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Stem Cells, Vol. 17, No. 6, 327-338, November 1999
© 1999 AlphaMed Press

Inhibition of Thymopoiesis of CD34+ Cell Maturation by HIV-1 in an In Vitro CD34+ Cell and Thymic Epithelial Organ Culture Model

Alan P. Knutsena, Stanford T. Roodmanb, John J. Freemanc, Kathleen R. Muellera, John D. Bouhasinc

a Division of Allergy/Immunology,
b Department of Pathology,
c Department of Pediatrics, St. Louis University Health Sciences Center, St. Louis, Missouri, USA

Key Words. Thymopoiesis • CD34+ stem cells • Fetal thymic epithelial organ culture • HIV

Dr. Alan P. Knutsen, Division of Allergy/Immunology, Pediatric Research Institute, St. Louis University Health Sciences Center, 3662 Park Avenue, St. Louis, Missouri 63110, USA.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The mechanisms by which HIV-1 affects thymopoiesis were determined by preincubating CD34+ cells or cultured thymic epithelial (CTE) cells with lymphotropic (T-) and monotropic (M-) strains of HIV-1 in an in vitro CTE organ and CD34+ cell coculture model that allows for analysis of development of thymocytes and mature T cells.

When purified CD34+ cells were precultured with either T- or M-tropic strains of HIV-1, thymopoiesis was impaired in a two-week coculture manifested by decreased cell number of thymocytes generated. However, the percentages of thymocyte subpopulations were comparable to control uninfected cocultures. Furthermore, HIV infection of thymocytes was predominantly observed in the CD44+CD3– population. However, in a four-week coculture experiment, HIV infection and depletion of more mature thymocytes were also observed.

When CTE cells were preincubated with T- and M- tropic strains of HIV before addition of CD34+ cells, the number of thymocytes and subpopulations of thymocytes at early and later stages of maturation were markedly decreased. Furthermore, CD34+ and CD44+ CD3– cells become HIV-infected.

In summary, HIV-1 infection inhibited thymocyte maturation at early stages of thymocyte maturation CD44+CD25–CD3–. In addition, HIV also depleted later stages of CD4+ thymocyte subpopulations.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
A central issue in the treatment of AIDS is the failure for full T cell immune reconstitution following highly active antiretroviral therapy (HAART). Regeneration of T cells following HAART may come from expansion of T cells in the peripheral lymphoid compartment and/or from de novo synthesis from the central thymic compartment. Ho et al. [1] reported that the regeneration of CD4+ T cells in patients treated with an HIV protease inhibitor came exclusively from the peripheral compartment and none from the central thymic compartment, which they attributed to either age-related thymic involution and/or HIV infection in the thymus. Recently, Douek et al. [2] reported that thymopoiesis declined significantly with age but was still evident in patients as old as 73 years. Using a recently developed technique, they identified that T cells, before being released from the thymus, generated circular fragments of DNA, called T-cell receptor rearrangement excision circles (TREC), which are present in naïve T cells but lost by dilution in memory T cells. In 10 HIV-infected individuals, the levels of TREC were significantly decreased. However, following HAART therapy, 9 of 10 patients showed a rapid and sustained increase in TREC levels, though not to age-adjusted HIV-uninfected controls.

In addition, Boldt-Houle et al. [3] reported that HIV infection of the thymus also caused depletion of selected T-cell receptor (TCR)-Vß repertoires. Since the thymus is the principal source of CD4+TCR{alpha}ß+ T cells, reconstitution of normal thymic function in HIV infection will be critical for effective T cell immune reconstitution with either HAART, immunomodulation or gene therapy. However, there appears to be decreased thymopoiesis for de novo generation of new T cells in HIV infection of adults or children. The mechanism by which HIV impairs thymopoiesis could occur as a result of direct HIV infection of thymocytes and mature T cells before release from the thymus, thymic epithelia, and/or stromal element impairment and/or HIV-1 infection of CD34+ cells and/or altered CD34+ cell thymopoietic potential.

