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Stem Cells, Vol. 14, No. 6, 702-713, November 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Development of a Method of Thymocyte Differentiation of Bone Marrow-Enriched CD34+CD38– Cells in Postnatal Allogeneic Cultured Thymic Epithelia to Evaluate Immunodeficiency Disorders

Alan P. Knutsena, Standford T. Roodmanb, Margaret E. Ruiza, Kathleen R. Muellera, John D. Bouhasina

a Pediatric Research Institute and
b Department of Pathology, St. Louis University Health Sciences Center, St. Louis, Missouri, USA

Key Words. CD34+ • Thymocyte maturation in vitro • Cultured thymic epithelia • Human immunodeficiency virus

Dr. Alan P. Knutsen, Pediatric Research Institute, St. Louis University Health Sciences Center, 3662 Park Avenue, St. Louis, MO 63110, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
An in vitro model of CD34+CD38– stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCR{alpha}ß+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25–CD3– thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Hematopoietic stem cell (SC) therapy is increasingly being used to correct T cell immune deficiencies and genetic disorders, and as rescue therapy in treatment regimens of malignancies which also ablate the bone marrow [1-4]. Recently, gene therapy of SC has been proposed for the treatment of AIDS in order to reconstitute the T lymphoid and myeloid cell populations [5-7]. We [8, 9] and others [10-12] have hypothesized that in order for the T cell immune system to be reconstituted in patients with AIDS, thymic transplantation or augmentation of residual thymic function would also have to be performed concomitantly.

The thymus gland, essential for T cell differentiation and maturation of bone marrow SC, is affected by HIV-1 infection. Thymic dysplasia has been described in AIDS where the thymic epithelial architecture is adversely affected [13]. HIV has been shown to directly infect thymocytes, although it is less clear to what extent HIV infects thymic epithelial cells [14-20]. However, HIV does cause thymic epithelial dysfunction and destruction either through direct infection or bystander inflammatory process, resulting in thymocyte depletion [21-25]. The pathophysiology is manifested by loss of thymocytes and corticomedullary differentiation, and calcification of Hassall's corpuscles. Recently, Ho et al. [26] and Wei et al. [27] reported that in HIV-infected adult patients, HIV production and clearance occurred continuously through HIV infection, driving a high continuous turnover of CD4+ T cells primarily from the peripheral lymphoid pool without any discernible contribution from the thymic compartment. Thus, in order to reconstitute the T cell immune system with a full repertoire of antigen specificity in patients with AIDS using gene therapy of SC, augmentation or replacement of the patient's thymic function would seem imperative. In order to test this hypothesis, we developed an in vitro model system using bone marrow-derived CD34+ SC and postnatal allogeneic cultured thymic epithelia cocultures to evaluate these issues [8, 9, 28]. In this report, we describe the methods that allow differentiation and/or maturation of CD34+CD38– bone marrow-derived SC in cultured thymic epithelia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Isolation of CD34+ SC
CD34+ SC were obtained from bone marrow filter screens of normal controls who donated bone marrow transplantation. The screens were washed extensively to obtain residual trapped cells, and the nucleated cells were obtained by Ficoll-Hypaque density centrifugation [9, 28]. After Ficoll-Hypaque density centrifugation, soybean agglutinin (SBA)-positive cells were removed by adherence on SBA-coated flasks (Applied Immune Sciences, Inc.; Menlo Park, CA). Residual T cells in the nonadherent fraction were removed by adherence on CD5/CD8– coated flasks (Applied Immune Sciences, Inc.). CD34+ cells were positively selected by panning using CD34+ monoclonal antibody (mAb)-coated flasks (Applied Immune Sciences, Inc.). Finally, CD38 cells were depleted from the CD34+ population by immuno-magnetic purification. One mg magnetic microbeads (Immunotech; Cedex, France) were precoated with anti-CD38 mAb (Immunotech), 5 mg per 1 mg microbeads, for 1 h at 23°C with gentle mixing, and washed. For negative depletion of CD38 cells, 1 to 10 x 106 CD34+ cells were incubated with 1 mg of anit-CD38-coated magnetic microbeads for 30 min with gentle rotational mixing at 23°C. CD38+ cells were removed using a magnetic separation apparatus (Dynal; Oslo, Norway). Anti-CD38 depletion was repeated to further deplete residual CD38+ cells.

