Thymocyte differentiation from lentivirus-marked CD34+ cells in infant and adult human thymus

Journal of Immunological Methods 245 (2000) 31–43 www.elsevier.nl / locate / jim

Thymocyte differentiation from lentivirus-marked CD34 1 cells in infant and adult human thymus Jay T. Evans a , Yukari Okamoto a , Daniel C. Douek a , Richard D. McFarland b , a a a, Joel Gatlin , Richard A. Koup , J. Victor Garcia * a

Division of Infectious Diseases Y9.206, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 -9113, USA b Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 -9072, USA Received 1 May 2000; received in revised form 28 June 2000; accepted 2 July 2000

Abstract Changes in thymic function and immune system homeostasis associated with HIV infection or chemotherapy have significant effects on the ability of patients to maintain a complete T cell receptor repertoire. Therefore, the development of in vitro systems to evaluate thymic function in children and adults may aid in the understanding of thymopoiesis and the development of new therapies to improve thymic output. Here we use a lentivirus-based gene transfer system to mark CD34 1 cells with EGFP and follow their differentiation into CD4 1 and CD8 1 single positive thymocytes in human thymic organ cultures. Lentivirus-marked cells entered the thymus and were detected in both the cortex and medulla. Pretreatment of the thymus with 2-deoxyguanosine depleted resident thymocytes and significantly increased the percentage of EGFP 1 thymocytes. High frequency gene transfer into CD34 1 cells and maintained expression throughout differentiation allows for the in vitro assessment of thymic function. In thymuses ranging in age from fetal to adult we observed EGFP 1 thymocytes at all stages of development suggesting that thymuses of all ages are capable of accepting new T cell progenitors and contributing to the maintenance of T cell homeostasis.  2000 Elsevier Science B.V. All rights reserved. Keywords: Thymus; Stem cells; Gene therapy; AIDS / HIV

1. Introduction Abbreviations: dGuo, 2-deoxyguanosine; DP, CD4 1 CD8 1 double positive cells; EGFP, enhanced green fluorescence protein; FTOC, fetal thymic organ culture; HAART, highly active antiretroviral therapy; MOI, multiplicity of infection; SCZ, subcapsular zone; SP, CD4 1 and CD8 1 single positive cells; TREC, T cell receptor rearrangement excision circles; VSV-G, vesicular stomatitis virus envelope glycoprotein *Corresponding author. Tel.: 11-214-648-9970; fax: 11-214648-0231. E-mail address: [email protected] (J.V. Garcia).

The human thymus serves as the predominant site for the development of precursor thymocytes into TCR-ab T cells (Picker and Siegelman, 1993). In normal individuals, the thymus is fully developed by 6 months of age; however, involution of the thymus with age is associated with decreased thymic function in adults (Steinmann, 1986; Douek et al., 1998). Removal of the thymus from adults has little to no

0022-1759 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-1759( 00 )00270-2

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effect on gross T cell function (Haynes et al., 1983), whereas removal of the thymus during the first year of life has been shown to decrease immune function and alter T cell subsets (Rubinstein et al., 1975; Brearley et al., 1987; Rocha et al., 1989). While thymic function may not be necessary after the first year of life in most individuals, there is significant thymocyte and T cell depletion that can occur as a result of chemotherapy and bone marrow transplant, or HIV infection after which the thymus may be required to play a role in complete immune recovery (Pantaleo et al., 1993; Mackall et al., 1995; Mackall and Gress, 1997). During HIV infection, there is a profound loss of CD4 1 cells which is thought to be due to direct (viral-mediated) and indirect cell death, and a failure to replace CD4 1 cells through the thymus (Grody et al., 1985; Pantaleo et al., 1993). Recently, we and others have shown that HIV infection decreases thymic output that can be restored following highly active anti-retroviral therapy (HAART) (Douek et al., 1998; Zhang et al., 1999). These studies relied on the measurement of T cell receptor rearrangement excision circles (TREC) to quantify thymic output. We have developed a lentivirus-based gene transfer system to analyze the development of hematopoietic precursor cells in the thymus. The advancements presented here describe a highly sensitive in vitro model system for the analysis of de novo thymopoiesis. Fetal thymic organ cultures (FTOC) have been used extensively to study thymopoiesis (Jenkinson and Anderson, 1994). In some cases, a piece of fetal liver is co-cultured with a piece of fetal thymus on a membrane that permits the exchange of nutrients while suspending the tissue on the surface of a liquid medium. In this system, the fetal liver provides a source of progenitor cells that can enter the thymus and differentiate into mature thymocytes. An alternative approach is to isolate CD34 1 cells from the fetal liver and incubate them with fetal thymus where they enter the thymic fragments and differentiate into all major subgroups of thymocytes including CD4 1 CD8 1 double positive (DP) as well as CD4 1 and CD8 1 single positive (SP) cells (Barcena et al., 1993, 1994). The identification of CD34 1 derived T cells in fetal thymic organ cultures can be greatly facilitated

