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JOURNAL OF VIROLOGY, July 1998, p. 5852–5861 0022-538X/98/$04.0010 Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Vol. 72, No. 7

Contact with Thymic Epithelial Cells as a Prerequisite for Cytokine-Enhanced Human Immunodeficiency Virus Type 1 Replication in Thymocytes ˆ NE, MARIE-THE ´ RE ` SE NUGEYRE, JOSE ´ PHINE BRAUN, MAURICE ROTHE, LAURENT CHE ¨ ´ FRANC ¸ OISE BARRE-SINOUSSI, AND NICOLE ISRAEL* Unite´ de Biologie des Re´trovirus, Institut Pasteur, 75724 Paris Cedex 15, France Received 28 October 1997/Accepted 2 April 1998

We report here that human immunodeficiency virus type 1 (HIV-1)-infected human thymocytes, in the absence of any exogenous stimulus but cocultivated with autologous thymic epithelial cells (TEC), obtained shortly (3 days) after thymus excision produce a high and sustained level of HIV-1 particles. The levels and kinetics of HIV-1 replication were similar for seven distinct viral strains irrespective of their phenotypes and genotypes. Contact of thymocytes with TEC is a critical requirement for optimal viral replication. Rather than an inductive signal resulting from the contact itself, soluble factors produced in the mixed culture are responsible for this effect. Specifically, the synergistic effects of tumor necrosis factor, interleukin-1 (IL-1), IL-6, and granulocyte-macrophage colony-stimulating factor may account by themselves for the high level of HIV-1 replication in thymocytes observed in mixed cultures. In conclusion, the microenvironment generated by TEC-thymocyte interaction might greatly favor optimal HIV-1 replication in the thymus. The progressive and irreversible decline of CD41 T cells observed during human immunodeficiency virus type 1 (HIV-1) infection is clearly associated with a progressive increase of the plasma viral load (23, 39). This progression in viral load correlates with the virus dissemination in lymph nodes, resulting in the destruction of the architecture of this secondary lymphoid organ. In contrast, little is known about the dynamic of virus replication in primary lymphoid organs, which are the sites of T-cell development and regeneration. This is an important question, since HIV-1 infection of primary lymphoid organs, and particularly the thymuses of infants, might participate in the disruption of CD41 T-cell homeostasis by preventing the regeneration of these cells in vivo. The hypothesis of thymus infection was first based on the rapid progression to AIDS of some children infected by their mothers (9, 11). Histological studies of thymic organs from some of these HIV-1-infected children or from infected fetuses showed profound alterations of both the cortex and medulla, characterized by T-cell depletion and disorganization of the network of thymic epithelial cells (TEC) (35, 38). Similar observations were reported for thymic tissues from infected macaques or small animal models (SCID-hu mice), which also attest to the clear presence of virus particles, confirming that the thymus is a target of HIV-1 infection (33). The main target cells of the virus are the thymocytes at different stages of maturation, as shown in vitro (13, 40, 49) and in vivo with the SCID-hu mouse model (1, 42, 44). In the SCID-hu mouse model impairment of CD41 cell renewal in response to a high viral burden was demonstrated (52). Infection of stromal TEC was also shown in vitro but appears to be restricted to certain HIV-1 isolates (10). The destruction of the thymus architecture is reminiscent of that observed in lymph nodes and thus might similarly be linked to active replication of the virus within the tissue (26,

