhibitor and AZT to prevent viral spread or de novo infection in vitro (5). As can be seen in Fig. 2a, culturing induces expression of low levels of virus. However, following 3 days of costimulation with activating antibodies to CD3 and CD28, more than 96% of thymocytes expressed CD25 (data not shown) and there was a substantial increase in expression of the HIV-encoded reporter, independent of cell size or granularity (Fig. 2a), indicating the presence of latent virus in the initial population. Since approximately 9% of CD4 SP thymocytes from HIV-infected implants harbored latent virus, virus-induced phenotypic changes should be evident by flow cytometry of the bulk population. Comparison of infected and uninfected mature thymocytes immediately following isolation revealed no phenotypic differences in expression of a variety of cell surface proteins (Fig. 2b). Expression levels of CD11a, CD38, CD40, CD40L, CD54, Fas, and HLA-DR were similarly unchanged (data not shown). Furthermore, there was no difference in the mean fluorescence intensity of any of the proteins analyzed except MHC-I, the intensity of which increased approximately sevenfold on total thymocytes from infected implants and twofold on mature thymocytes (Fig. 2c). The two- to sevenfold increase in the mean fluorescence intensity of MHC-I expression on thymocytes from an infected thymus has been described previously and may be due to indirect consequences of infection involving cytokine dysregulation (22). The lack of gross phenotypic alteration between uninfected thymocytes and thymocytes containing latently infected cells implies that latent infection does not quantifiably induce major external changes in cell surface protein expression. Furthermore, any phenotypic changes that may have occurred in these molecules during the initial productive infection must be reversed following the transition into viral latency.



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  		FIG. 1. Phenotype of SCID-hu thymocytes and peripheral blood lymphocytes (PBL). Thymocytes and peripheral blood lymphocytes from uninfected and HIVNL-r-HSAs-infected SCID-hu (Thy/Liv) mice were harvested 5 weeks postinfection and were stained with monoclonal antibodies to CD45, CD4, and CD8 on the day of biopsy. CD45+ (human) cells were gated and analyzed for CD4 and CD8 expression. Human thymocytes and lymphocytes from two experiments (tissue donors 67 and 30, respectively) are shown.

 





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  		FIG. 2. Flow cytometry analysis of cells harboring latent HIV. (a) Presence of latent HIV in CD4 SP thymocytes. At 5 weeks postinfection with HIVNL-r-HSAs Thy/Liv implants (tissue donor 67) were pooled and cultured in medium containing a viral protease inhibitor and AZT (same experiment as Fig. 1a, experiment 1). CD4 SP, muCD24-negative thymocytes (day 0) were then isolated by panning. The left panels were gated on CD45+ cells to exclude any murine cell contamination and show that less then 1% of the isolated thymocytes expressed CD8 or muCD24. These thymocytes were either left unstimulated or costimulated for 3 days, and HIV (muCD24) expression was quantitated by flow cytometry. The right panels show day 3 expression of HIV as a result of each condition. HIV (muCD24) expression is illustrated based on size and granularity following costimulation (upper panels). Quadrants are set according to isotype controls for each condition. (b) Phenotype of infected thymocytes. Uninfected and HIVNL-r-HSAs-infected CD4 SP, muCD24-negative thymocytes (from Fig. 2a, day 0) were stained for the indicated proteins on day 0 and analyzed by flow cytometry. Following activation approximately 10% of these cells showed evidence of latent infection (Fig. 2a). The y axis indicates the percentage of cells expressing each protein. Similar results were observed in multiple experiments. (c) MHC-I expression on thymocytes and peripheral blood lymphocytes (PBL) in response to HIV infection. CD4 SP, muCD24-negative, CD45-positive thymocytes and total-CD45-positive peripheral lymphocytes from uninfected and HIVNL-r-HSAs-infected mice (tissue donor 75) were stained on day 0 for MHC-I (HLA-ABC) expression and were analyzed by flow cytometry. The gate indicates MHC-I-positive cells and is based on an isotype control. The mean fluorescence intensity (MFI) of MHC-I-positive cells is shown above each gate. Similar results were observed in multiple experiments. (d) Phenotype of human peripheral blood lymphocytes. Peripheral blood lymphocytes from uninfected and HIVNL-r-HSAs-infected mice (tissue donor 67) were stained on day 0 with an antibody to the indicated human protein and CD45. CD45-positive (human) lymphocytes were analyzed by flow cytometry for the percentage of cells expressing each protein (shown as y axis). These results were consistent over multiple experiments.

