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Stem Cells, Vol. 14, No. 1, 132-145, January 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

T Cell Differentiation/Maturation of CD34+ Stem Cells from HIV-Seropositive Hemophiliacs in Cultured Thymic Epithelial Fragments

Margaret Ruiza, Stanford T. Roodmanb, John D. Bouhasina, Alan P. Knutsena

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

Key Words. AIDS • HIV • CD34+ stem cells • Cultured thymic epithelial fragments • Thymocyte differentiation/maturation

Dr. Alan P. Knutsen, Pediatric Research Institute, St. Louis University Health Sciences Center, 3662 Park Avenue, St. Louis, MO 63104, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 MLCs
 Discussion
 References
 
The clinical manifestations of AIDS are predominantly due to the cellular and humoral immune dysfunction caused by HIV infection, and thymic dysplasia caused by HIV infection probably contributes to the T cell lymphopenia. In the present study, T cell differentiation and/or maturation was assessed when enriched CD34+ stem cells (SCs or SC) purified from bone marrow of HIV-seropositive hemophiliacs were cocultured with allogeneic cultured thymic epithelial fragments (CTEFs).

When HIV-seropositive hemophiliacs' enriched CD34+ SC were cocultured with allogeneic CTEFs, acquisition of the T cell phenotypic markers CD7, CD2, CD3, CD4, CD8 and T cell receptor for antigen (TCR){alpha}ß was observed from cells harvested from the culture media peaking at approximately 28 days. Origin of the differentiated and matured T cells from the CD34+ SC was confirmed by labeling the SC with 5-(and -6)-(((4-chloromethyl) benzoyl)amino)tetra-methyl-rhodamine (CMTMR), a fluorescent cytoplasmic dye, and detecting fluorescence in the differentiated and matured T cell by flow cytometry. In one experiment, CMTMR labeling was omitted and double positive CD4+CD8+ and triple positive CD3+CD4+CD8+ thymocytes were identified. These studies confirmed that thymocyte differentiation/maturation from SC had occurred.

In addition, T cells obtained from the CD34+ SC and CTEF cocultures proliferated to phytohemagglutinin stimulation maximally with stem cell donor antigen-presenting cells (APCs) and also proliferated to pooled B cells in a mixed lymphocyte culture (MLC). Furthermore, the T cells produced were tolerant to thymus donor B cell HLA antigens (p < 0.025); though there was slight MLC reactivity to autologous stem cell donor B cell HLA compared to thymic B cells (p < 0.025). These T cells demonstrated positive self-alloreactivity to stem cell HLA antigens in four of nine persons, though decreased compared to pool B cell alloantigens. Furthermore, in three experiments, responsiveness to stem cell donor B cells subsequently disappeared upon further duration of CD34+ SC-CTEF coculture. These studies suggested that CD34+ SC gave rise to accessory cells populating the thymus that contributed to HLA restriction. To further evaluate this hypothesis, two different donors of CD34+ SC were cultured simultaneously with thymic epithelial fragments and MLC reactivity was then examined toward APC of the stem cell donors. In these experiments, T cells responded to stimulation with HLA antigens of the pool B cells and did not respond to thymus donor B cells. In six of eight experiments, the chimeric SC-CTEF T cells did not respond to stimulation with B cells of either stem cell donor. These studies suggest that HLA restriction and tolerance were induced by cells of the stem cell donor as well as the thymic epithelial cell HLA antigens

In summary, these studies demonstrated that HIV-infected hemophiliac bone marrow-derived nonadherent CD34+ SC were capable of differentiating and/or maturing into T cells when cocultured in a normal allogeneic thymic environment. Furthermore, the T cells derived from derived CD34+ SC were capable of differentiating into T cells when cocultured in a normal allogeneic thymic environment, proliferated maximally with APCs from the stem cell donor and were tolerant of thymic HLA class II antigens, and to a lesser degree to stem cell donor B cell HLA antigens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 MLCs
 Discussion
 References
 