In the present study an in vitro model of CD34+ cell maturation in a thymic epithelia organ culture (CTE) was utilized to determine the effects of HIV infection on thymopoiesis. By coculturing a small percentage of HIV-infected CD34+ cells or coculturing uninfected CD34+ cells with HIV-infected thymic epithelia and tracking HIV p24 staining of thymocyte subpopulations, at different stages of maturation, it could be determined whether and where HIV inhibited thymocyte maturation. In our model these early steps of thymocyte maturation can be analyzed as well as later stages of thymocyte maturation. As depicted in Figure 1Go, the following model of the stages of thymocyte maturation was assessed [4].



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  		Figure 1. Model of CD34+ cell thymocyte maturation. NK = natural killer.

 

   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
HIV Strains
In these studies we utilized T-tropic HIV strains HIV-3b (TCID50 108/ml) and HIV-MN (TCID50 106.83/ml) and the M-tropic HIV strain HIV-BaL (TCID50 107/ml) obtained from Advanced Biotechnologies (London, UK). The effects on thymopoiesis of titering HIV from 100-100,000 vp/ml (viral particles per ml) were examined by the addition of HIV strains to CD34+ cell and CTE cocultures. Loss of CD4+ and CD44+CD25+ expression in the coculture was observed after 14 days for HIV-3b as low as 100 vp/ml, HIV-MN at 676 vp/ml, and HIV-BaL at 100 vp/ml. In subsequent experiments approximately 105 TCID50 vp/ml were used for preincubation with CD34+ cells and CTE. Dr. Ramesh Akkina provided VSV-G pseudotyped HIV-1 viral stocks for infection of CD34+ cells. These latter viruses include macrophage tropic (JR-CSF) and lymphotropic (NL4-3) strains that are envelope-deficient and therefore do not spread to other cells post-infection.

Isolation of CD34+ Cells
CD34+ cells were isolated from cord blood obtained from the Cord Blood Stem Cell Bank at Cardinal Glennon Children's Hospital (St. Louis University Health Sciences Center; St. Louis, MO). Nucleated cells were isolated from cord blood by Ficoll-Hypaque density centrifugation [5-8]. CD34+ cells were positively selected using the MiniMACS system (Miltenyi; Auburn, CA) with CD34+ monoclonal antibody (mAb) and magnetic microspheres. Purity of the fractionated population was monitored using flow cytometry. Overall, >98% of purified cells were CD34. Typically, CD3+ T cells were undetectable as analyzed by flow cytometry.

Preincubation of CD34+ Cells with HIV
Approximately 1.0 to 1.5 x 105 CD34+ cells were precultured with approximately 105 TCID50 vp/ml of T-tropic or M-tropic strains of HIV-1, HIV-3b, HIV-MN and HIV-BaL, or with pseudotyped HIV in 1 ml culture media for 48 h and washed 4x with 10 ml phosphate buffered saline (PBS) containing 2% fetal calf serum (FCS) prior to coculture with normal CTE cells.

CTE
Fetal thymus (gestational age 17-24 weeks) were obtained from Advanced Bioscience Resources Incorporated (Gaithersburg, MD). CTE cultures were established by an explant technique and subcultured as previously described [5-8]. The thymic capsule was removed, and the tissue minced into 1 mm fragments and agitated gently in Dulbecco's modified Eagle's medium (GIBCO BRL; Grand Island, NY) supplemented with 5% FCS to wash out as many thymocytes as possible. Thymocytes and other hematopoietic cells, including dendritic cells, were depleted by incubation in 1.35 mM 2'-deoxyguanosine (Sigma; St. Louis, MO). The CTE cells were incubated on sterile gelfoam sponges, 1 cm x 3 cm, in 6 cm Petri dishes containing 12 ml of 1.35 mM 2'-deoxyguanosine and Ham's F-12 medium supplemented with 10% FCS, 25 mM HEPES, 2 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 µg/ml amphotericin at 37°C in a 5% C02 atmosphere for 12-14 days. Subsequently, the CTE cells were transferred to 24-well collagen-coated transwell culture plates (Nunc; Swedesboro, NJ) and incubated for two to four days in Iscove's/Ham's supplemented with 5% FCS, 10 ng/ml epidermal growth factor (Collaborative Research; Bedford, MA), 5 µg/ml insulin (Sigma; St. Louis, MO), 1.8 x 10–4 M adenine, 10–4 g/ml sodium pyruvate, 50 U/ml penicillin, and 50 µg/ml streptomycin before being seeded with CD34+-enriched cells.