by retroviral marking with an easily detectable marker such as enhanced green fluorescence protein (EGFP) (Verhasselt et al., 1998). However, currently available retrovirus vectors have two major shortcomings: (1) poor transduction of human CD34 1 cells, and (2) relatively low levels of marker expression in human cells (An et al., 1997; Verhasselt et al., 1998). We have recently described a lentivirus-based gene transfer system that efficiently transduces human CD34 1 cells (Douglas et al., 1999; Evans et al., 1999). Using this vector we have developed a rapid and sensitive method to track the differentiation of hematopoietic progenitor cells into mature SP thymocytes. Similar levels of thymopoiesis were observed in fetal, newborn, infant, and adult thymuses showing that thymuses of all ages are capable of accepting new T cell progenitors and producing mature thymocytes.

2. Materials and methods

2.1. Vector production and characterization The vectors used in this study were produced by co-transfecting 293T cells with the plasmids pRtatpEGFP, pBH10C 2 env 2 , and pLVSVG by calcium phosphate transfection as previously described (Douglas et al., 1999; Evans et al., 1999). The vector containing supernatant was collected, pooled, filtered through a 0.45-mm filter, and concentrated once by centrifugation at 100,0003g for 90 min. The viral pellet was resuspended in serum-free medium and stored at 2808C until used. Vector preparations were titered on HeLa cells as previously described (Evans et al., 1999). The titers of the viral preparations used in this study ranged from 6310 7 to 5310 8 infectious units / ml. Vector supernatants were tested for the presence of replication competent retroviruses using a CD4 1 Hut78 cell infection assay as previously described (Douglas et al., 1999; Evans et al., 1999). No replication competent retroviruses were detected in any of the vector preparations used. Vector preparations were incubated with neutralizing rabbit-antiVSV-G polyclonal antiserum (Lee Biomolecular Research Inc., San Diego, CA) for 30 min prior to transduction to rule out the presence of contaminat-

J.T. Evans et al. / Journal of Immunological Methods 245 (2000) 31 – 43

ing plasmid DNA or protein that might result in artificially elevated transduction efficiencies (pseudotransduction).

2.2. Fetal liver CD34 1 cell isolation and transduction CD34 1 cells were isolated from fetal livers obtained from Advanced Bioscience Resources (Alameda, CA). Livers were disrupted in RPMI supplemented with 10% FBS, 50 U / ml penicillin, 50 mg / ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 1 mg / ml collagenase / dispase and 0.5 U / ml DNAseI. Following disruption, the cells were filtered through a 70-mm membrane and the mononuclear cell fraction was isolated by ficoll separation. CD34 1 cells were purified by positive selection using the Miltenyi MACS CD34 1 cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotech, Germany). Purified CD34 1 cells were cultured in serum-free Iscove’s modified Dulbecco’s medium (IMDM) plus 1% bovine serum albumin, 5 mg / ml human insulin, 100 mg / ml human transferrin, 10 mg / ml low density lipoproteins, 0.1 mM bmercaptoethanol, 300 ng / ml SCF, 300 ng / ml Flt-3 ligand, 10 ng / ml IL-3 and 10 ng / ml IL-6 (R&D Systems, Minneapolis, MN). Transductions were performed on Retronectin (Takara Biomedicals, Japan) coated plates (20 mg / cm 2 ) at 0, 24, and 48 h post isolation using a multiplicity of infection (MOI) of 2 to 10. Transduced and mock transduced CD34 1 cells were either used immediately for FTOC or cryopreserved for use with infant and adult thymic organ cultures.