35). Therefore, in order to better understand AIDS pathogenesis, particularly in infants, it may be important to clarify how virus replication is controlled in thymocytes within the particular microenvironment of the thymic tissue. Thymocytes need an activation process to achieve HIV-1 replication (21, 45–47). Activation, proliferation, and maturation during their normal development are dependent upon a permanent crosstalk with stromal cells. Among the cells constituting the thymic stroma, both fibroblasts and TEC (2) were shown to be involved in this crosstalk. A physical contact between the maturating T cells and the TEC is thought to be important for T-cell development (3, 50). This crosstalk between thymocytes and TEC also involves secretion of many cytokines inducing activation and/or proliferation signals. We particularly focused our interest on interleukin-1b (IL-1b), IL-6, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), since these cytokines play a pivotal role both in T-cell development and in HIV-1 replication as shown with lymphocytic T cells or monocytic cells. Both TEC and thymocytes express the mRNAs for IL-1, IL-6, and TNF (53). However, TEC express considerably higher levels of IL-1b and IL-6 than thymocytes (30, 31). Production of IL-1b in the human thymus was shown to be activated by a specific contact between TEC and thymocytes (32). GM-CSF is produced mainly by TEC in the thymus (30). Both IL-1b and GM-CSF were demonstrated to specifically activate the proliferation of immature thymocytes (12). IL-6 was also reported to be a cofactor of proliferation of various subpopulations of thymocytes (20). TNF and IL-1a were shown to induce activation of immature thymocytes, leading to their differentiation in a thymus reconstitution assay (54). Furthermore, these different cytokines were shown to stimulate HIV-1 replication. TNF and IL-1b were demonstrated to enhance HIV-1 replication by directly increasing the transcription level through induction of NF-kB in T-cell lines (15, 24, 34). In resting circulating T lymphocytes, TNF and IL-1b instead act as cofactors to strengthen the long terminal repeat transactivation triggered by antigen recognition (22). IL-6 was specifically shown to enhance HIV-1 replication in monocytes/

* Corresponding author. Mailing address: Unite´ de Biologie des Re´trovirus, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: 33 1 4568 8944/8733. Fax: 33 1 4568 8957. E-mail: [email protected]. 5852