AIDS is manifested by susceptibility to recurrent microbial infections and malignancies as the result of cellular and humoral immune dysfunction caused by HIV infection [1]. In particular, the T cell immune deficiency is characterized by CD4+ T helper cell dysfunction and eventual depletion. A number of different mechanisms by which HIV infection causes CD4+ T cell loss and dysfunction have been identified, including direct lysis, syncytia formation, autoimmune processes, apoptosis and disruption of cellular signaling [1]. In addition, the thymus gland, essential for differentiation of progenitor thymocytes into mature T cells, is affected by HIV infection. This has been described as a thymic dysplasia in pediatric AIDS [2]. However, the thymic epithelial architecture is adversely affected by HIV infection, which is unlike the thymic dysplasia described in primary T cell immunodeficiencies. HIV directly infects thymocytes and possibly epithelial cells as well, resulting in their dysfunction and destruction [3-9]. This is demonstrated by loss of thymocytes, loss of corticomedullary differentiation and calcification of Hassall's corpuscles. Recently, Ho et al. [10] reported in adults that HIV production and clearance occurs throughout HIV infection and drives a continuous high turnover of CD4+ T cells. Furthermore, they calculated that the generation of CD4+ T cells was primarily from the peripheral lymphoid pool with none derived de novo from the central thymic compartment when patients were treated with an HIV protease inhibitor. They attributed the lack of thymus-derived CD4+ T cells to age-related thymic involution and/or HIV infection of the thymus. In addition, several investigators have reported that bone marrow hematopoietic progenitor cells of lymphocytes, erythrocytes and myeloid cells are affected by HIV infection, contributing to a decrease in lymphopoiesis. However, it is less clear whether bone marrow stem cells (SC) identified by the cell surface membrane protein CD34 are already infected with HIV [11-15]. Thus, in addition to mature T cells and thymocytes, SC and thymic epithelial cells may be infected with HIV, or at least affected by HIV infection resulting in both loss and decreased production of thymus-derived T cells.

The purpose of this study was to determine whether CD34+ SC from HIV-seropositive hemophiliacs were capable of differentiation and/or maturation into functional T cells when provided a normal thymic microenvironment. Immune enhancement or reconstitution of the T cell compartment will probably be necessary in patients with AIDS to prevent susceptibility to infections and malignancies. A novel approach prior to the advent of antiretroviral medication was the transplantation of cultured thymic epithelial fragment (CTEF) tissue in patients with AIDS to replace thymic function [16-18]. Although one study reported transient increase of T cell numbers following CTEF transplantation, no sustainable increase of T cell numbers was observed. The reasons for the graft failure were not clear, though may have been due to rejection or HIV infection of thymic epithelia, HIV infection of newly differentiated thymocytes and T cells, and/or lack of adequate numbers of bone marrow SC. In this study, we observed that bone marrow-derived SCs from HIV-seropositive hemophiliacs differentiated and/or matured into functional T cells. The T cells derived from the stem cell and normal allogeneic cultured thymic fragment cocultures were tolerant of thymic HLA antigens. This suggests that cultured thymic epithelia transplantation in conjunction with effective antiretroviral therapy might be a feasible modality in the T cell immune reconstitution in patients with AIDS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 MLCs
 Discussion
 References
 
Patients
Hemophiliac patients who were HIV-seropositive and had peripheral blood CD4+ numbers less than 200 cells/mm3 were recruited from the St. Louis University Health Sciences Center Regional Hemophilia Center (St. Louis, MO). Bone marrow, 50 to 100 ml, was collected from the posterior iliac crest in the outpatient Hematology Clinic under local anesthesia. The patients were free of acute illness when they donated bone marrow. Patient #2 had atypical mycobacterium identified in the bone marrow aspirate and was excluded. This study was approved by the St. Louis University Health Sciences Center Institutional Review Board and informed consent was obtained from the subjects.

Isolation of CD34+ SCs
SCs were obtained from bone marrow aspirates of HIV-infected hemophiliacs. Bone marrow was collected in heparinized tubes, diluted in normal saline, and the nucleated cells obtained by Ficoll-Hypaque density centrifugation [19]. Typically, a mean of 5 x 106 mononuclear cells/ml of bone marrow were recovered, with the nonadherent CD34+ cells representing 11.1% ± 7.9% of the mononuclear cells. CD2+ lymphocytes were then depleted by rosette formation with sheep RBC treated with 2-amino-ethylisothiouronium bromide (AET-SRBC) and a second Ficoll-Hypaque density centrifugation [20]. Adherent cells were removed by adherence on plastic Petri dishes [21]. Last, the CD34+ cells were positively selected by panning, using CD34+ monoclonal antibody (mAb)-coated flasks (Applied Immune Sciences, Inc., Santa Clara, CA) [21]. Purity of the fractionated population was monitored using flow cytometry. A mean of 4 x 104 enriched CD34+ cells with a range of 1 to 10 x 104 cells were isolated from one ml bone marrow.