Preincubation of CTE with HIV
CTE cells were precultured with approximately 105 TCID50 vp/ml of HIV-1, HIV-3b, HIV-MN and HIV-BaL, in 1 ml culture media for 48 h and washed 6x with 10 ml PBS containing 2% FCS prior to coculture with normal CD34+ cells. Though HIV was undetectable in the CTE cell supernatant by polymerase chain reaction (PCR), HIV may have been trapped within the thymic organ culture matrix.

CD34+ Cell and CTE Cocultures
The CTE cell cultures were seeded with normal CD34+ stem cells by infusion of 1-1.5 x 105 CD34+ cells into each well of a 24-well transwell plate containing five fragments of the CTE in Iscove's/Ham's medium at a 1:1 ratio containing 5% FCS, 25 U/ml interleukin 2 (IL-2) at 37°C in a 5% CO2 atmosphere [5-8]. Fresh medium was replaced every four days. In parallel cocultures CD34+ stem cell maturation in CTE cell cultures infected with HIV or pseudotyped HIV and CTE cells derived from AIDS thymus were compared to uninfected CTE cells. At two to four weeks of CD34+ and CTE coculture, the mononuclear cells and CTE cell organ culture were harvested for examination of thymocyte maturation of the cells free in the culture media and within the thymic epithelia compartment.

Detection of Thymocytes
T cell surface phenotypes of the differentiated CD34+ cells were determined by reacting mAb conjugated with either fluorescein isothiocyanate (FITC), phycoerythrin (PE), or peridinin chlorophyll protein (Per-CP) and then analyzed by flow cytometry [5-8]. Combinations of mAbs obtained from Becton Dickinson (San Jose, CA) were used to identify different stages of thymocyte maturation, including CD44+CD25+ populations of CD3–CD4–CD8– triple negative (TN) cells, immature CD3–CD4+CD8+ double positive (DP) and CD3+CD4+CD8+ triple positive (TP) thymocytes, and mature CD3+CD4+ and CD3+CD8+ single positive (SP) T cells, and CD45RA+ expression. Detection of HIV infection of thymocytes was performed using FITC p24 mAb (KC57, Coulter; Hialeah, FL). Mononuclear cells were aspirated from the culture wells, washed, and resuspended in PBS containing 5% FCS. A range of 5-10 x 103 events was collected per sample. Negative controls (murine IgG1-FITC; IgG1-PE; and IgG1-Per-CP; Becton Dickinson) were used to set the positive gate. The lymphocyte region was selected by CD45+CD14– gating for the lymphocyte population and fluorescence was measured in a fluorescence-activated cell sorter Caliber flow cytometer (Becton Dickinson).

Scanning Laser Immunofluorescence Confocal Microscopy
These studies were performed to determine T cell maturation from CD34+ cells within the CD34+ and CTE organ culture model. HIV infection of thymocytes was performed by FITC-labeled anti-p24 HIV mAb. Preparation of the CTE thymic tissue for immunofluorescence was performed in the Histopathology Laboratory (St. Louis University Health Sciences Center; St. Louis, MO). The CTE tissue culture was fixed in 1% buffered paraformaldehyde, washed in PBS, imbedded in optimal cutting temperature compound and snap frozen. The frozen CTE tissue was sectioned at 8 microns using a cryostat. The slides were then air-dried at 4°C for 1 h, fixed in cold acetone for five min and then air-dried for 15 min. The slides were stored at –20°C until ready for staining. Immunofluorescence staining was performed by rehydrating the tissue slides in Tris buffered saline for seven min, incubation with the appropriate fluorescent-labeled mAb diluted in FCS, washed, and sealed with Paramount aqueous mounting media (Dako; Carpinteria, CA) and coverslip. Fluorescence detected by the scanning laser confocal microscope was performed as previously described [5-8]. Background staining was determined with negative isotype controls that included appropriate murine IgG mAb of parallel CD34+ cell/CTE coculture (nonspecific IgG1 monoclonal) and a CTE culture not seeded with CD34+ cells to subtract nonspecific background reactivity. Thymocytes were stained with mAbs. Combinations of mAbs were used to identify different stages of thymocyte maturation similar to the mononuclear cells obtained from the supernatant. Detection of HIV infection of thymocytes was performed using FITC p24 (KC57) mAb. The confocal system used in these studies was a Zeiss 410 LSM system built around a Zeiss Axiovert 135 inverted microscope. Samples were observed with Zeiss 63X oil, NA 1.25 objective. The detector gain and background (dark current) subtract were set on a sample exhibiting the brightest dual fluorescence, and these detector gain/background subtract settings were then held constant when recording confocal images of all subsequent samples. Quantitative analyses of the fluorescence intensity of single spots or defined areas were made for the fluorophors using the Zeiss software LSM (ver. 3.92).