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macrophages through induction of the transcription factor NF–IL-6 (37). The role of GM-CSF in HIV-1 replication was demonstrated with chronically infected promonocytic cell lines (19) and primary mononuclear monocytes (29). Several reports showed that the positive role of GM-CSF in HIV-1 replication is related to its capacity to fully differentiate monocytic cells to macrophages (27). For a better understanding of the control of HIV-1 replication in thymocytes within the thymic environment, we therefore addressed the following questions: (i) do the TEC favor HIV-1 replication in thymocytes, which, unless they are activated, do not produce the virus in vitro, and (ii) what stimuli are implicated in such an interaction? Using a new procedure for enrichment of the human TEC population, we were able to set up an autologous mixed culture with thymocytes at times shortly after surgical excision of the thymus. We show that under these conditions, a cell-to-cell interaction is a requirement for promotion of a high and sustained level of HIV-1 replication in thymocytes. Furthermore, we provide evidence that TNF, IL-1, IL-6, and GM-CSF, produced in the mixed culture, act synergistically and account for the high level of HIV-1 replication in thymocytes. MATERIALS AND METHODS Reagents. (i) Antibodies. To characterize both TEC and thymocyte populations, monoclonal antibodies (MAbs) against CD1a (B17.20.9), CD2 (39C1.5), CD3 (X35), CD14 (RMO52), CD16 (3G8), CD19 (J4 119), CD45 (ALB12), cytokeratin (KL1), and vimentin (Vim3B4) were used. MAbs against immunoglobulin G1 (IgG1) (MARK1) and IgG2a (U7.27) were used as negative controls. All of these MAbs were purchased from Immunotech (Marseille, France), except Vim3B4 was from Boehringer-Mannheim (Meylan, France). A rabbit polyclonal serum against cytokeratin (BT571) from Biomedical Technologies Inc. (Stoughton, Mass.) was also used. Conjugated MAbs, including CD4-phycoerythrin (13B8.2), CD8-fluorescein isothiocyanate (CD8-FITC) (B9.11), IgG1phycoerythrin (679.1Mc7), and IgG1-FITC (679.1Mc7), were purchased from Immunotech. The procedure for isolation of TEC was with MAbs against CD3 (OKT3) (Ortho Diagnostic Systems Inc., Raritan, N.J.), against CD14 (Leu-M3) and CD45 (HLe-1) (Becton Dickinson & Co., San Jose, Calif.), and against thymic fibroblasts (1B10) (Valbiotech, Paris, France). Goat anti-rabbit–FITC and goat anti-mouse–FITC were purchased from Immunotech. Goat IgG against human GM-CSF and normal goat IgG were purchased from R & D Systems (Minneapolis, Minn.). Rabbit polyclonal serum against TNF was kindly provided by J. M. Cavaillon (Institut Pasteur, Paris, France). Normal rabbit polyclonal serum was obtained from F. Traincard (Institut Pasteur). IL-1 receptor antagonist (IL-1 ra) was purchased from R & D Systems. (ii) Cytokines. The human recombinant cytokines macrophage colony-stimulating factor (M-CSF), GM-CSF, IL-1b, and TNF were purchased from Genzyme Corp. (Cambridge, Mass.). IL-6, IL-3, and stem cell factor (SCF) were from R & D Systems. Immunostaining and cytofluorometry. Indirect immunostaining with antibodies raised against cytokeratin (KL1 or BT571) and vimentin (Vim3B4) was performed in Lab Tek chambers (Corning Costar Corp., Cambridge, Mass.) on cells made permeable by acetone-methanol treatment (10). Other direct or indirect immunostaining was analyzed by cytofluorometry with an EPICS ProFile II (Coulter). Cells and culture conditions. (i) Isolation of an enriched population of TEC from human thymus biopsies. Fresh thymus fragments were obtained during elective cardiac surgery on HIV-1-seronegative children (age 6 days to 24 months). The TEC enrichment procedure was adapted from a technique previously used to enrich cytotrophoblasts from human placentas (28). After a gentle teasing of thymic tissue, nondispersed cells were trypsinized during 2 h at 37°C (four cycles of 30 min each) in saline buffer (13 Hanks balanced salt solution [GIBCO]) containing 5 mg of trypsin per ml, 10 mg of DNase IV D per ml, 25 mM HEPES, 2 mM L-glutamine (GIBCO), and antibiotics. Dispersed cells were washed and separated by centrifugation through a discontinuous 10 to 70% Percoll gradient (Pharmacia) at 2,000 rpm for 20 min. Fractions containing TEC (20 to 40%) were collected, pooled, and washed. Thymocytes, macrophages, B lymphocytes, natural killer (NK) cells, and dendritic cells were then removed by negative selection with MAbs directed against CD45 (HLe-1), CD14 (Leu-M3), CD19 (J4 119), CD16 (3G8), and CD3 (OKT3) at a 1/50 dilution and magnetic beads coated with sheep anti-mouse IgG (Dynabeads M-450; Dynal A. S, Oslo, Norway) at a concentration corresponding to two or three beads for one cell in each round of depletion. The cells obtained were cultured in 23 selective medium consisting of McCoy’s 5A (GIBCO) supplemented with 20% fetal calf serum (FCS), 1 mg of hydrocortisone per ml, and 1028 M cholera toxin (10).