CTEFs
Thymus glands were obtained from children undergoing corrective cardiac surgery who had portions of their thymus removed as part of the surgical process. CTEF cultures were established by an explant technique and subcultured as previously described [22-26]. Three to five grams of thymus were obtained, the thymic capsule removed, and the tissue minced into one mm fragments and agitated gently in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, BRL, Grand Island, NY) supplemented with 5% fetal calf serum (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) [22]. The CTEF were incubated on sterile gelfoam sponges, 1 cm x 3 cm, in 6 cm Petri dishes in 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, 1 µg/ml gentamicin, 50 µg/ml streptomycin and 2 µg/ml amphotericin (complete medium) at 37°C in a 5% CO2 atmosphere for two weeks. Subsequently, the CTEF were transferred to 24-well transwell culture plates (Nunc, Naperville, IL) and incubated for three to seven days in 67% DMEM and 33% Ham's medium supplemented with 5% FCS, 0.4 µg/ml hydrocortisone (Calbiochem Behring, La Jolla, CA), 11 ng/ml epidermal growth factor (Collaborative Research, Bedford, MA), 1 x 10–10 M cholera enterotoxin (Sigma), 5 µg/ml insulin (Sigma), 1.8 x 10–4 M adenine, 10–4 g/ml sodium pyruvate, 50 µg/ml gentamycin and 0.2 µg/ml fungizone.

Coculture of CTEF and SCs
The CTEF cultures were seeded with SCs by infusion of 5 to 10 x 104 SCs onto the surface of the CTEF in 24-well transwell plates in Iscove's/Ham's medium at a 1:1 ratio containing 3% FCS, 10% serum-free media substitute HL-1 (Ventrex, Irvine, CA), 25 U/ml of interleukin 2 (IL-2) at 37°C in a 5% CO2 atmosphere. Fresh medium was replaced every four days. Cord blood T cell conditioned media (CB-TCM), prepared as previously described [24], was added to parallel SC-CTEF cocultures. The CB-TCM was found to contain stem cell factor, IL-7, IL-3, IL-1b, transforming growth factor ß (TGF-ß), and GM-CSF-ß by immunoblot analysis (data not shown).

Detection of T Cell Phenotypes
T cell surface phenotypes of the differentiated SC were determined by reacting mAbs conjugated with fluorescein (FITC) and analyzed by flow cytometry [19, 20]. Cells were aspirated from the culture wells, washed, resuspended in RPMI containing 5% FCS and then subjected to Ficoll-Hypaque density centrifugation to remove dead cells. The mononuclear cells isolated from the interface were washed, resuspended in phosphate-buffered saline (PBS) containing 5% FCS and mixed with optimal concentrations of mAb. Ten to 20 x 106 cells were stained with mAb and 5000 events recorded. Negative controls (murine IgG1-FITC; Becton Dickinson, San Jose, CA) emitted only a small percentage of positive fluorescence and were used to set the positive gate. The lymphocyte region was selected by CD45 gating for the lymphoid population. FITC, phycoerythrin (PE) and peridinin chlorophyll protein (Per-CP) fluorescence was measured using the appropriate bandpass filters in a FACScan flow cytometer (Becton Dickinson) and the data analyzed using WinList software (Verity, Topsham, ME). mAbs used included: CD45, CD14, CD34, CD44, CD7, CD2, CD3, CD4, CD8, T cell receptor for antigen (TCR){alpha}ß, and TCR{gamma}{delta} obtained from Becton Dickinson.

In order to ensure that the SC-CTEF T cells originated from the donor SC population and not the cultured thymic fragment, two methods were employed: HLA typing and labeling the SC with a cytoplasmic immunofluorescence probe. HLA-A phenotype analysis was performed in the HLA Laboratory (kindly performed by Dr. Graff) using standard HLA typing techniques on peripheral blood lymphocytes obtained from the SC and CTEF donors, which were compared to the T cells generated from the SC-CTEF cocultures. In the second method, the SCs were labeled with 5-(and -6)-(((4-chloromethyl)benzoyl)amino) tetra-methyl-rhodamine (CMTMR; Molecular Probes, Inc., Eugene, OR) prior to coculture with the CTEF. CMTMR is retained in living cells through several generations and is not transferred to adjacent cells. Fluorescence of CMTMR within the cells of the cell suspension from the SC-CTEF cocultures was measured at 585 nm (FL2) by flow cytometry. These two methodologies demonstrated that the SC-CTEF T cells arose from the stem cell donor. Fluorescence of CMTMR precluded use of dual and triple labeling.