 
We next assessed the phenotype of human cells in the periphery of HIVNL-r-HSAs-infected SCID-hu mice. We observed a decrease in the relative number of CD4+ T cells and an increase in the relative number of CD8+ T cells, consistent with CD4+ cell depletion in the thymus (Fig. 1); however, no detectable change in other cell surface proteins was seen (Fig. 2d). Likewise, expression of the RA and RO isoforms of CD45 was unchanged on human peripheral blood lymphocytes from infected animals (data not shown). Latent HIV infection did not alter the mean fluorescence intensity of any of the proteins analyzed, including MHC-I, the intensity of which was increased on thymocytes from HIV-infected implants of the same mice (Fig. 2c). We are unable to quantitate expression of the viral reporter gene in the peripheral blood lymphocytes due to the high levels of endogenous muCD24 expression on contaminating murine cells in the periphery (5); however, these peripheral T cells were clearly infected with latent virus, as costimulation induced substantial release of viral p24 into the medium, whereas no p24 was detected in the medium of unstimulated cells (600 pg of p24/ml for costimulated cells versus quantities below the 8-pg/ml detection level of the enzyme-linked immunosorbent assay for nonstimulated cultures). The immense increase in viral p24 following costimulation, coupled with the fact that more than 90% of peripheral human cells contain full-length HIV reverse transcripts (data not shown), implies a high level of latent infection in the periphery. Together, these data indicate that latently infected T cells are exported from the thymus and that these cells are phenotypically indistinguishable from uninfected lymphocytes.

Reactivation of latent HIV induces down-regulation of CD4 and MHC-I. To ascertain if viral reactivation from latency results in CD4 and MHC-I down-regulation, we isolated CD4 SP, HIV reporter-negative thymocytes by fluorescence-activated cell sorter. The enriched population contained less than 1% CD8 or muCD24 positive cells, and more than 98 percent of the cells expressed high levels of CD4. Costimulation of uninfected thymocytes had no effect on CD4 expression, while costimulation of the latently infected thymocytes resulted in a loss of CD4 cell surface expression from now productively infected cells (Fig. 3a). The down-regulation of CD4 occurred only on cells that coexpressed the virally encoded muCD24, indicating that viral replication was associated with the loss of CD4 expression. We are unable to specifically analyze peripheral blood lymphocytes for CD4 down-regulation by HIV because of the high endogenous levels of muCD24; however, Fig. 1 illustrates that the fluorescence intensity of CD4 or CD8 is unchanged on human peripheral blood lymphocytes in infected animals. To assess if viral reactivation caused a similar decrease in MHC-I expression, we utilized donor fetal tissue that expressed the HLA-A2 antigen and an antibody that specifically recognizes this molecule. As seen with CD4 in the previous experiment, MHC-I was expressed at high levels on latently infected thymocytes (similar to mock; not shown); however, following HIV reactivation, there was a down-regulation of MHC-I on infected cells expressing newly reactivated virus (Fig. 3b). This down-regulation is specific for CD4 and MHC-I as costimulation increases the expression of CD25 on both uninfected and previously latently infected thymocytes (Fig. 4), and levels of CD45 are not changed following HIV reactivation (data not shown). Thus, although latent HIV infection does not influence cell surface protein expression, following reactivation, virus protein levels are sufficient to influence, directly or indirectly, the down-regulation of CD4 and MHC-I.



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  		FIG. 3. HIV reactivation from latency causes CD4 and MHC-I down-regulation on infected cells. (a) CD4 down-regulation. Thymocytes from a single Thy/Liv implant (tissue donor 187) were stained with monoclonal antibodies to CD4, CD8, CD45, and muCD24 and were then isolated by fluorescence-activated cell sorting at 5 weeks postinfection with HIVNL-r-HSAs (day 0, top). Uninfected thymocytes from the same tissue donor were sorted in parallel (day 0, bottom). Thymocytes were cultured in the presence of protease inhibitor and AZT. The left panels were gated on CD45-positive cells and indicate that more than 98% of the sorted cells were CD4 SP and muCD24 negative postsort. Less than 1% of these thymocytes expressed CD8 (data not shown). Thymocytes were then costimulated for 3 days and were analyzed for CD4 and muCD24 expression by flow cytometry. The right panels are gated on CD45-postive cells and show HIV (muCD24) and CD4 expression following costimulation (Costim). Unstim, unstimulated. The level of p24 expression from costimulated and unstimulated cultures is shown at the right of the figure. (b) MHC-I down-regulation. CD4 SP thymocytes from HIVNL-r-HSAs-infected implants (tissue donor 62) were negatively selected as done for Fig. 2a. Isolated cells contained less than 2% CD8-positive and less than 1% muCD24-expressing cells (day 0). Isolated thymocytes were cultured either unstimulated or costimulated for 2 days in the presence of a viral protease inhibitor and AZT and were analyzed for HLA-A2 and HIV (muCD24) expression by flow cytometry. The mean fluorescence intensity (MFI) of HLA-A2 expression from muCD24-positive and -negative cells on day 2 is shown. Uninfected thymocytes isolated and cultured in the same manner for 2 days are shown in the right panels for comparison. The level of p24 expression for each condition is shown below each dot plot.