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Hydrocortisone and cholera toxin eliminate macrophages, fibroblasts, and thymocytes which might contaminate the culture. After 48 h, the culture supernatant was removed, cells were washed, and thymic fibroblasts were lysed by using a specific fibroblast antibody (1B10) at a 1/50 dilution with rabbit complement (Valbiotech) (41) at a 1/10 dilution. Adherent cells were cultured overnight in 13 selective medium, washed, trypsinized, and grown in 12-well plates at a density of 75,000 cells/well in 1 ml of culture medium consisting of McCoy’s 5A (GIBCO) supplemented with antibiotics, L-glutamine, 10 mM HEPES, and 10% FCS. Twelve hours later, adherent cells were washed. Cells obtained by this procedure at day 3 were then characterized by using different selective markers (surface and cytokeratin markers as shown in Table 1), which indicated that more than 95% of the cells were TEC. (ii) Isolation of an enriched population of thymocytes from human thymus biopsies. Fresh thymus fragments were finely minced, and dispersed cells were separated on a Ficoll-Hypaque gradient (Pharmacia) as previously described (49). In some experiments, thymocyte preparations were further purified by passage through nylon wool columns (Valbiotech) to eliminate undetectable (in cytofluorographic analysis) contaminating antigen-presenting cells such as macrophages, TEC, B cells, and dendritic cells. Thymocytes were stored at 4°C in RPMI 1640 supplemented with antibiotics, L-glutamine, 10 mM HEPES, and 20% FCS. At day 3, thymocytes showed a viability of .99%. They were phenotypically characterized just after isolation from the thymus environment (day 0) and 3 days later (day 3) by immunostaining with antibodies against CD1a, CD4, CD2, and CD8. (iii) Mixed culture of TEC and thymocytes. Autologous coculture of TEC and infected or control thymocytes was carried out at 3 days after thymic excision either under conditions permitting cell-to-cell contact or in transwell chambers (0.45 mm). Different ratios of TEC to thymocytes were first tested, under conditions of cell-to-cell contact, to determine which would be the most suitable for observation of the expected effect of this interaction on HIV-1 replication. The most efficient ratio was 8 3 106 infected thymocytes/7.5 3 104 to 10 3 104 TEC, which then was used for all the experiments. The coculture medium was McCoy’s 5A (GIBCO) supplemented with antibiotics, L-glutamine, 10 mM HEPES, and 10% FCS. Mixed cultures were performed in 12-well plates, and every 3 or 4 days 400 ml of supernatant was replaced by 400 ml of fresh culture medium. All cytokines (IL-1, IL-6, TNF, GM-CSF, IL-3, M-CSF, and SCF) were used at a concentration of 20 ng/ml and added at days 0, 4, and 7 of the coculture. Cytokine inhibitors were added at the start of the coculture. A MAb against IL-6 was used at a concentration of 10 mg/ml, and a polyclonal antibody against TNF was used at a serum dilution of 1/300. IL-1 ra was used at 2.5 ng/ml. Antibody raised against GM-CSF was used at various concentrations in a range from 1 to 25 mg/ml. In some coculture experiments, chemical fixation of TEC was performed with paraformaldehyde (PFA) prior to coculture. PFA was used at a concentration of 2% in phosphate-buffered saline during 15 min. In some other experiments, cultures or cocultures were supplemented with 50% (vol/vol) of noninfected conditioned medium obtained at day 10 from either TEC culture or TEC-thymocyte coculture and then centrifuged and filtered through a 0.45-mm-pore-size membrane. The conditioned medium was added at day 0 and replaced every 3 or 4 days. (iv) Other cells. Human primary thymic fibroblasts were obtained by the explant technique and propagated in a nonselective medium consisting of RPMI 1640 supplemented with antibiotics, L-glutamine, and 10% FCS. The human thymic fibroblast cell line HS67 and human choriocarcinoma cell line JAR were obtained from the American Type Culture Collection. They were propagated in McCoy’s 5A (GIBCO) supplemented with antibiotics, L-glutamine, 10 mM HEPES, and 10% FCS. The human gut epithelial cell line HT29 (18) was kindly provided by J.-G. Guillet (ICGM, Paris, France) and propagated in Dulbecco modified Eagle medium containing 4.5 mg of glucose (GIBCO) per ml and supplemented with antibiotics, L-glutamine, 1 mM sodium pyruvate (GIBCO), and 10% FCS. HIV-1 strains and isolates. Syncytium-inducing (SI) laboratory strains HIV1B-LAI and HIV-1D-NDK and SI primary isolate HIV-1B-LAIp (p is for primary isolate, to distinguish this from the laboratory strain HIV-1B-LAI) were obtained in our laboratory (Pasteur Institute) (7, 16). The SI primary HIV-1E-4039 isolate and the non-SI HIV-1G-4068 isolate were obtained from the Pasteur Institute of Bangui (RCA). Other SI isolates were HIV-1B-DH12, kindly provided by M. A. Martins (National Institutes of Health, Bethesda, Md.), and HIV-1A-92UG029 from the World Health Organization Network for HIV Isolation and Characterization (5). The primary isolates HIV-1B-LAIp, HIV-1E-4039, HIV-1B-DH12, HIV-1G-4068, and HIV-1A-92UG029 were propagated on phytohemagglutininstimulated peripheral blood mononuclear cells as previously described (7) to obtain viral stocks. HIV-1B-LAI and HIV-1D-NDK were propagated on the permissive CD4 T-cell line CEM. HIV-1 infection of human thymocytes. Thymocytes (25 3 106 cells) were infected with each virus, used at a multiplicity of infection of about ;0.001, for 1 h at 37°C. The cells were then washed three times with RPMI 1640 containing 10 mM HEPES, resuspended in culture medium, and cultured alone or cocultured with TEC (or with other adherent cells in some experiments). HIV-1 p24 antigen concentrations determined in culture supernatants were measured at various times postinfection by using a p24 antigen detection kit