Lymphoproliferative Responses to Phytohemagglutinin (PHA)
The purpose of these studies was to determine whether differentiated SC-CTEF T cells have acquired the capacity of mature T cells to proliferate when nonspecifically stimulated with PHA. T cell proliferation to PHA stimulation is dependent on antigen-presenting cells (APCs), but is not HLA-restricted. Parallel studies were performed using APCs of stem cell and thymus donors to determine whether the newly differentiated T cells cooperated optimally with APCs of the stem cell or thymic donors. B cells, transformed with Epstein Barr virus (EBV-B), served as APCs as previously described [27-29]. Five x 105 CTEF T cells cocultured with 20% B cells derived from either the stem cell or thymic donor were stimulated with PHA for three days at 37°C in a 5% CO2 humidified atmosphere [28, 29]. Tritiated thymidine 1 µCi (ICN) was added to each well for the final 18 h of culture. The cultures were harvested onto glass filter paper and the tritiated-thymidine incorporated into DNA was counted in a Beckman beta scintillation counter. Data were expressed as geometric mean net cpm of stimulated (E) minus unstimulated (C) cultures and as stimulation index (SI) calculated as E divided by C.

Mixed Lymphocyte Cultures (MLC)
To evaluate whether the SC-CTEF-derived T cells were tolerant to allogeneic thymic HLA antigens, a one-way MLC to EBV-B cells from the thymic donor as the target cells was performed [27]. Previous studies in mice and humans have demonstrated that T cells produced in the allogeneic thymus were tolerant to thymic HLA antigens. As a positive control, MLC reactivity to pooled B cells was simultaneously performed. CTEF T cells 0.5 x 105 cells were cultured with mitomycin C-treated EBV-B cells 0.5 x 105 obtained from the stem cell, thymic or pooled B cell donors for six days at 37°C in a 5% CO2 atmosphere. Tritiated thymidine 1 µCi (ICN) was added to each well for the final 18 h of culture. The cultures were harvested onto glass filter paper and the tritiated-thymidine incorporated into DNA was counted in a Beckman beta scintillation counter.

Statistics
T cell phenotype data were expressed as mean ± SD and lymphoproliferative responses were expressed as geometric mean times/divide (x/÷) SE. Comparison of group differences was performed by Student's paired t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
MLCs
 Discussion
 References
 
Characterization of SCs
As shown in Table 1Go, prior to the addition of AET-SRBC depletion of CD2+ cells in the separation process (patients 1 and 3), CD3+ T cells were found to significantly contaminate the CD34+ cell preparation. Subsequently, this step was added and CD3+ T cells in the CD34+ enriched bone marrow cell preparations ranged from 0% to 4%. In five of the CD34+ enriched cell preparations, CD3+CD4+TCR{alpha}ß+ T cells were not detectable and were <1% in Patient 9. The SCs were enriched to 97.1% ± 4.0% (Table 1Go). SCs comprised 11.1% ± 7.9% of the bone marrow mononuclear cells obtained after Ficoll-Hypaque density centrifugation (Table 1Go). This is higher than reported to be isolated from normal individuals, which is reported to range between 1% to 3% [30-32].


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  		Table 1. Recovery of CD34+ cells in bone marrow of HIV-seropositive hemophiliacs
 
The enriched SCs represented a heterogeneous population as demonstrated by flow cytometry (Fig. 1Go). Gating on forward and side scatter, the R1 region defined small resting cells with little cytoplasmic granularity and contained approximately 52% of the CD34+ cells. The R2 region, containing larger cells with more cytoplasmic granularity and usually considered dividing cells, contained approximately 38% of the CD34+ cells. In both the R1 and R2 regions, nearly all the cells were CD34+, 98.3% and 98.6%, respectively, in this representative example (Fig. 1Go).



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  		Fig. 1. Isolation of bone marrow CD34+ SCs. Flow cytometry analysis of purified SCs using light scatter properties revealed a heterogeneous population. CD34+ cells represented 95% and 98% of the cells in gates R1 and in R2, respectively.