Cultured Thymic Epithelial Fragments (CTEF)
Thymus glands were obtained from children undergoing corrective cardiac surgery who had portions of their thymus removed as part of their surgical process. This study was approved by the St. Louis University Health Sciences Center Institutional Review Board, and informed consent was obtained from the subjects' parents. CTEF cultures were established by an explant technique and subcultured as previously described [9, 28]. The thymic capsule was removed from 3 to 5 grams of thymus, and the tissue was minced into 1 mm fragments and agitated gently in Dulbecco's modified Eagle's medium (GIBCO BRL; Grand Island, NY) supplemented with 5% fetal calf serum (FCS) to wash out as many thymocytes as possible. Thymocyte and other hematopoietic cells, such as dendritic cells, were depleted from the fragments by incubation in 1.35 mM 2'-deoxygauanosine (Sigma; St. Louis, MO) [9, 28]. The CTEF 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, 25mM HEPES, 2 mM glutamine, 50 U/ml penicillin, 1 µg/ml gentamicin, 50 µg/ml streptomycin and 2 mg/ml amphotericin (complete medium) at 37°C in a 5% CO2 atmosphere for 11 to 14 days. Subsequently, three CTEF per well were transferred to 24-well collagen-coated transwell culture plates (Nunc; Naperville, IL) and incubated for three to five days in Iscove's/Ham's medium at a 1:1 ratio containing 5% FCS, 10% HL-1 serum-free media (Ventrex; Portland, ME), 0.4 µg/ml hydrocortisone (Calbiochem Behring; La Jolla, CA), 11 ng/ml epidermal growth factor (Collaborative Research; Bedford, MA), 5 mg/ml insulin (Sigma) and 0.1 mg/ml sodium pyruvate before being seeded with CD34+CD38– enriched SC.

Coculture of CTEF and CD34+CD38– SC
The cultures were seeded with 5,000 to 50,000 CD34+CD38– SC in 24-well transwell plates containing three CTEF per well in Iscove's/Ham's medium at a 1:1 ratio containing 5% FCS, 10% HL-1 serum-free media (Ventrex), 11 ng/ml epidermal growth factor (Collaborative Research) and 25 U/ml of interleukin 2 (IL-2) at 37°C in a 5% CO2 atmosphere. Transwell inserts were used for media changes without disrupting the SC and CTEF coculture. Negative control cultures included CD34+CD38– SC and CTEF cultured each alone in 24-well transwell plates under similar culture conditions to the SC and CTEF cocultures. Fresh medium with IL-2 was replaced every four days.

Coculture of CTEF-CD34+ SC and HIV
The CTEF cultures were seeded with CD34+ SC by infusion of 5 to 10 x 104 CD34+ SC to parallel cocultures, cell-free HIVIIIB or HIVSF2 stocks obtained from Dr. Maurice Green were added weekly. The titers of the HIV stocks were measured by Dr. Green using reverse transcriptase activity and had been previously determined to infect T cells.

Detection of T Cell Phenotypes
T cell surface phenotypes of differentiated SC were determined by reacting mAb conjugated with either fluorescein (FITC), phycoerythrin (PE) or peridinin chlorophyll protein (Per-CP) and then analyzed by flow cytometry [9, 28]. mAbs used are listed in Table 1Go. Combinations of mAbs were used to identify immature thymocytes, such as double-positive (CD3–CD4+CD8+) and triple-positive (CD3+CD4+CD8+) thymocytes, and mature CD3+CD4+CD8– and CD3+CD4-CD8+ T cells. Mononuclear cells and CTEF were aspirated from the culture wells, washed, resuspended in phosphate-buffered saline (PBS) containing 10% FCS, and then subjected to Ficoll-Hypaque density centrifugation to remove dead cells. The mononuclear cells at the interface were washed twice and resuspended in PBS containing 10% FCS. One to 5 x 104 cells were mixed with optimal concentrations of mAb and analyzed by cytofluorometric analysis using a FACScan flow cytometer (Becton Dickinson; San José, CA). Negative controls (murine IgG1-FITC; IgG1-PE and IgF1-Per-CP; Becton Dickinson) emitted only a small percent of positive fluorescence and were used to set the positive gate. Analysis of three-color populations was performed using the WinList program (Verity; Topsham, ME).