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TABLE 1. Phenotypic characterization of TEC- or thymocyte-enriched populationsa % Positive: Marker

CD1a CD2 CD4 CD8 CD14 CD16 CD19 CD45 Cytokeratin Vimentin

TEC

Thymocytes

Day 0

Day 0

Day 14

,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 .95 ,5

91.5 .98 71.5 82 ,1 ,1 ,1 97 ,1 ,1

91 .98 83 85 ,1 ,1 ,1 99.5 ,1 ,1

a The phenotypes were determined at day 3 after thymus excision, which corresponds to day 0 of the culture or coculture. The phenotype of the thymocyte population was also determined at day 14. Direct or indirect immunostaining assays and cytofluorographic analysis are detailed in Materials and Methods. The data represent the means from three independent experiments. The standard deviation never exceeded 5%.

(ELAVIA Ag; Diagnostics Pasteur, Marnes la Coquette, France) according to the instructions of the manufacturer.

RESULTS Development and characterization of a TEC-thymocyte autologous mixed culture. To test the effect of TEC-thymocyte interaction on HIV-1 replication in thymocytes, mixed cultures of these two human cell populations were first established as follows: (i) autologous mixed cultures were carried out to prevent any allogenic mixed lymphocyte reaction which might itself promote HIV-1 replication and would not be physiologically relevant, and (ii) these mixed autologous cultures were done shortly after surgical excision of the thymus, since the expression of cell surface markers, as well as the distribution of the different subpopulations of thymocytes, is modified after a prolonged period of time. In preliminary experiments, we observed that thymocytes lose their capacity to sustain virus replication in coculture when used after 4 to 5 days (data not shown). This led us to set up a new technical approach to obtain an enriched TEC population in a shorter time (3 days after thymus excision) than that required with the classical explant technique, which leads to TEC being obtained after 15 to 17 days. This novel technique is described in detail in Materials and Methods. A panel of antibodies raised against cell surface molecules has been used to further characterize TEC or thymocyte populations at day 3 after thymus excision, which corresponds to day 0 of the culture or coculture. The results of indirect immunofluorescence assays are presented in Table 1. An anticytokeratin antibody stained more than 95% of cells in the TEC-enriched population, confirming their epithelial characteristics, whereas an antivimentin MAb stained fewer than 5% of the cells, indicating that contamination by thymic fibroblasts was low. Fewer than a threshold level of 1% of the TEC were positively stained by MAbs raised respectively against CD1a, CD2, CD14, CD16, CD19, and CD45, indicating little, if any, contamination with dendritic cells, thymocytes, macrophages, NK cells, or B cells. The thymocyte population (day 0) was enriched at least to 98% as indicated by specific CD2 staining. No detectable level (,1% in each case) of macrophages (CD14-positive cells), NK cells (CD16-positive cells), B cells (CD19-positive cells), TEC (cytokeratin-positive cells), or thymic fibroblasts (vimentin-positive cells) was observed in the