 
T Cell Differentiation of SCs
Enriched SC preparations when cocultured in CTEFs differentiated/matured into T cells as assessed by the acquisition of the T cell surface molecules CD7, CD2, CD3, CD4, CD8, TCR{alpha}ß and TCR{gamma}{delta} (Table 2Go). Mononuclear cells obtained from the SC-CTEF cocultures were predominantly CD45+ and were within the lymphocyte region as demonstrated by flow cytometry (Fig. 2Go). The majority of the T cells were found in the R1 region. Results of SC-CTEF coculture T cell phenotype analysis reported was made on the lymphoid R1 region, as illustrated for Patient #6 in Figure 2Go.


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  		Table 2. Peak T cell differentiation of CD34+ SCs fom HIV-seropositive hemophiliacs in CTEF cocultures
 


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  		Fig. 2. Flow cytometry of differentiated T cells from SC obtained from a HIV-seropositive hemophiliac (Patient #6) and CTEF coculture at 10 days of coculture. Forward and side scatter analysis of the SC-CTEF T cells revealed a heterogeneous population with most cells contained in the lymphoid gate R1. Gating on the lymphoid cells within R1 showed that cells were CD45+CD14– cells. The majority of cells were CD7+, CD2+, CD3+, CD4+ and TCR{alpha}ß+ with a minor population of CD8+ and no TCR{gamma}{delta}+.

 
The time course of T cell differentiation/maturation was variable. Sampling cultures for phenotype analysis was attempted weekly; although due to limited culture cell number, this was not always possible. Using the available data, maximum expression of T cell phenotypes of CD7+, CD2+, CD3+, CD4+, CD8+ and TCR{alpha}ß+ T cells appeared to peak between 17 to 28 days with a mean of 28 ± 16 days in most experiments, as seen for Patients 1, 7, 8, 10 and 11. However, T cells were detected as early as 6 to 10 days for Patients 6, 7, 8, 9 and 11. In Patients 3, 5 and 12, peak T cell differentiation occurred later at 45 days. CD4+ and TCR{alpha}ß+ T cells predominated, though there were smaller populations of CD8+ and TCR{gamma}{delta}+ T cells. The peak mean SC-CTEF T cell expression was CD44+, 28%; CD7+, 54%; CD2+, 56%; CD3+, 65%; CD4+, 32%; CD8+, 15%; TCR{alpha}ß+, 39% and TCR{gamma}{delta}+, 9% phenotypes (Table 2Go).

Though CTEF were seeded with SC which were >95% CD34+, the CD34+ cells represented only a small fraction of the cells after coculture in the thymic epithelial fragments after 6 to 10 days (Table 2Go). CTEF cultures which were not seeded with SC had <1% expression of any of the thymocyte/T cell surface markers (data not shown), significantly different compared to SC-CTEF cocultures. Similarly, enriched CD34+ cell preparations when cultured in media alone did not reveal any CD2+CD4+TCR{alpha}ß+ T cell phenotypes (data not shown). Dual labeling was not performed in these experiments due to the fluorescence of the CMTMR dye. However, the ratio of (CD4+ + CD8+)/CD3+ was >1.5 in several studies, suggesting the presence of dual positive CD4+CD8+ thymocytes. To confirm this, Patient #11 was repeated and the SC were not prelabeled with CMTMR. In this experiment, cells harvested from the SC-CTEF coculture revealed 58.9% CD4+CD8+ double positive thymocytes and 59.6% CD3+CD4+CD8+ triple positive thymocytes (Fig. 3Go). There were 21.0% CD3+CD4+ and 7.1% CD3+CD8+ single positive T cells. This study indicated thymic differentiation/maturation of SCs and not outgrowth of contaminating T cells.



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  		Fig. 3. Flow cytometry of differentiated thymocytes from SC obtained from Patient #11 and CTEF coculture. Double positive CD4+CD8+ thymocytes were 58.9% and triple positive CD3+CD4+CD8+ thymocytes were 59.7% at 12 days of coculture. Single positive CD3+CD4+ T cells were 21.0% and CD3+CD8+ T cells were 7.1%.