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  		Table 1. mAb identifying mononuclear cells
 
In order to ensure that the CS-CTEF T cells originated from the donor CD34+ SC population, two different methods were employed. In some cultures, HLA-A phenotype analysis of the thymocytes obtained from the SC-CTEF coculture was determined in the HLA Laboratory (kindly performed by Dr. Graff) using standard serologic HLA-A typing techniques. This was then compared to that of the SC donors. In other experiments, a portion of CD34+CD38– SC was labeled with 5-(and –6)-(((4-chloromethyl) benzoyl)amino)tetramethylrhodamine (CMTMR; Molecular Probes, Inc.; Eugene, OR) prior to coculture with the CTEF [9, 29]. CMTMR is retained in living cells through several generations and is not transferred to adjacent cells. Fluorescence of CMTMR within CD2+ and CD4+ FITC-stained thymocytes from the SC-CTEF cocultures was then measured at 585 nm by flow cytometry, confirming the SC origin of the thymocytes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
CD34+CD38– SC Isolation
The isolated CD34+ SC comprised 0.9% of the bone marrow mononuclear cells, with CD34+CD38– representing 16.0% ± 4.2% of the total CD34+ SC population as analyzed by flow cytometry. After two steps of CD38 depletion, the CD38+ cells contaminating the cell preparation were not detectable in three preparations and <5% in the remainder (Table 2Go). The percentage of CD34+ cells detected was variable depending upon recovery of CD34 expression after obtaining the cells from the CD34+-coated flasks. Staining for CD34+CD38– cells was performed for 18 h at 4°C according to manufacturer's recommendations (Applied Immune Sciences, Inc.) to allow resynthesis of the cell surface CD34 molecule. CD3 expression was <1% CD3+ cells; thus there were no contaminating T cells in the SC preparation. Furthermore, when CD34+CD38– SC were cultured alone, the cells did not increase in number or express T cell phenotype surface markers and were predominantly dead cells based on forward and side scatter flow cytometric properties (Figure 1Go, right panel).


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  		Table 2. Kinetic studies of CD34+CD38– SC differentiation in cultured thymic epithelia cocultures
 


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  		Figure 1. Photomicrographs of SC and CTEF cocultures at one week (20x). SC and CTEF cultured in media alone did not expand in number nor express T cell surface markers. There was marked proliferation of mononuclear cells in the SC-CTEF coculture with skirting of thymic epithelial cell around the thymic fragment. The cell skirt around the CTEF fragment's only control was mainly fibroblasts.

 
Kinetics of CD34+CD38– Differentiation
When SC and CTEF was cocultured together, there was an exuberant growth or skirting of thymic epithelia cells surrounding the CTEF and proliferation of mononuclear cells, as illustrated in Figure 1Go (left panel). This was in marked contrast to when SC or CTEF was cultured alone. CTEF cultured alone displayed a fibroblastic skirt surrounding the thymic epithelial fragment with some mononuclear cells that were dead by forward and side scatter properties (Figure 1Go, middle panel). However, when enriched CD34+CD38– SC were cocultured with cultured thymic epithelia, the appearance of thymocyte phenotype expression was observed during one to four weeks of coculture (Table 2Go). Early thymocyte phenotype markers, CD7 and CD2, were expressed in 79% and 82% of thymocytes at one week of coculture and increased to 96% and 95% of cells at two weeks. Concomitantly, CD34+ cells decreased from 100% at time zero to 5% at week 1, but remained at approximately 1.3% to 2.3% between two to three weeks of coculture (Table 2Go).