2.3. Thymic organ culture Fetal thymuses were obtained from Advanced Bioscience Resources (Alameda, CA) and were processed within 24 h of harvest. Postnatal thymuses were obtained from patients undergoing either repair of congenital cardiac abnormalities with incidental partial thymectomy (newborn and infants) or thymectomy for myasthenia gravis (adult) (Children’s Medical Center or Parkland Memorial Hospital, Dallas, TX). Newborn, infant and adult thymuses were processed within 6 h of harvest. Thymuses were cut into |2-mm 3 pieces and cultured on 0.4-mm mem-

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branes suspended in RPMI medium (supplemented with 10% FBS, 50 U / ml penicillin, 50 mg / ml streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate). For depletion studies, the culture medium was supplemented with 0.06, 0.3 or 1.5 mM 2deoxyguanosine (dGuo). Thymus fragments were disrupted at 3, 5 and 7 days after the addition of dGuo and analyzed by flow cytometry for DP cells and for the apoptosis marker Annexin V (Pharmigen, San Diego, CA). For thymic reconstitution with fetal liver CD34 1 cells, thymuses were cultured with or without 0.3 mM dGuo for 72 h and washed for 3 to 4 h with fresh medium to remove residual drug. RtatpEGFP or mock transduced CD34 1 cells (3310 4 cells / fragment) were extensively washed to remove any remaining vector particles and added to thymus fragments. The thymus fragments were cultured for 6–7 days after the addition of CD34 1 cells and disrupted for flow cytometry analysis. For infant and adult thymuses, cryopreserved RtatpEGFP transduced and mock transduced CD34 1 cells were used. Frozen CD34 1 cells were thawed and cultured for 12–18 h in serum-free Iscove’s modified Dulbecco’s medium (IMDM) plus 1% bovine serum albumin, 5 mg / ml human insulin, 100 mg / ml human transferrin, 10 mg / ml low density lipoproteins, 0.1 mM bmercaptoethanol, 300 ng / ml SCF, 300 ng / ml Flt-3 ligand, 10 ng / ml IL-3 and 10 ng / ml IL-6 (R&D Systems, Minneapolis, MN) prior to co-culture with thymus fragments.

2.4. Immunohistochemistry and confocal laser scanning microscopy Fetal thymic fragments were incubated with or without 0.3 mM dGuo for 3 days to deplete resident thymocytes. Thymic fragments were washed to remove residual dGuo and cultured with mock or RtatpEGFP transduced CD34 1 cells. Six days after the addition of CD34 1 cells, individual thymic organ culture wells were harvested, fragments mounted whole in tissue freezing medium (Triangle Biomedical Sciences, Durham, NC), and 5-mm serial cryosections cut. Sections were immediately fixed for 5 min in ice-cold 1% paraformaldehyde in PBS, and then for an additional 5 min in ice-cold methanol. Sections were stored in the dark at 2808C until staining. For immunofluorescent staining, sections

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were air dried and then incubated with the rabbit anti-keratin antibody A575 (Dako) which stains human thymic medullary epithelium strongly, but cortical epithelium weakly, as previously described (Douek et al., 1996). Sections were then washed and incubated with human Ig-adsorbed Texas Red-conjugated (Fab)2 anti-rabbit Ig second layer antibody (Jackson ImmunoResearch, West Grove, PA). Sections were washed, mounted and visualized by confocal laser scanning microscopy (Leica, Heidelberg, Germany). Negative control staining with nonspecific rabbit Ig produced no specific fluorescence signal (data not shown).

2.5. TREC assay Fetal thymic organ cultures were depleted with 0.3 mM dGuo and cultured with mock or RtatpEGFP transduced CD34 1 cells as described above. After disruption, the cells were stained with mouse antiCD3-APC (Clone SK6, Becton Dickinson, San Jose, CA). Cells from mock thymic fragments were sorted for CD3 1 cells using a FACStar cell sorter (Becton Dickinson). Cells from thymuses receiving EGFP 1 CD34 1 cells were sorted for EGFP 1 cells (total) or EGFP 1 CD3 low / 1 thymocytes. Sorted and non-sorted cells from mock and EGFP 1 thymuses were assayed for TREC levels by real-time quantitative-PCR using the 59-nuclease (TaqMan) assay with an ABI7700 system (Perkin-Elmer, Norwalk, CT). Cells were lysed in 100 mg / ml proteinase K (Boehringer, Indianapolis, IN) for 1 h at 568C, then 10 min at 958C. Real-time quantitative-PCR was performed on 5 ml of cell lysate with the primers: 59-cacatccctttcaaccatgct-39 and 59-gccagctgcagggtttagg-39, and probe FAMacacctctggtttttgtaaaggtgcccact-TAMRA (MegaBases, Chicago, IL). PCR reactions contained 0.5 U Platinum taq polymerase (Gibco, Grand Island, NY), 3.5 mM MgCl 2 , 0.2 mM dNTPs, 500 nM each primer, 150 nM probe and Blue-636 reference (MegaBases, Chicago, IL). Amplification conditions were 958 for 5 min then 958 for 30 s, and 608 for 1 min for 40 cycles. A standard curve was plotted and TREC values for samples were calculated using the ABI7700 software. Samples were analyzed in duplicate.