 
The origin of the differentiated T cells from the SC-CTEF cocultures was determined by HLA-A typing. HLA-A phenotype of the differentiated T cells was the same as the SC donor and disparate from the CTEF donor. In addition, a fluorescent cytoplasmic probe, CMTMR, was added to the SC prior to SC-CTEF coculture. Analysis of the differentiated T cells demonstrated the presence of the fluorescent probe by flow cytometry analysis in each case (Fig. 3Go). Furthermore, based on the fluorescent intensity decrease, the probe could be detected in a subpopulation of cells through eight divisions (mean channel peak 12; arrow in Fig. 3Go), indicating that at least for some cells, many cell divisions had occurred in the coculture, whereas the majority of cells had divided once (mean channel peak 500; arrow head Fig. 4Go).



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  		Fig. 4. CD34+ SCs were labeled with CMTMR and then cocultured with the CTEFs. Fluorescence of the T cells from the CTEF cultures was measured at 585 nm for CMTMR and at 530 nm for CD34 and TCR{alpha}ß by two-color flow cytometry analysis using FL2 and FL1 channels, respectively, of the single-cell suspension from the SC-CTEF cocultures. The top panels illustrate that at the time of initiation of the coculture, the CD34+ cells represented 94% of purified bone marrow-derived SC within the R1 gate, and that 100% of the cells were labeled with the CMTMR probe. After eight days of coculture, SC-CTEF T cells showed that the CD34+ cells had decreased to 39% (middle panels), and CMTMR fluorescence was detected with diminished intensity in CD34+ cells with repeated cell divisions. Cells dividing the least had a mean channel 500 (arrow head) and cells dividing the most had a mean channel 12 (arrow). The lower panels demonstrated that 42% of SC-CTEF T cells were TCR{alpha}ß+, and that the CMTMR fluorescence was detected in TCR{alpha}ß+ T cells.

 
Lymphoproliferative Responses
As shown in Table 3Go, T cells obtained from SC of HIV+ hemophiliacs which had been cocultured with CTEFs were assayed at multiple times for PHA stimulation in order to obtain the period of maximal stimulation. The time course of optimal PHA lymphoproliferative responses paralleled expression of mature T cell surface molecules. In paired experiments, responses to PHA stimulation were observed when APCs derived from either the stem cell or thymic donors were used. However, maximal response to PHA stimulation was significantly increased with APC of the stem cell donors compared to APC of thymus donors, geometric mean of 11530 x/÷ 1.4 (SI 9.3 x/÷ 1.3) versus 1553 x/÷ 4.7 net cpm (SI 3.1 x/÷ 1.6) (p < 0.03) (Table 3Go).


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  		Table 3. Lymphoproliferative responses to PHA stimulation by T cells obtained from SCs from HIV-seropositive hemophiliacs' CTEF cocultures
 

   MLCs
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 MLCs
Discussion
 References
 
To examine the acquisition of HLA restriction and tolerance, SC-CTEF T cells were examined for MLC reactivity to B cell HLA antigens from both thymic epithelia and stem cell donors. As seen in Table 4Go, T cells obtained from the stem cell and thymic epithelial cocultures responded to allogeneic HLA antigens of pooled B cells, geometric mean net cpm of 6096 x/÷ 1.6 (SI 8.5 x/÷ 1.3). In paired cultures, these T cells failed to respond to HLA antigens of B cells from the thymus donor, geometric mean net cpm of 36 x/÷ 2.7 (SI 1.2 x/÷ 1.3) (p < 0.05) and had reduced reactivity to HLA antigens from B cells of the stem cell donor, geometric mean net cpm of 269 x/÷ 3.1 (SI 2.7 x/÷ 1.4) (p < 0.05). These SC-CTEF T cells demonstrated some self-alloreactivity to stem cell HLA antigens in four of nine patients. However, the response was decreased compared to the reactivity to pool B cell alloantigens in most patients. Furthermore, in three patients who were tested at a later time point, MLC response to B cells of the stem cell donor subsequently disappeared upon further duration of the SC-CTEF coculture (data not shown). These studies suggested that SCs gave rise to T cells and accessory cells populating the thymus which contributed to HLA restriction. To further evaluate this hypothesis, two HLA disparate donor populations of SCs were cultured in the same thymic epithelial fragment and MLC reactivity was then examined toward APCs from each stem cell donor (Table 5Go). In these experiments, SC-CTEF T cells responded to stimulation with HLA antigens of the pool B cells and did not respond to the thymus donor B cells as expected (Table 5Go). In addition, the chimeric SC-CTEF T cells also did not respond to stimulation with B cells of either stem cell donor in six of eight experiments. These studies suggested that HLA tolerance was being induced by cells originating from the stem cell donors as well as the thymic epithelial HLA antigens.