Both double-positive CD4+CD8+ and triple-positive CD3+CD4+CD8+ thymocytes were detected by flow cytometric analysis at one week of coculture (Table 2Go). This was concomitant with a decrease of triple-negative CD3–CD4–CD8– thymocytes from 19% at one week to 6% at two weeks of coculture, where it remained between 2% to 8% for the remainder of culture. Double-positive CD4+CD8+ thymocytes were rapidly produced peaking at two weeks at 30%, although they were decreased compared to thymocytes freshly obtained from a thymus (column 1), and then decreased again to 14% at four weeks. Triple-positive CD3+CD4+CD8+ thymocytes detected by flow cytometry were 19% at one week of coculture and increased to 27% at two weeks of coculture as illustrated in one experiment in Figure 2Go. The percentages of triple-positive thymocytes remained approximately the same between two to four weeks of coculture, and were slightly increased to that observed from freshly isolated thymocytes. Concomitantly, mature single-positive CD4+T cells increased from 29% at one week of culture to 54% at four weeks, and this was increased compared to freshly isolated thymocytes. Nearly all of the freshly isolated thymocytes and T cells obtained from the coculture were TCR{alpha}ß+, 67% to 79%, with only a small percentage being TCR{gamma}{delta}+ cells, approximately 1%. CD45RO+ and CD45RA+ expression underwent changes with length of coculture. CD45RO+ thymocytes predominated between one to three weeks of culture. However, CD45RA+ cells increased between two and three weeks of coculture with decreasing percentages of dual-positive CD45RO+CD45RA+ thymocytes from 30% at one week to 9% at three weeks.



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  		Figure 2. Phenotypes of cells obtained from 5,000 SC and cultured thymic epithelia cocultured for one week demonstrating dual expression of CD3, CD4, Cd8 cells. Analysis of live cells within the gate (in left panel) demonstrated double-positive CD4+CD8+ (8%), triple-positive CD3+CD4+CD8+ (5%) thymocytes and mature single-positive CD4+ (44%) and CD8+ (4%) T cells using WinList program.

 
In this coculture model system, coexpression of CD44 and CD25 underwent sequential kinetic changes, as reported in Table 2Go. Expression of both CD44 and CD25 was increased compared to freshly isolated thymocytes. At one week of coculture, CD44+CD25+ cells were maximally expressed at 63% and CD44–CD25+ cells were 9%, as illustrated in Figure 3Go, when triple-negative CD3–CD4–CD8– thymocytes were 19%. However, at two weeks of coculture, CD44+CD25+ and CD44–CD25+ cell populations were equally expressed, 48% and 42%, respectively, and triple-negative thymocytes had decreased to 6%. This remained stable at three weeks of coculture, but then changed at four weeks of coculture so that CD44+CD25– cells predominated at 32%, which was comparable to freshly isolated thymocytes. In subsequent experiments, CD44+CD3– early thymocytes can be identified as well, confirming the kinetic changes of CD44+ thymocyte populations. Likewise, HLA-DR expression was increased from 13% at one week to 30% at four weeks of coculture.



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  		Figure 3. CD44 and CD25 phenotype of cells obtained from SC and cultured thymic epithelia coculture at one week. The cultured cells were predominantly CD44+CD25+ (83%) with a minor CD44–CD25+ (11%) population.

 
Myelomonocytic cell lineages were also increased in the cocultures compared to freshly isolated thymocytes (Table 2Go). In particular, promyelocytes, CD33+ cells, were increased up to 23% at one week of coculture. Mature CD14+ myeloid cells were also increased to 8% cells, and CD16+ natural killer cells were increased to 20% at one week of coculture. At time zero, CD33+, CD14+ and CD16+ cells were negligible in the purified CD34+CD38– population; however, both of these cell populations decreased with increasing length of coculture (Table 2Go). CD19+ B cells represented only a maximum of 3% of the cells.

In order to demonstrate that thymocyte and T cell populations were derived from the allogeneic CD34+CD38– donor cells, SC were prelabeled with CMTMR and placed in parallel cultured thymic epithelia cocultures. As illustrated in Figure 4Go, CD2+CMTMR+ and CD4+CMTMR+ cells were observed at one week of coculture. This represented 47% and 39%, respectively, of all cells present at one week (Table 2Go). As expected, the CMTMR fluorescence decreased with increasing length of coculture as each generation divided the intensity of the CMTMR label per cell, which could be quantified on the flow cytometric histogram (not shown).