 
{alpha}CD3 and {alpha}CD28 costimulation of CD4 SP, muCD24-negative thymocytes dramatically increased the expression of the {alpha} chain of the interleukin 2 receptor (CD25) on both latently infected and uninfected thymocytes (Fig. 4). Backgating analysis of cells that expressed HIV reporter following costimulation indicated that the intensities of HIV and CD25 expression were not necessarily linked, as we observed in multiple experiments similar levels of CD25 expression on cells expressing high or low levels of muCD24 (Fig. 4). Furthermore, in all experiments, virus expression could be identified in thymocytes that did not express detectable CD25. Moreover, {alpha}CD3 stimulation alone was able to reactivate latent virus without an increase in CD25 expression (5; data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data suggest that latent infection does not substantially perturb cellular surface protein expression on thymocytes or on peripheral blood lymphocytes. Analysis of CD4 and MHC-I expression indicates that any residual HIV protein expression during latency is insufficient to impart the phenotypic cellular changes associated with productive infection. Whether this is due to a low level of or a complete lack of viral protein expression during latency is unclear. Following viral reactivation, both the surface expression of both CD4 and MHC-I is down-regulated on cells that express HIV but not on cells in the same culture that are not expressing virus, illustrating that viral reactivation is necessary for this phenotype. Since latently infected cells express equivalent levels of homing (CD11a, CD44, CD54, and CD62L) and chemotactic receptors (CXCR4 and CCR5) to those of their uninfected, resting counterparts, they would circulate and distribute through the body in a normal fashion, which is consistent with previous studies that observed latently infected cells in both the peripheral blood and lymph nodes of infected individuals (8).

The low levels of viral RNA (5), the undetectable viral reporter expression, and the lack of CD4 and MHC-I down-regulation observed during HIV latency suggest that viral protein expression is insufficient for adequate MHC-I presentation of HIV peptides to cytotoxic T lymphocytes (CTL). Similarly, the normal levels of MHC-I expressed on latently infected cells would not trigger an NK cell response. These factors could explain in part why latently infected cells are able to persist for such a long period of time in the presence of an HIV-specific CTL response (15, 30). Unfortunately, this would also suggest that therapeutic attempts to eradicate HIV by boosting the CTL response would not affect the latent population, as without an increase in viral protein production, these cells would remain invisible to the immune response. On the other hand, our data indicate that, once the latent virus is reactivated, infection proceeds with the normal phenotypic consequences, suggesting that the infected cell could then be targeted by HIV-specific CTL.

Interestingly, it appears that the level of HIV infection is not precisely controlled by the degree to which the cell is activated. By analyzing costimulated thymocytes, we found that both CD25-positive and CD25-negative cells express HIV and that CD25 expression, although generally signifying a more activated cell, did not correlate with a higher level of HIV expression. Our previous studies have demonstrated that HIV gene expression can be induced within 24 h following reactivation from latency (5). As CD25 is a relatively late T-cell activation marker, HIV may be expressed prior to CD25 acquisition, suggesting that immunotherapies targeting CD25 (4) may not eliminate cells recently activated to produce virus. However, our data suggest that strategies designed to reactivate HIV expression from a latently infected cell, such that it could be targeted by the immune system, may only need to minimally stimulate a cell, potentially avoiding the problems associated with nonspecific immune activation.

It is interesting that destruction of thymocytes by HIV can lead to an inversion of the human CD4-to-CD8 cell ratio in the periphery of these mice (Fig. 1). This depletion in the periphery is correlated with the HIV-induced depletion of thymocytes in the Thy/Liv implant, which give rise to the peripheral cells. The inversion of the CD4/CD8 cell ratio suggests that the depletion of thymocytes can cause a quantitative change in the export of lymphocytes from the thymus. In the setting of pediatric HIV infection and the developing immune system, the impaired export of lymphocytes to the periphery could enhance the CD4+ T-cell deficiency associated with HIV infection. It appears that, if any phenotypic aberrations occur with active infection prior to latency, they are reversed following the loss of active viral replication. Accordingly, our data indicate that some cells that survive differentiation in the infected thymus can be exported to the periphery and appear phenotypically normal yet harbor latent HIV. Consequently, it is important to target the thymus and the latent pool generated therein with novel therapeutic approaches in order to eliminate these reservoirs of infection.


   ACKNOWLEDGMENTS
 
We thank G. Bristol, R. Cortado, and A. Kacena for expert technical assistance.

This work was supported by NIH grants nos. AI 36554 and AI 36059 and the UCLA CFAR.


   FOOTNOTES
 
* Corresponding author. Mailing address: UCLA School of Medicine, Department of Medicine, 11-934 Factor Bldg., Mail Code 167817, 10833 Le Conte Ave., Los Angeles, CA 90095. Phone: (310) 794-7765. Fax: (310) 825-6192. E-mail: jzack{at}ucla.edu. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, February 2002, p. 1673-1681, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1673-1681.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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