2.6. Flow cytometry Samples processed for flow cytometry were stained with one or a combination of the following directly labeled mouse monoclonal antibodies or similarly labeled isotype controls: anti-CD34 (clone My10), anti-CD3 (clone SK6), anti-CD4 (clone SK3), anti-CD8 (clone SK1) (Becton Dickinson). Stained samples were fixed in PBS plus 1% paraformaldehyde and analyzed by flow cytometry within 4 h. Annexin V staining was done according to manufacturers instructions (Pharmigen). Four-color flow cytometry was performed using a FACSCalibur flow cytometer and the data were analyzed using the CellQuest software package (Becton Dickinson). When using the optimal emission wavelength of 530 nm (FL1) for EGFP expression, RtatpEGFP transduced cells were consistently off scale when calibrated against the autofluorescence of mock (untransduced) cells. This made it necessary to use an alternate emission wavelength of 575 nm (FL2) for our analysis of EGFP expression (Evans et al., 1999).

3. Results

3.1. Purification and transduction of CD34 1 cells Fetal hematopoietic stem and progenitor cells reside primarily in the liver until 22–24 weeks of gestation when they migrate to the bone marrow. The relative abundance of CD34 1 cells in fetal liver makes this source of cells ideal for marking studies in thymic organ culture. Fetal liver CD34 1 cells were purified by disrupting liver tissue in a collagenase / dispase / DNAse mixture followed by mononuclear cell isolation (1–4310 8 cells). Positive selection for CD34 1 cells resulted in the recovery of 1–3310 7 cells which were .90% CD34 1 (Fig. 1a) and contained undetectable levels of contaminating CD4 1 or CD8 1 cells (Fig. 1a). The high level of CD34 1 cell purity is essential for these marking studies because the presence of any contaminating T cells may obscure the identification of marked thymocytes derived from the input CD34 1 cells. Transductions of CD34 1 cells were performed within 2 h of isolation without any prior expansion

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Fig. 1. EGFP expression in fetal liver CD34 1 cells. (a) Flow cytometry analysis and characterization of fetal liver CD34 1 cells. Freshly isolated CD34 1 cells were stained with mouse monoclonal isotype control (left); mouse anti-CD34-FITC (center); or double stained with mouse anti-CD8-FITC and mouse anti-CD4-PE (right). (b) Analysis of EGFP expression in untransduced CD34 1 cells stained with mouse monoclonal isotype control (left) or mouse anti-CD34-PerCP (center) and RtatpEGFP transduced CD34 1 cells stained with anti-CD34PerCP (right).

or activation. CD34 1 cells were transduced three times with the RtatpEGFP vector and analyzed by flow cytometry for EGFP and CD34 expression. Cell surface phenotype analysis of these cultures indicated that 81% of the cells maintained their CD34 1 phenotype during this 4–5-day period (Fig. 1b) and 74% of the CD34 1 cells expressed EGFP. The high levels of fetal liver CD34 1 cell transduction demonstrates the utility of lentivirus-based vectors for the efficient transduction of human hematopoietic cells under conditions that maintain their CD34 1 phenotype. The presence of contaminating plasmid DNA or marker protein in vector preparations may result in artificially elevated transduction efficiencies (pseudotransduction) and therefore, lower levels of marked thymocytes in organ cultures. The RtatpEGFP vector used in these studies was pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) and transduction can be inhibited with neutralizing antibodies against VSV-G. Therefore,

pseudotransduction was ruled out by incubating the vector with neutralizing rabbit-anti-VSV-G polyclonal antiserum for 30 min prior to transduction. Flow cytometric analysis showed that incubation with rabbit polyclonal antiserum against VSV-G reduced the transduction efficiency by .95% (data not shown). These results indicate that the high level of EGFP expression seen in RtatpEGFP transduced cells was the result of vector transduction and not pseudotransduction from protein or plasmid DNA contamination in the vector preparations.