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  		Figure 4. CD34+CD38– SC were labeled with CMTMR prior to coculture with CTEF. After one week coculture, mononuclear cells obtained from the cocultures were stained for CD2 and CD4. As illustrated, CD2+CMTMR+ (47%) and CD4+CMTMR+ (38%) cells were identified, confirming SC origin of the thymocytes obtained from the coculture.

 
Dosage of CD34+CD38– SC Differentiation
In these dose response studies, 5,000 to 50,000 CD34+CD38– SC were cocultured with a constant amount of CTEF, and thymocyte and T cell phenotypes were measured at three weeks. As seen in Table 3Go, mononuclear cells obtained from the cocultures increased from 122,000 cells/fragment when 5,000 stem cells per well were used to seed the CTEF cocultures, and increased linearly at the other three SC dosages so that 358,400 cells/fragment were isolated when 50,000 SC per well were added. However, the expression of cell surface markers was nearly identical at the four SC seeding dosage schedules, although there were increased percentages of double-positive CD4+CD8+ and triple-positive CD3+CD4+CD8+ thymocytes and decreased percentage of mature single-positive CD4+ T cells with increasing CD34+CD38– seeding dosages. In addition, when 25,000 and 50,000 enriched CD34+CD38– SC were added to the CTEF, subpopulations of CD4+ thymocytes were observed that expressed either high or low densities of CD4 molecule. Double and triple-positive CD4+CD8+ thymocytes and mature CD4+ T cells were predominately CD4hi. However, more immature single-positive CD3–CD4+CD8– cells comprising approximately 9% of the cells expressed predominantly CD4lo cells (Fig. 5Go). Expression of other cell surface phenotypes was unchanged and comparable to that found at three weeks of coculture.


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  		Table 3. Dosage studies of CD34+CD38– SC differentiation in cultured thymic epithelia cocultures
 


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  		Figure 5. Phenotypes of cells obtained from 50,000 SC and cultured thymic epithelia cocultured for three weeks demonstrating dual expressions of CD3, CD4, and CD8 cells. Analysis of double-positive CD4+CD8+, triple-positive CD3+CD4+CD8+ thymocytes and mature single-positive CD4+ and CD8+ T cells was performed using WinList program. Subpopulations of CD4+ thymocytes expressing low and high densities of CD4 were observed. Immature CD3–CD4+ thymocytes comprised 9% of the cells and were predominantly CD4lo. Double-positive and triple-positive CD4+CD8+ thymocytes (24% and 22%, respectively), and single-positive CD4+ (41%) T cells were predominantly CD4hi

 
Coculture of CTEF-CD34+ SC and HIV
This is vitro model was extended by infection of the SC and CTEF coculture with two different strains of HIV, HIVIIIB and HIVSF2. CD4+ thymocyte populations were markedly decreased in HIV-infected cocultures (Fig. 6Go, Table 4Go). In addition, HIV infection also impaired early thymocyte differentiation in the subcortical region, based on increased expression of CD44+CD25–CD3– thymocytes and decreased expression of CD44+CD25+ thymocytes (Fig. 7Go, Table 4Go).



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  		Figure 6. Flow cytometry study of SC-CTEF coculture infected with HIVIIIB (top) and HIVSF2 (middle) compared to uninfected control (bottom) of CD3+, CD4+, and CD8+ thymocytes. CD4+ thymocytes form HIV-infected cocultures were markedly decreased compared to control.

 

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  		Table 4. CD34+CD38– SC differentiation in HIV-infected cultured thymic epithelia coculture
 