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  		Table 4. MLC reactivity by T cells obtained from CD34+ SCs from HIV-seropositive hemophiliacs in CTEF cocultures
 

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  		Table 5. MLC reactivity by chimeric T cells obtained from two CD34+ SC donors cultured in the same thymic epithelial fragment
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 MLCs
 Discussion
References
 
T cell immune enhancement or reconstitution will be necessary in patients with AIDS with severe T cell depletion to prevent recurrent infections with opportunistic microorganisms and the development of lymphoreticular malignancies. T cell lymphopenia in HIV infection is due to a number of mechanisms, probably including diminished regeneration of mature T cells from bone marrow progenitor cells. The etiology of this may include HIV infection or suppression of bone marrow progenitor cells [11-15], of thymocytes [6, 8] and/or of thymic epithelial cells [2-5].

In this study, enriched SC populations from bone marrow of HIV-infected hemophiliacs were capable of differentiating or maturing into T cells when cocultured in a normal allogeneic thymic environment. The total CD34+ cell population contains predominately lineage-committed cells, in addition to the primitive hematopoietic progenitor cell capable of multilineage differentiation and self-renewal [30-36]. Thus, maturation of a thymocyte precursor may have occurred in our thymic fragment cocultures. Indeed, Groh et al. [37] reported that thymocyte precursors were capable of maturing into mature CD3+TCR+ thymocytes when cocultured in the presence of IL-7 or IL-2. However, the predominant thymocyte phenotype in these studies was CD3+TCR{gamma}{delta}+, and the CD3+CD4+ T cells accounted for only a minor population (4%). They concluded that there was maturation from these thymocyte precursors. However, the predominant phenotype in our studies was CD3+CD4+TCR{alpha}ß+ thymocytes. Furthermore, SCs cultured in conditioned media in the absence of thymic fragments did not express CD2+CD4+TCR{alpha}ß+ T cells. In addition, we observed in an experiment, in which the cell marker was omitted, double triple positive and double positive thymocytes. Thus, thymocyte differentiation of SCs is likely; though maturation of committed precursors cannot be excluded from these studies. Recently, we have begun to replicate these studies using CD34+CD38– lineage negative SC and cultured thymic epithelia cocultures and observed double positive CD4+CD8+ and triple positive CD3+CD4+CD8+ thymocytes and single positive CD3+CD4+ T cells in the thymic epithelia tissue. When CD34+CD38– SC are cocultured with CTEFs, definite phenotypic changes are observed with maximal expression of mature single positive CD4+ T cells at three weeks culture (manuscript in preparation). The results of our study are similar to that reported by Barcena et al. [38]. In their study, they isolated CD34+CD38– lineage SC from fetal liver and cultured them with fetal thymus in vitro. However, in addition to T cells, their culture conditions also supported differentiation of B cells and monocytes, cells not observed in this study. The culture conditions are different and probably account for this disparity.

The results of our study support the hypothesis that T cell differentiation in HIV-positive individuals could occur following allogeneic thymic transplantations similar to that attempted in vivo in patients with AIDS [16-18]. However, in these in vivo studies, there was only transient increased T cell numbers and function observed. The reasons for failure of the thymus grafts were not clear. Although thymic graft rejection was postulated to be a possibility, neither Danner et al. [16] nor Dwyer et al. [17] observed evidence of graft rejection on thymic graft biopsy specimens. Thymic biopsy specimens revealed either the absence of thymic epithelial tissue with the presence of HIV-infected T cells in the graft site, or demonstrable thymic epithelial tissue with a multinucleated giant cell inflammatory response. These biopsy results suggest that thymic epithelial tissue was being damaged by a T cell-mediated inflammatory response due to HIV infection rather than being rejected. Supporting this hypothesis are in vitro studies that demonstrated that HIV-infected thymocytes and T cells induce thymic epithelial cell injury [4-6, 8]. Unfortunately, these cultured thymic epithelia transplants were performed prior to the use of antiretroviral therapy, which might have prevented the degeneration of the thymic tissue.