3.2. EGFP 1 CD34 1 cells differentiate into CD3 1 thymocytes within the fetal thymus To demonstrate that the transduced CD34 1 cells can differentiate into thymocytes while maintaining high levels of transgene expression, EGFP 1 CD34 1 cells were washed to remove remaining vector particles and added to FTOC under conditions that allow CD34 1 cell differentiation. Six days after the

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addition of CD34 1 cells to the thymus, samples were disaggregated and the total percentage of EGFPexpressing cells in the thymus was determined by flow cytometry (Fig. 2 and Table 1). In addition, cells from the FTOC were stained for T cell surface markers to determine the percent of thymocytes generated from the input CD34 1 cells and their phenotypic distribution along the differentiation

pathway. Our results show that when EGFP 1 CD34 1 cells were cultured with fetal thymus for 6 days, 16% of the total cells were EGFP 1 and 7% of the CD3 1 cells express EGFP (Fig. 2a). In addition, EGFP-expressing thymocytes were detected at all stages of development including early CD4 low CD3 low and more mature DP and SP thymocytes (Table 1 and Fig. 2b, respectively).

Fig. 2. Development of EGFP 1 thymocytes in FTOC after the addition of exogenous EGFP 1 CD34 1 cells. Analysis of FTOC 6 days after the addition of mock or EGFP 1 CD34 1 cells. (a) EGFP expression in total live cells and CD3 1 cells. (b) FTOC derived cells stained with mouse anti-CD8-PerCP and mouse anti-CD4-APC (left); analysis of EGFP expression in gates R2 (CD4 1 CD8 1 double positive cells) and R4 (CD4 1 single positive cells) (right). Markers indicate percentage of EGFP positive cells in each histogram.

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Table 1 EGFP expression in FTOC 6 days after the addition of EGFP 1 fetal liver CD34 1 cells Age of thymus

Fetal liver EGFP 1 a

0.3 mM dGuo b

Fetal (18 weeks) Fetal (19 weeks) 5 Months

80%

3 Years

72%

20 Years

72%

2 1 2 1 2 1 2 1 2 1

85% 72%

Fetal thymic organ culture c Live cells

CD4 low CD3 low

CD3 1

CD4 1 CD8 1

CD4 1 CD8 2

CD4 2 CD8 1

16% 38% 13% 34% 4% 9% 8% 12% 6% 12%

17% 32% 17% 29% 15% 34% 14% 34% ND d

7% 24% 4% 11% 4% 6% 3% 6% ND

5% 13% 3% 14% 6% 10% 3% 13% 12% 16%

4% 8% 3% 6% 7% 13% 11% 9% 5% 15%

4% 13% 4% 11% 2% 1% 1% 2% 2% 7%

CD34 1 cells were isolated as described in Section 2 and transduced (MOI of 2–10) three times with RtatpEGFP. The table indicates percentage of cells that are EGFP positive by FACS analysis 24 h after the last transduction. b Thymus fragments were incubated for 3 days with (1) or without (2) 0.3 mM dGuo prior to the addition of CD34 1 cells. c FACS analysis for cell surface phenotype and EGFP expression was performed 6–7 days after the addition of CD34 1 cells to the thymus. The table indicates the percentage of EGFP positive cells in each cell subset. d Not done due to limiting amounts of thymus tissue. a

These results show that lentivirus vector-transduced CD34 1 cells expressing EGFP can differentiate into thymocytes and that this model system closely recapitulates the major stages of thymocyte differentiation.

3.3. Depletion of human thymocytes using 2 deoxyguanosine (dGuo) and reconstitution with EGFP 1 CD34 1 cells A complication often encountered when evaluating thymic function using exogenous CD34 1 cells in human FTOC is the low levels of thymocytes produced from the input CD34 1 cells. As indicated above, even with an input of .80% EGFP positive cells only a fraction of the thymocytes obtained from the organ culture were derived from EGFP 1 cells. In a system with high intrinsic variability such as FTOC, low levels of CD34 1 -derived thymocytes represent a significant complication. Human and mouse thymus fragments can be depleted of thymocytes with dGuo (Jenkinson et al., 1982; Markert et al., 1997). Depletion with dGuo creates a niche for immigrant thymocyte precursors increasing the percentage of new CD34 1 -derived thymocytes. However, a detailed flow cytometric analysis of apoptosis and thymocyte depletion as a result of dGuo has not been described. Therefore the in vitro