Detection of HIV Infection
Identification of thymocytes infected with HIV-1 was determined by imaging HIV using anti-p24 immunofluorescence by flow cytometry and scanning laser confocal microscopy. The detection of cytoplasmic HIV p24 antigen was performed by immunofluorescence using methods previously described [5, 9]. Briefly, prior to staining with anti-p24, the cells or tissue were fixed and permeabilized with Perm Fix (PharMingen; San Diego, CA) for 20 min at 40°C, then incubated with FITC-conjugated anti-p24 (KC57) 0.1 µg/ml (Coulter) 1:20 dilution for 30 min 4°C, and then fixed with 1% paraformaldehye for 10 min [10]. Appropriate murine IgG isotype mAbs were used as controls for nonspecific staining. Confirmation and quantification of HIV infection of the coculture model were determined by quantitative PCR measurement of HIV using the Amplicor HIV-1 monitor test (kindly performed by Dr. Arens).

Statistics
Groups were compared using paired and unpaired Student's t-test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Preincubation of CTE Cells with HIV
In these experiments CTE cells were preincubated with T-tropic and M-tropic strains of HIV and the effect on normal CD34+ cell maturation was determined. As shown in Table 1Go, HIV-infection of CTE markedly inhibited thymocyte maturation. When CTE cells were preincubated either with HIV-3b, -MN and -BaL (data were combined as there was no difference between the different HIV strains), the cell number of thymocytes produced in 14 days significantly decreased to 0.48 x 106 cells/ml (p < 0.05) or a 3.5-fold increase (p < 0.05) compared to the initial input of the number of the CD34+ cells relative to 1.29 x 106 cells/ml or a 9.2-fold increase in the control uninfected coculture. When the stages of thymocyte maturation were examined, both early and late stages of thymocyte maturation were affected by preincubation of CTE cells with HIV compared to control cocultures. In an early thymocyte maturational stage, CD44+CD25+ cells were decreased 12.6% versus 39.7% (p < 0.01), comparing CTE HIV-infected versus control, respectively (Table 1Go and Fig. 2Go). In later stages of thymocyte maturation, total CD4+, DP CD4+CD8+, and mature SP CD4+ cells were decreased compared to the control coculture, 6.8% versus 29.1% (p < 0.05), 4.3% versus 20.6% (p < 0.05), and 2.5% versus 8.4% (p < 0.05), respectively. Though HIV p24+ staining of mononuclear cells was low in the cells obtained from the supernatant coculture, CD34+ and CD4+ cells within the CTE cells were p24+ when measured by laser confocal immunofluorescence (Fig. 3Go). This suggested that preinfection of CTE cells with HIV allowed infection of CD34+ cells and early thymocytes migrating within the CTE and/or inhibited maturation of thymocytes at multiple stages of maturation by effecting the thymic epithelia.


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  		Table 1. Thymocyte maturation of CD34+ cells preinfected with HIV compared to CTE pre-infected with HIV in an in vitro CD34+ cell and CTE organ coculture model.
 


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  		Figure 2. FACS analysis of CD45, CD4, CD8, CD25 and CD44 expression of thymocytes obtained from coculture of CD34+ cells with CTE. Either CTE or CD34+ cells were preincubated with HIV-3b for 48 h prior to coculture, yielding [(CTE + HIV) + CD34] and [(CD34 + HIV) + CTE] cocultures, respectively. Preincubation of CTE with HIV resulted in a marked decrease of cell number reflected as decreased CD45+bright expression. DP CD4+CD8+ and mature SP CD4+ thymocyte populations were decreased. In addition, CD44+CD25+ thymocytes were also decreased.