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  		Figure 7. Flow cytometry study of SC-CTEF coculture infected with HIVIIIB (top) and HIVSF2 (middle) compared to uninfected control (bottom) of CD3+, CD44+, and CD25+ thymocytes. CD44+CD25–CD3– thymocytes were markedly increased and CD44+CD25+ were decreased from HIV-infected cocultures compared to control, suggesting inhibition of early thymocyte maturation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
In this study, we report an in vitro model of CD34+CD38– SC differentiation in postnatal allogeneic cultured thymic epithelia cocultures. Our results are similar to that reported by Barcena et al. [30, 31] and Scadden's group [32, 33] who used fetal liver as the SC source and fetal thymus organ coculture. However, there were several important differences noted. In particular, Barcena's group irradiated the fetal thymus organ culture which may have damaged thymic epithelial cells in addition to depleting thymocytes, and Scadden's group generated thymic epithelia monolayers eliminating the three-dimensional cortical and medullary thymic structure. Also, cocultures of the fetal liver SC and fetal thymic lobules were performed on the gelfoam islands, and the thymocytes isolated were then extracted from the thymic organ cultures. Importantly, CD34+CD38– SC cultured in growth media alone did not proliferate, indicating that any possible contaminating CD3+ T cells in the SC donor preparation probably did not account for the T cells observed in the coculture. In our model, the precentages of CD7 and CD2 cells were comparable to that reported by Barcena et al. [30, 31], but the percentage of triple-positive CD3+CD4+CD8+ thymocytes was slightly decreased. We speculate that further proliferation of T cells in the coculture supernatant accounts for this difference. In addition, there were fewer B cells, although approximately the same percentages of promyelocytes and natural killer cells in our model system compared to other studies [30.31].

We also evaluated CD44 and CD25 expression in this model system, since CD44 expression appears early in differentiation in the subcapsular cortex prior to expression of T cell receptor for antigen (TCR), CD3, CD4 and CD8, i.e., triple-negative thymocytes. Other groups have reported that CD44 plays an important role in thymocyte migration and maturation [34-37]. As thymocytes further mature through double and triple-positive thymocyte stages, CD44 disappears but then reappears in more mature thymocytes. The ligands for CD44 include hyaluronate and fibronectin, matrix proteins, which promote cell adhesion. In this model of SC differentiation, there were sequential changes of CD44 and CD25 expression, so that the percentage of CD44+CD25+ cells were maximal at one week of coculture when triple-negative thymocytes were maximal. Subsequently, the percentage of CD44–CD25+ cells increased at two and three weeks, and finally CD44+CD25– cells predominated at four weeks of coculture, thus paralleling in principle thymic maturation. Recently, we have reported a deficiency of CD44 expression and decreased CD3+CD4+CD8+ cells when CD34+ SC from a patient with severe combined immunodeficiency were cocultured with allogeneic CTEF using this in vitro model system [28]. Furthermore, Vanhecke et al. [38] recently reported six different stages of differentiation of CD4+CD8+ double-positive thymocytes to mature functional CD4+ T cells. In stage four, CD4+ thymocytes expressed CD45RO before becoming CD45RO–, and subsequently CD45RA+ resting naive T cells in the final stage. The results of our study paralleled their findings, but not a recent study that reported only CD45RA+ T cells generated in vitro using CD34+ SC and thymic epithelia monolayer model [33]. We observed decreasing percentages of CD45RO+CD45RA+ and of CD45RO+ cells with concomitant increasing percentages of CD45RA+ cells between two and three weeks of culture.

In one experiment, the SC and cultured thymic epithelia in vitro model was infected with HIV. Similar to other reports, CD4+ T cells were decreased [32, 33]. In addition, there were increased triple-negative thymocytes, increased CD44+CD25–CD3– thymocytes and decreased CD44+CD25+ thymocytes, suggesting that HIV may inhibit early thymocyte maturation steps in addition to depleting mature CD4+ T cells. Inhibition of early maturation steps prior to expression of CD4 may be related HIV proteins, such as TAT, and not due directly to HIV infection. The model system described in this study should be useful in determining the suppressive effects of HIV infection on thymopoiesis. In addition, this model system will be useful in the evaluation of antiretroviral medications in reversing the effects of HIV.

In summary, an in vitro model of CD34+CD38– SC differentiation into thymocytes using CTEF cocultures is described. This model system may be beneficial in evaluation of thymocyte differentiation of primary T cell immunodeficiency diseases, gene therapy of SC and pharmacological augmentation of thymic function.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors express their gratitude to the patients who donated thymus and bone marrow, and to Dr. A Fiore who collected thymus tissue for us.

These studies were partly supported by grants from Greater St. Louis Hemophilia Foundation and Fleur de Lis.


   Footnotes
 
Provisionally accepted July 29, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received June 14, 1996; accepted for publication August 27, 1996.




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