enriched population of thymocytes. Since the experiments we describe in the following sections required culture of the thymocytes for 10 to 14 days, we also determined the number of viable cells and their phenotype after this prolonged period of time. Of 8 3 106 at the onset of the culture, 1 3 106 to 1.4 3 106 thymocytes were still alive after 14 days in the absence of TEC as determined by trypan blue exclusion. The phenotype of the thymocyte population did not significantly change after 14 days in culture alone, as shown in Table 1. TEC-thymocyte interaction is required to promote a high level of HIV-1 replication in thymocytes. To test whether the TEC-thymocyte interaction is required for HIV-1 replication in thymocytes, we infected freshly isolated unfractionated thymocytes with distinct HIV-1 strains in vitro. These viruses were the two laboratory strains HIV-1B-LAI and HIV-1D-NDK and the phenotypically and genotypically characterized primary isolates HIV-1B-LAIp (p is for primary isolate), HIV-1B-DH12, HIV-1E-4039, and HIV-1A-92UG029 used as SI isolates and HIV1G-4068 used as a non-SI isolate. Infected thymocytes were cultivated alone or cocultivated with autologous TEC. HIV-1 replication was evaluated by measuring the p24 antigen concentration in thymocyte culture or TEC-thymocyte coculture supernatants at various times postinfection. As shown in Fig. 1, autologous mixed cultures of infected thymocytes with TEC led to active replication from day 5 to day 14 for both the HIV-1 laboratory strains and the primary isolates tested irrespective of their phenotypic or genotypic characteristics. It is interesting that the level of replication obtained at day 14 postinfection was of the same order of magnitude for all the viruses tested and particularly for the laboratory strain HIV1B-LAI or the primary isolate HIV-1B-LAIp. This excludes the possibility of coactivation by any residual mitogenic reagents used to grow the primary isolate on peripheral blood mononuclear cells. In contrast, no replication was detectable when thymocytes were cultivated alone (Fig. 1). Since the lack of detectable replication in thymocytes cultured alone might be related to a reduced life span when they are not protected by their interaction with TEC, we determined the number of viable thymocytes at day 14 under both culture conditions. As already mentioned, of 8 3 106 thymocytes used at the onset of the culture, 0.5 3 106 to 1 3 106 thymocytes were still alive after 14 days in the absence of TEC. In coculture with TEC, the number of thymocytes alive was 0.5 3 106 to 2 3 106. This difference in thymocyte viability under the two culture conditions is not sufficient to explain the lack of detectable replication in the absence of TEC. In addition, the measurement of HIV replication corresponds to the accumulation of p24 antigen in the culture starting early on, when the number of viable thymocytes was still high under both culture conditions. To verify that activation of HIV-1 replication was not due to any component of FCS which might interact with factors present in the coculture, human serum was used in parallel experiments. No difference was observed for the two serum conditions (data not shown). Eight different thymuses were used to perform these cocultures under autologous conditions. All of the cocultures supported HIV replication in thymocytes, with some differences in p24 production (from 1 to 9 ng/ml was detected at day 14 for the infection with HIV-1B-LAIp, for example). Heterologous cocultures were also performed with thymuses from 20 different donors. HIV replication in thymocytes was also induced under these conditions, to slightly higher levels of p24 production (from 5 to 20 ng of p24 per ml at day 14) than under autologous conditions (data not shown). Induction of HIV-1 replication in thymocytes is limited to mixed culture with TEC. We further determined whether the

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FIG. 1. Interaction with TEC is a requirement for promotion of HIV-1 replication at a high level in thymocytes. Thymocytes were infected by two laboratory strains, HIV-1B-LAI and HIV-1D-NDK, or five primary isolates, HIV-1B-LAIp, HIV-1E-4039, HIV-1B-DH12, HIV-1A-92UG029, and HIV-1G-4068, at a multiplicity of infection of 0.001 as described in Materials and Methods. They were cultivated alone (squares) or with autologous TEC (circles). The p24 antigen concentration in supernatants harvested at days 5, 8, 11, and 14 postinfection was determined. The results shown here are from a representative experiment of three independent experiments performed for each HIV-1 isolate. A total of eight experiments with eight different thymuses were performed with HIV-1B-LAIp.