 


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  		Figure 3. Laser confocal immunofluorescence microscopy of CD3+, CD4+, CD8+, CD44+, CD25+, CD34+ and HIV-p24 (antibody combinations used are listed on the left in the order of FITC, PE, PerCP) of CD34+ cells and CTE cocultures. Either CTE or CD34+ cells were preincubated with HIV-3b for 48 h prior to coculture, yielding [(CTE + HIV) + CD34] and [(CD34 + HIV) + CTE] cocultures, respectively, as described in Figure 1Go (top). In the figure FITC appears green, PE red and PerCP blue. Coexpression of FITC/PE appears yellow, FITC/PerCP blue-green, PE/PerCP purple and FITC/PE/PerCP white. In cocultures of [(CTE + HIV) + CD34+ cells], there was decreased CD44+CD25+ expression, and HIV p24+ cells in CD34+ were increased compared to the uninfected control. HIV p24+ CD34+ was greatest with HIV-3b and HIV-MN and less so with HIV-BaL. In contrast, in cocultures of [(CD34+ + HIV) + CTE cells], there was similar CD44+CD25+ expression compared to the control, and HIV p24+ expression was variable, present in some but not as marked as in the CTE + HIV model. (Magnification x 63, Zoom 0.822).

 
Preincubation of CD34+ Cells with HIV
Similarly, when CD34+ cells were precultured with HIV, the cell number present after 14 days of coculture with CTE was decreased to 0.59 x 106 or a 4.2-fold increase in cell number, compared to control cocultures (Table 1Go). In the inoculated HIV-infected CD34+ cell preparation, the percentage of p24+CD34+ cells after 48-h incubation ranged from 1.1% to 1.9% and was similar for both T- and M-tropic HIV strains (Fig. 4Go). Since the majority of CD34+ cells were uninfected with HIV, this allowed for thymocyte maturation of these uninfected CD34+ cells. This is evident in a two-week coculture, as the expression of total CD44+ CD25+, CD4+, DP CD4+CD8+, and SP CD4+ populations were comparable to the control cocultures, as seen in Table 1Go and Figure 2Go.



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  		Figure 4. FACS analysis of HIV p24 expression in CD34+ cells after 48 h incubation with HIV-3b, HIV-MN, HIV-BaL.

 
The model would predict that in longer coculture of CD34+ cells preinfected with HIV and CTE, HIV in the coculture would spread and infect CD4+ thymocyte populations disrupting all stages of thymocyte maturation. As seen in Table 2Go, early and late stages of thymocyte maturation were evaluated in two and four-week cocultures. In particular, at four weeks all thymocyte maturation stages were decreased when CD34+ cells were preinfected with HIV compared to control cocultures: early subcortical stage CD44+CD2+CD3– cells (32.9% versus 56.1%, p = ns), cortical stage CD44+CD25+CD3– cells (37.8% versus 66.7%, p < 0.05) and CD4+CD8+ (16.2% versus 42.0%, p = ns) and SP CD4+CD8– cells (9.8% versus 17.4%, p = ns). When CD34+ cells were preinfected with HIV and cocultured for four weeks compared to two weeks, there were decreased CD4+CD8+ cells, 8.5% versus 16.2% (p < 0.05), decreased CD44+CD25+CD3– cells, 16.9% versus 37.8% (p = ns), and decreased CD44+CD2+CD3– cells, 32.9% versus 44.9% (p < 0.05).


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  		Table 2. Thymocyte maturation of CD34+ cells preinfected with HIV in an in vitro CD34+ cell and CTE organ coculture model comparing two versus four week cocultures.
 
When measurement of cytoplasmic HIV p24+ staining of various thymocyte maturation stages was performed, p24+CD34+ and p24+CD4+cells were 1.2% and 1.9%, respectively (Table 1Go); however, p24+CD44+ cells were increased, 11% of total lymphoid cells at two weeks and 9% at four weeks (Table 2Go). Furthermore, as seen in Figure 5Go, p24+CD44+ cells were increased mainly within the CD3– population, but also in some p24+CD3+CD44+ cells. This suggested that when CD34+ cells are HIV-infected, maturation is inhibited initially at CD44+ CD3– cells, an early stage of thymocyte maturation.



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  		Figure 5. FACS analysis of CD3, CD4, CD44 and HIV p24 expression of thymocytes obtained from coculture of CD34+ cells with CTE. CD34+ cells were preincubated with HIV-3b for 48 h prior to coculture. Though, the percentage of p24+CD4+ cells was low when CD34+ cells were preincubated with HIV, the percentage of p24+CD44+ cells was increased. Furthermore, when this population was examined by gating on positive and negative CD3 cells, the p24+CD44+ population was predominantly CD3–. This suggested that HIV infection of CD44+CD3– cells resulted in inhibition of maturation past this stage.