There is conflicting data regarding whether SCs are infected with HIV in patients with AIDS [11-15]. CD4 is found on a subpopulation of SCs and may indicate that a subpopulation of SCs are susceptible to infection with HIV [39]. In our study, the percentage of CD34+ cells recovered from the bone marrow mononuclear cell population was increased approximately 10-fold in these HIV-seropositive hemophiliacs compared to the values reported for normal individuals [30]. This increased CD34+ percentage may reflect a depletion of T cells with a relative increased percentage of SCs in the bone marrow. Our study indicates that HIV-infected hemophiliacs have adequate numbers of bone marrow SCs which were capable of differentiating into functional T cells in vitro (Table 2Go). Furthermore, differentiated T cells from the stem cell and thymic epithelia fragment cocultures were tolerant of thymic HLA antigens as expected. This further supports the observations that graft rejection was not mediated by newly differentiated T cells from the thymic graft [16, 17]. However, these studies do not eliminate the possibility that thymic graft rejection was mediated by preexisting mature T cells at the time of transplant. Our studies also suggested that HLA restriction or tolerance was also mediated via bone marrow-derived accessory cells. Thymocytes and accessory cells, such as dendritic cells, were largely eliminated by 2'-deoxyguanosine treatment, prior to seeding with SC; yet differentiated T cells generated were tolerant to B cells of the thymic donor. Van de Kerckhove et al. [40] reported that SC from fetal liver infused in conjunction with allogeneic thymic graft transplantations into severe combined immune deficiency mice differentiated into T cells and thymic dendritic cells. Furthermore, these T cells were found to be tolerant to thymic HLA antigens by clonal anergy and to self-antigens by clonal deletion. Though the mechanism of tolerance was not investigated in our studies, SC-CTEF T cells were observed to be tolerant of thymic HLA antigens and demonstrated reduced MLC reactivity to self-antigens. Furthermore, tolerance was observed to both stem cell donor HLA antigens when chimeric SC cultures were performed. However, MLC reactivity to self-stem cell donor HLA antigens observed in some cases may reflect incomplete clonal deletion. Paul et al. [41] probably observed this phenomena in a patient with complete DiGeorge anomaly who received a partially HLA-matched thymus transplantation. This patient developed a graft-versus-host reaction following transplantation. However, the T cells were of host origin, indicting that this was self-alloreactivity. In keeping with this idea, we observed that with increasing duration of SC-CTEF coculture, reactivity to self-antigens was lost in several experiments.

Thus, failure of previously cultured thymic epithelia transplantation attempts in patients with AIDS was probably mediated by HIV infection and not due to inadequate stem cell numbers or graft rejection. This would indicate that effective antiretroviral therapy would be necessary to protect the thymic graft, as well as protect newly differentiated thymocytes and T cells. Since conventional antiretroviral medications, such as zidovudine, are only partially effective, more effective therapy will be necessary. Recently, transfection of mature T cells and SC with mutant transdominant HIV genes has been proposed as an alternative modality to resist active HIV infection [42-44]. Based on the results of our studies, transfection of SCs with transdominant HIV genes in conjunction with cultured thymic epithelial transplantations would appear to be the next step in order to protect the thymocyte and monocyte lineages, as well as the thymic microenvironment in order to immune reconstitute patients with AIDS.

In summary, these studies indicate that HIV-seropositive hemophiliacs have increased percentages of bone marrow nonadherent SCs which are capable of differentiating and proliferating into mature and functional T cells when cocultured in a normal allogeneic thymic microenvironment. Furthermore, the differentiated T cells demonstrate tolerance and restriction for thymic and stem cell donor HLA antigens. However, alloreactivity to stem cell donor HLA antigens was sometimes observed. These studies suggest that autologous SC therapy, in conjunction with allogeneic thymic epithelial transplantations, might be a useful method for T cell immune reconstitution in patients with AIDS. However, protection from HIV infection of newly differentiated thymocytes and T cells will be necessary to protect the thymic graft.


   Acknowledgments
 
The authors express their sincere respect and gratitude to the patients who participated in this study. The authors thank Dr. Dennis O'Connor, who performed the bone marrow aspirates, to Dr. Glenn Pennington for thymus tissue and to Lisa Elam for assistance with the polymerase chain reaction analyses. The authors are also extremely appreciative to Dr. Richard Hong for technical advice.

These studies were partly supported by Greater St. Louis Hemophilia Foundation and Quantum Health Division.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 MLCs
 Discussion
 References
 

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Received August 30, 1995; accepted for publication October 3, 1995.




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