induction of HIV-1 replication in thymocytes was strictly specific to the interaction of thymocytes with TEC and did not require other cell types of thymic origin. We first excluded the participation of antigen-presenting cells such as macrophages, B cells, or dendritic cells, which still could be present at a nondetectable level in thymocyte preparations, by retaining them on nylon wool columns. As shown in Fig. 2A, the same level of replication of HIV-1B-LAIp or HIV-1D-NDK was obtained whether or not a nylon wool column was used to achieve thymocyte isolation prior to cultivation with TEC. We also considered the possibility that other cell types of

thymic origin, such as fibroblasts, might interact positively to induce HIV-1 replication in thymocytes. Neither the thymic fibroblastic cell line HS67 nor thymic primary fibroblasts in coculture with infected thymocytes were efficient at inducing HIV-1B-LAIp or HIV-1D-NDK replication (Fig. 2B). We also asked whether it is a general property of cells with epithelial characteristics, irrespective of the tissue from which they originate, to interact with thymocytes to induce HIV-1 replication. Coculture of infected thymocytes with either a human gut intestinal (HT29) or a human choriocarcinoma (JAR) cell line did not lead to HIV-1 replication (Fig. 2B). Thus, efficient

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FIG. 2. Activation of HIV-1 replication is limited to thymocyte-TEC cocultures. (A) Thymocytes obtained after Ficoll-Hypaque separation were purified or not (control) through nylon wool columns (NW) and then infected with either the primary isolate HIV-1B-LAIp (white bars) or the laboratory strain HIV-1D-NDK (dark bars) and cultured alone or with autologous TEC. (B) Thymocytes infected with the primary isolate HIV-1B-LAIp (white bars) or the laboratory strain HIV-1D-NDK (dark bars) were cultured alone or with autologous TEC, human primary thymic fibroblasts, the human thymic fibroblast cell line HS67, the human gut epithelial cell line HT29, or the human choriocarcinoma cell line JAR. Each bar represents the p24 level determined in supernatants harvested at day 14 (A) or 10 (B) postinfection. The results shown here are from one representative experiment of three carried out with three different thymuses.

signals necessary for HIV-1 replication in thymocytes seem to be restricted to the TEC-thymocyte interaction. HIV-1 replication in thymocytes requires cell-to-cell contact with TEC, and this effect is mediated by soluble factors. To further determine whether physical TEC-thymocyte contact is required for induction of a high level of HIV-1 replication in thymocytes, a coculture of TEC and infected thymocytes was performed in double-chamber dishes to prevent direct cell-tocell contacts but to allow for exchange of soluble factors. Under these culture conditions, no replication was detected in thymocytes infected with three distinct HIV-1 strains (HIV-1BLAIp, HIV-1D-NDK, and HIV-1A-92UG029), whereas in parallel experiments, cell-to-cell contact of TEC and thymocytes led to a high level of virus replication irrespective of the virus tested (Fig. 3A). The absence of detectable HIV-1 replication in thymocytes cultured in chambers did not result from a loss of inducibility, since they were still able to replicate the virus after mitogen stimulation (data not shown). We further studied how cell-to-cell contact resulted in induction of HIV-1 replication. HIV-1B-LAIp-infected thymocytes were cocultivated with TEC previously fixed with PFA. Under these experimental conditions, no replication was observed, suggesting that HIV-1 replication might be mediated by soluble factors secreted during the cell-to-cell contact rather than by an inductive signal resulting from the interaction between cell surface ligands (Fig. 3B). This hypothesis was further confirmed by the fact that HIV-1B-LAIp-infected thymocytes cultivated alone in conditioned medium obtained from an uninfected mixed culture eliciting contact allowed virus replication (Fig. 3B). However, the replication level obtained with this conditioned medium alone was lower than that obtained in the mixed culture of infected thymocytes with TEC. Conditioned medium from either isolated thymocytes or TEC was