 
In order to eliminate secondary HIV infection of thymocytes in the CD34+ and CTE coculture, pseudotyped HIV was utilized. In these experiments, when CD34+ cells were preincubated with pseudotyped HIV and added to CTE cocultures, this resulted in a decreased number, 0.8 x 106 versus 1.8 x 106 (p < 0.05), and decreased fold increase, 5.8-fold increase versus 12.7 (p < 0.05), at two weeks of coculture compared to control coculture, and fold increase (Table 3Go). The major thymocyte stage affected by HIV infection was significantly decreased CD4+CD8+ cells, 19.5% versus 27.2% (p < 0.05) and slightly decreased CD25+, 54.1% versus 71.5% (p = ns) and CD44+CD25+ cells, 50.7% versus 61.0% (p = ns) compared to the control coculture. When CD34+ cells were preincubated with HIV-3b, there was significantly (p < 0.05) decreased CD4+, CD4+CD8+, CD4–CD8–, CD44+CD25+ cell populations compared to control coculture. When CTE cells were preincubated with pseudotyped HIV, there were no decreased thymocyte subpopulations compared to the control coculture, unlike the result when CTE cells were preincubated with HIV-3b.


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  		Table 3. Thymocyte maturation of CD34+ cells preinfected either with HIV-3b or pseudotyped HIV compared to CTE preinfected with HIV-3b or pseudotyped HIV in an in vitro CD34+ cell and CTE organ coculture model.
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
HIV may disrupt thymopoiesis by affecting both thymic epithelia and thymocytes. In pediatric patients who have died of AIDS, histological examination of autopsy specimens revealed a thymic dysplasia manifested by marked involution with depletion of cortical and medullary thymocytes, decreased cortical epithelia, abnormal fibronectin cortical matrix, obscured corticomedullary differentiation, and microcystic changes of Hassall's corpuscles [11-14]. In addition, a lymphomononuclear infiltrate and giant cell formation has been described in the HIV-infected thymus. Antibody cross-reactive to core p17 and p24 HIV antigens and thymic epithelial proteins has been observed in the HIV-infected thymus, suggesting an autoimmune component to HIV damage. In addition are the observations that human fetal thymus/liver organs transplanted into severe-combined immunodeficient (SCID) mice (SCID-hu thy/liv) in in vitro human models of thymopoiesis demonstrate that both HIV-infected thymocytes and T cells induce thymic epithelial cell injury [13-23]. Abnormal thymic epithelia synthetic function has been demonstrated by decreased synthesis of thymic hormones, such as thymosin-{alpha}1 and thymosin-ß4 [14]. In previous studies we evaluated thymopoietic function of the HIV-infected thymus from two children with AIDS by coculturing normal CD34+ cells in an in vitro cultured CTE organ culture model [5]. Thymocyte maturation was markedly abnormal, with decreased CD4+ populations and an arrest of differentiation at the CD3–CD4–CD8– TN CD44+CD25– stage, suggesting aberrant cortical function [5]. In these children the thymic architecture was markedly abnormal with a guirlande architectural pattern and disruption of thymic epithelia and vimentin mesenchymal components, probably representing end-stage HIV infection of the thymus. In addition there were clusters of HIV-infected CD34+ stem cells identified in the subcortical regions of these thymuses.

HIV also disrupts thymopoiesis by direct infection of thymocytes [12-26]. Utilizing the SCID-hu thy/liv model that was then challenged with HIV-1, investigators demonstrated that thymopoiesis was impaired by direct infection of thymocytes. Uittenbogaart et al. [9] and Kitchen et al. [25] reported that HIV-1 infects both CD4+-bearing thymocytes and CD4– thymocytes. Both CD34+ cells and thymocytes express the HIV-1 coreceptors chemokine receptor 4 (CXCR-4) and CCR-5 [27-29]. Indeed, CXCR-4 expression is high in immature thymocytes.