effect of dGuo on the viability of thymocytes in human thymic organ culture was evaluated (Fig. 3). Our data show that dGuo induces thymocyte depletion in a time and concentration dependent manner (Fig. 3). Annexin V staining showed a direct correlation between dGuo concentration and apoptosis in dGuo-treated thymuses (Fig. 3a). Significant increases in apoptosis were observed at all concentrations of dGuo tested (0.06 to 1.5 mM). In addition, the increased levels of apoptosis in dGuotreated thymuses correlated with a decrease in DP thymocytes (Fig. 3b). The highest levels of apoptosis and thymocyte depletion were observed with 0.3 mM dGuo 5 days after the addition of dGuo. Treatment with dGuo beyond 5 days showed a decrease in Annexin V staining and little increase in the depletion of DP cells indicating that most of the dGuo sensitive cells had been depleted by day 5 (Fig. 3). To determine if the pretreatment of human thymus fragments with dGuo would increase the number of CD34 1 -derived T cell progenitors in the thymus, EGFP 1 CD34 1 cells were added to fetal thymus fragments pretreated with 0.3 mM dGuo as described above. Our results show a 2–3-fold increase in the number of thymocytes derived from the exogenous CD34 1 cells in preconditioned thymuses (Fig. 4 and Table 1). Significant increases can be seen in total EGFP expressing cells, immature CD4 low CD3 low

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Fig. 3. Depletion of endogenous thymocytes by dGuo. Flow cytometry analysis of thymus fragments from a 12-month-old patient. Thymus was cut into 2-mm 3 pieces and cultured with 0.06 mM, 0.3 mM, or 1.5 mM dGuo for 7 days. At the indicated times (0, 3, 5 and 7 days, x-axis) thymus fragments were disrupted and analyzed by flow cytometry for Annexin-V, CD4 and CD8 expression ( y axis). (a) Percentage of total cells expressing Annexin-V after the addition of dGuo. (b) Decrease in DP cells after the addition of dGuo.

thymocytes and more mature DP and SP thymocytes (Fig. 4 and Table 1). Immature thymocyte populations contained a higher percentage of EGFP 1 cells than the more mature SP thymocytes (Table 1). This result was expected due to the short incubation time (6 days) between the addition of CD34 1 cells and disruption for analysis.

3.4. Distribution of EGFP 1 cells in thymic cortex and medulla The thymus consists of distinct lobules that con-

tain three major compartments; the subcapsular zone (SCZ), cortex, and medulla (Picker and Siegelman, 1993). Each of these compartments plays an important role in thymopoiesis. Immigrant T cell progenitors must enter the SCZ and migrate to the cortex, the primary site of TCR rearrangement. DP thymocytes undergo positive and negative selection as they move into the medulla and mature into SP thymocytes prior to release into the periphery (Picker and Siegelman, 1993). Immunohistochemistry was used to determine if lentivirus-marked T cell progenitors were entering the thymic fragments and migrating into both the cortex and medulla. For these experiments dGuo-depleted thymic fragments were incubated with EGFP 1 or mock CD34 1 cells, as described above, and co-cultured for 6 days prior to sectioning. Cortex and medulla can be readily distinguished by staining with anti-keratin antibody A575 which stains human thymic medullary epithelium strongly, but cortical epithelium weakly (Douek et al., 1996). EGFP cells were easily identified in sections when comparing mock and EGFP 1 sections (Fig. 5 panels a and b, respectively). Closer examination of the sections containing EGFP 1 cells identified lentivirus-marked cells in both the medulla (panel c) and cortex (panel d) showing clearly that marked cells were capable of migrating into the thymus and moving into these compartments. The majority of marked cells were found in the cortex rather than in the medulla consistent with our flow cytometry data showing higher percentages of marked immature CD3 low CD4 low and DP thymocytes relative to the more mature SP thymocytes (Table 1).

3.5. EGFP 1 CD3 1 cells contain high levels of TREC TCR rearrangement during thymopoiesis produces episomal DNA fragments representing excisional products of the TCR gene (Bogue and Roth, 1996; Kong et al., 1998). TCR rearrangement excision circles (TREC) are stable, episomal DNA fragments that are not duplicated during mitosis (Takeshita et al., 1989; Livak and Schatz, 1996). Therefore, higher TREC levels are observed in early thymocytes that have recently rearranged their TCR than in more mature thymocytes and T cells that have undergone multiple rounds of cell division. PCR can be used to quantify accurately the number of TREC in a

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Fig. 4. Differentiation of EGFP 1 CD34 1 cells into thymocytes in FTOC after depletion with dGuo. Flow cytometry analysis of FTOC pretreated with or without 0.3 mM dGuo prior to the addition of EGFP 1 CD34 1 cells. Panels show EGFP expression in total live cells (left), CD4 low CD3 low cells (center), and CD3 1 cells (right) from untreated thymus fragments (top) or dGuo-treated thymus fragments (bottom). Markers indicate the percentage of EGFP positive cells in each histogram.

population of cells (Douek et al., 1998; Kong et al., 1998). Table 2 shows TREC levels in thymocytes sorted for CD3 and / or EGFP as well as non-sorted cells from a representative fetal thymus 6 days after the addition of EGFP 1 or mock CD34 1 cells. The TREC levels in the unsorted thymocytes are in the normal range for thymuses in both the mock and EGFP samples (Douek et al., 1998; Jamieson et al., 1999), and thus represent the expected levels of TCR rearrangement. The TREC levels in sorted CD3 1 cells from the thymus fragments that received mock CD34 1 cells were lower than for the unsorted population because the more mature CD3 1 cells in the sorted population have undergone several rounds of replication and thus TREC were diluted. The EGFP 1 CD3 1 / low sorted cells derived from the thymus fragments receiving EGFP 1 CD34 1 cells have higher TREC than the unsorted cells. This is due to the fact that EGFP 1 CD3 1 / low thymocytes will include more of the immature CD3 low cells, since the fetal thymus was only in culture for 6 days after the addition of EGFP 1 CD34 1 cells. These immature cells contain higher levels of TREC since they have undergone fewer rounds of replication after TCR rearrangement. The EGFP 1 sorted cells have a low

level of TREC because that population is comprised primarily of myeloid cells derived from the original input CD34 1 cells (data not shown). The high levels of TREC observed in the EGFP 1 CD3 1 cells shows that lentivirus-transduced CD34 1 cells are capable of differentiating into thymocytes that have undergone TCR rearrangement.

3.6. Differentiation of mature thymocytes from EGFP 1 CD34 1 cells in fetal, newborn, infant, and adult thymic organ cultures To determine if age related involution of the thymus has a negative effect on thymic function, thymuses ranging in age from 18 weeks of gestation to 20 years old were evaluated for their ability to produce mature SP thymocytes from EGFP 1 CD34 1 cells. Thymuses were processed as indicated above and incubated with or without dGuo for 3 days prior to the addition of EGFP 1 or mock CD34 1 cells. The thymus fragments were cultured for 6 days, disrupted, and stained for thymocyte-specific markers. Pretreatment of fetal, newborn, infant and adult thymus fragments with dGuo resulted in similar increases (2–3-fold) in the percentage of both total

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Fig. 5. Analysis of EGFP 1 cells in the cortex and medulla of a dGuo depleted thymus fragment 6 days after the addition of EGFP 1 CD34 1 cells. Immunohistochemistry of fetal thymic fragments incubated with control or EGFP 1 CD34 1 cells for 6 days prior to mounting in tissue freezing medium and sectioning. Frozen sections were fixed and stained with a rabbit anti-keratin primary antibody and human Ig-adsorbed Texas Red-conjugated (Fab)2 anti-rabbit Ig secondary antibody. Panel a: medulla (m) and cortex (c) of a thymus fragment incubated with control CD34 1 cells. Panel b: medulla and cortex of thymus fragment incubated with EGFP 1 CD34 1 cells. Panel c: EGFP 1 cells in the medulla. Panel d: EGFP 1 cells in cortex / medulla junction indicated by box in panel b. Size bar550 mm is shown for panels a and b, 10 mm size bar is shown for panels c and d. Negative control staining with non-specific rabbit Ig produced no specific fluorescence signal (data not shown).

Table 2 TREC levels in FTOC 6 days after the addition of mock or EGFP 1 fetal liver CD34 1 cells Transduction a

Sorted b

TREC c

Mock Mock EGFP 1 EGFP 1 EGFP 1

No CD3 1 No EGFP 1 EGFP 1 / CD3

41,300 28,026 39,730 5589 49,440

a Control (mock) or EGFP 1 CD34 1 cells incubated with dGuotreated thymus fragments. b Thymus fragments were disrupted, stained with mouse antiCD3-APC and sorted for the indicated populations. c Numbers indicate TREC levels per 100,000 cells.

live cells and thymocytes expressing EGFP 1 (Table 1 and Fig. 6). There was a 2–4-fold decrease in the percentage of EGFP 1 live cells (total) between the fetal thymus fragments and the thymuses from older patients in both dGuo-treated and non-treated thymuses (Table 1). However, similar percentages of EGFP 1 thymocytes were observed in all thymuses tested (Table 1). The thymus from the oldest patient evaluated in this study (20 years old) contained in the highest percentage of EGFP expressing DP cells and was capable of producing SP thymocytes (Fig. 6 and Table 1). The production of DP and SP thymocytes in thymuses ranging in age from fetal to