In the present study we examined the separate effects of preinfecting either CD34+ cells or thymic epithelia and then evaluating thymopoiesis. The results of these studies indicate that HIV infection of CD34+ cells inhibits thymopoiesis at the precortical and cortical stages of maturation, e.g., at the CD44+CD25–CD3– and somewhat at the CD44+CD2–CD3– stage of maturation. With preincubation of thymic epithelia with HIV, all stages of thymopoiesis were affected; whereas when CD34+ cells were preincubated with HIV, thymocyte maturation of these cells was inhibited. When HIV-infected CD34+ cells were incubated longer in a four-week coculture, HIV infection spread to affect all maturation stages of thymopoiesis. Thus, these studies demonstrated that HIV infection not only depleted CD4+ thymocytes including TP CD4+CD8+CD8+, DP CD4+CD8+ thymocytes and mature SP CD4+ T cells, but also inhibited earlier thymocyte maturation steps at the TN stage. Our results are supportive of the results found in the SCID-hu thy/liv model that suggested HIV also inhibited early stages of thymocyte maturation [30]. Thus, HIV disrupts thymopoiesis by both direct infection of thymocytes as well as thymic epithelial dysfunction and destruction either through direct infection or a bystander inflammatory process, resulting in depletion of thymocytes and decreased de novo mature T cell generation [19-23]. Ultimate proof of thymopoietic potential in HIV infection will come from examination of thymopoiesis of HIV-infected thymuses from patients with AIDS and in clinical trials.

Although there have been reports of detection of low levels of HIV in bone marrow CD34+ cells, almost all recent studies have indicated that these cells appear to be largely uninfected [31-39]. However, the hypothesis that CD34+ cells might be susceptible to HIV infection is supported by the finding that these cells also express the HIV coreceptors CXCR-4 and CCR-5 [29, 40]. Furthermore, HIV infection may suppress CD34+ cell maturation via a number of mechanisms besides direct infection, including inhibitory cytokines (tumor necrosis factor-{alpha}), and induction of apoptosis [31, 41, 42]. An indirect effect on early hematopoietic progenitors was postulated in the recent study by Jenkins et al. [30] in the SCID-hu thy/liv model. In this study depletion of progenitors was demonstrated in SCID-hu mice grafts infected with HIV-1 and was followed by a decline in thymocyte cell number. Since proviral genomes were not detected in hematopoietic cells, they concluded that HIV acted indirectly on hematopoietic cells, resulting in reduced colony forming units and consequently in loss of mature thymocytes. However, CD34+ cells within the thymic microenvironment might be more susceptible to HIV infection. This was suggested by the findings of Ruiz et al. [5], who reported in autopsy specimens of children who had died of AIDS that CD34+ cells were HIV-infected. In the present study our group extended these findings using an in vitro model of thymopoiesis. When the CTE organ culture was preincubated with HIV-1 and subsequently CD34+ normal cells were added to the culture, CD4+ and CD4+CD8+ thymocytes were depleted and thymopoiesis was markedly arrested at the CD44+CD25– TN stage. Furthermore, CD34+ cells within the CTE organ culture were HIV-1 p24+. We hypothesize that subpopulations of CD34+ cells in the thymus and early thymocytes are susceptible to HIV infection that results in a maturation arrest of these CD34+ cells and early thymocyte maturation. In addition, HIV may cause a maturation arrest of non-HIV-infected cells by other inhibitory mechanisms.


   Acknowledgments
 
Supported by National Institutes of Health Grant AI42553.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Ho DD, Neumann AU, Perelson AS et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 1995;373:123-126.[Medline]

  2. Douek DC, McFarland RD, Kelser PH et al. Changes in thymic function with age and during the treatment of HIV infection. Nature 1998;396:690-695.[Medline]

  3. Boldt-Houle DM, Jamieson BD, Aldrovandi GM et al. Loss of T cell receptor Vbeta repertoires in HIV type 1-infected SCID-hu mice. AIDS Res Hum Retrovir 1997;13:125-134.[Medline]

  4. Godfrey DI, Zlotnik A. Control points in early T-cell development. Immunol Today 1993;14:547-553.[Medline]

  5. Ruiz ME, Freeman J, Bouhasin JD et al. Arrest of in vitro T cell differentiation of normal bone marrow derived CD34+ stem cells cocultured with thymic epithelia from children with AIDS. STEM CELLS 1996;14:533-547.[Abstract]

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accepted for publication September 9, 1999.



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