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Fig. 6. Thymocyte development in adult thymus after the addition of EGFP 1 CD34 1 cells. Flow cytometric analysis of thymus fragments from a 20-year-old patient. Thymus fragments were pretreated with or without 0.3 mM dGuo prior to the addition of EGFP 1 CD34 1 cells. (Left) Dot plot analysis of CD8 and CD4 expression 6 days after the addition of CD34 1 cells. (Right) Histograms showing EGFP expression in total live cells, CD4 1 CD8 1 cells (gate R2), and CD4 single positive cells (gate R4) from untreated (top) or dGuo-treated (bottom) thymus fragments. Markers indicate the percentage of EGFP positive cells in each histogram.

adult confirms that adult thymuses are capable of maintaining thymocyte differentiation and maturation similar to that seen in fetal thymuses. Therefore, decreased thymic output with age is probably due to a quantitative rather than qualitative difference between young and old thymuses reflecting mainly a decrease in thymic size with age.

4. Discussion Recent advances in the ability to purify and transduce human CD34 1 cells combined with current technologies in FTOC has facilitated the development of sensitive and reliable methods for the study of thymic function. Using a readily detectable marker (EGFP) we were able to follow the development of T cell progenitors in the thymus and their differentiation into mature SP thymocytes. To increase the percentage of marked T cell progenitors, thymus fragments were pretreated with dGuo to partially deplete existing thymocytes and create a niche for incoming T cell progenitors. Early thymocytes are highly sensitive to this drug because of their ability to uptake and phosphorylate deoxyguanosine to deoxyGTP (Cohen et al., 1980). Incubation of thymic fragments with dGuo resulted in a time- and concentration-dependent increase in apoptosis and depletion of resident thymocytes.

Pretreatment of the thymus with dGuo resulted in a 2–3-fold increase in the number of marked immigrant thymocytes. The high levels of TREC found in the sorted EGFP 1 CD3 1 cells and the presence of marked cells in the cortex and medulla of the thymic fragments provides further evidence that this model system closely recapitulates all stages of thymopoiesis. During HIV infection there is a profound loss of CD4 1 cells due to direct and indirect cell death and to a failure to replace CD4 1 cells (Pantaleo et al., 1993). Progressive destruction of the thymus and other secondary lymphoid organs in HIV infection can have severe long-term consequences (Grody et al., 1985; Pantaleo et al., 1993). Recently we have shown that thymopoiesis plays a central role in T cell homeostasis in HIV-infected individuals (Douek et al., 1998). Even in adults where thymic function was previously thought to be absent, significant increases in T cell receptor excision circles (TREC) were detected following HAART (Douek et al., 1998; Zhang et al., 1999). While TREC levels generally decline with age, thymocyte differentiation has been detected in adults up to 56 years of age (oldest patient tested) (Jamieson et al., 1999). The data presented here shows conclusively that thymuses ranging in age from fetal to adult are capable of accepting new T cell progenitors and producing mature SP thymocytes, and correlates well with

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J.T. Evans et al. / Journal of Immunological Methods 245 (2000) 31 – 43

recently published work (Douek et al., 1998; Jamieson et al., 1999; Zhang et al., 1999; Sempowski et al., 2000). While the process of age-related thymic involution is well documented (Steinmann, 1986), the ability of residual thymic tissue to contribute to the generation ¨ T cells throughout life is not fully underof naıve stood. Our data suggest that there is no significant decrease in function of the remaining thymus tissue with increasing age. Therefore, we hypothesize that decreased thymic output with age is due to a quantitative rather than qualitative difference in the remaining thymus tissue. In cases of immune depletion due to HIV infection or chemotherapy, the ability of the thymus to regenerate a complete TCR repertoire is important for full immune system recovery. The use of highly sensitive and rapid methods for the analysis of thymic function will greatly assist in the understanding of factors that influence thymopoiesis and in the identification of new drugs, cytokines, and protocols to improve thymic output in these patients.

Acknowledgements We thank Dr. J. Douglas for the development of the RtatpEGFP vector and Dr. M. Emerman for the VSV-G expression vector. The technical assistance of A. Mobley and B. Darnell with flow cytometry and B. Hill with TREC assays is greatly appreciated. We are especially grateful to the Department of Neurology at UTSW and the Cardiac Surgery Service at Children’s Medical Center of Dallas for supplying adult and infant thymuses. We also thank Dr. L.J. Picker and Dr. D. Sodora for their critical reviews of this manuscript. This research was supported by NIH grants AI-39416 (J.V.G.) R37AI35522, R21-AI44758 and R01-AI42397 (R.A.K.) and Leukemia Society of America grant 6540-00 (D.C.D.). R.A.K. is an Elizabeth Glaser Scientist of the Pediatric AIDS Foundation.

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