unable by itself to induce HIV-1 replication in thymocytes (Fig. 3B). These data strongly suggest that soluble factors secreted during the physical contact between TEC and thymocytes mediate HIV-1 replication in thymocytes. The cytokines TNF, IL-1, and IL-6 secreted in the mixed culture are necessary participants in HIV-1 replication in thymocytes. To identify the soluble factors secreted in the TECthymocyte mixed culture medium which lead to HIV-1 replication in thymocytes, we used a panel of cytokine inhibitors. These inhibitors were added at the start of the mixed culture of HIV-1B-LAIp-infected thymocytes with TEC. As shown in Fig. 4A, incubation of the mixed culture with antibodies against IL-6 or TNF or with IL-1 ra partially prevented the induction of replication, whereas no inhibition was observed with irrelevant control antibodies. The inhibition rates were 30, 50, and 81% for IL-6, IL-1, and TNF, respectively. These cytokine inhibitors, used in association, acted synergistically to decrease HIV-1 replication by 92%. Similar results were obtained with heterologous cultures and the same cytokine inhibitors (data not shown). Therefore, TNF, IL-1b, and IL-6 are produced in the coculture and are required for optimal HIV-1 replication in thymocytes. To further determine whether these cytokines are sufficient by themselves to induce optimal HIV-1 replication in thymocytes, we incubated infected HIV-1B-LAIp thymocytes, purified on nylon wool columns, with these cytokines used separately or in various combinations. IL-1b or IL-6 used separately led to a threshold level of HIV-1 replication. TNF induced a higher, although still low, level. In contrast, combined treatments with two cytokines, especially when TNF was included, or with the three cytokines together were much more efficient (Fig. 4B). However, even the combination of the three cytokines, which gave rise to the strongest effect, did not result

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FIG. 3. Optimal HIV-1 replication in thymocytes requires cell-to-cell contact with TEC, and this effect is mediated by soluble factors. (A) Thymocytes infected with the primary isolate HIV-1B-LAIp or HIV-1A-92UG029 or the laboratory strain HIV-1D-NDK were cultured alone or with autologous TEC either under conditions permitting cell-to-cell contact or in transwell chambers. (B) Thymocytes infected with the primary isolate HIV-1B-LAIp were cultured either alone (control thymocytes) or with autologous TEC treated or not (control coculture) with 2% PFA. Thymocyte cultures were also performed in medium supplemented with 50% of conditioned medium (CM) obtained from cultures of either thymocytes or TEC alone or a thymocyte-TEC mixed culture. Each bar represents the p24 level determined in supernatants harvested at day 14 postinfection. The results shown here are from one representative experiment of three carried out with three different thymuses.

in a level of replication similar to that observed for a mixed culture of infected thymocytes with TEC (Fig. 4B). Therefore, the combination of TNF, IL-1b, and IL-6 is able to promote by itself HIV-1 replication in thymocytes, but an additional factor(s) might also be required for optimal induction. GM-CSF, secreted mainly by TEC, strongly synergizes with the combination TNF–IL-1–IL-6 to increase HIV-1 replication in thymocytes. We showed that the conditioned medium from the mixed culture of TEC and thymocytes, but not that from TEC or thymocytes cultivated alone, was able to induce HIV-1 replication in infected thymocytes cultivated alone. However, this conditioned medium did not induce a replication level similar to that obtained in the mixed culture. The reasons for this partial inefficiency might be related to the preparation or dilution of the conditioned medium, which might result in nonsaturating concentrations of cytokines. However, the combined treatment with TNF, IL-1b, and IL-6, used at saturating concentrations (control cytokines), also did not entirely replace the coculture conditions (Fig. 4B and 5A). The coculture of HIV-1B-LAIp-infected thymocytes with PFA-treated TEC in the presence of this combination of these three cytokines did not significantly enhance the level of HIV-1 replication, indicating that cell-to-cell contact in addition to these cytokines did not improve the level of HIV-1 replication (Fig. 5A). In contrast, when this combination of cytokines was used together with conditioned medium obtained from TEC cultivated alone, a level of HIV-1 replication of the same order of magnitude as that of the coculture was attained (Fig. 5A). This indicates that an additional soluble factor secreted by TEC is able to strongly

synergize with the association of IL-1b, IL-6, and TNF. GMCSF, which is secreted mainly by TEC, was a potential candidate. We showed that conditioned medium from TEC lost its ability to potentiate the IL-1b, IL-6, and TNF effect when preincubated with an antibody against GM-CSF, indicating that GM-CSF was indeed an additional cofactor of activation of HIV-1 replication in thymocytes (Fig. 5B). To confirm this result, various concentrations (from 1 to 25 mg/ml) of a polyclonal goat antibody raised against GM-CSF were added to the mixed culture of infected thymocytes with TEC. We showed that HIV-1 replication in HIV-1B-LAIp- or HIV-1D-NDK-infected thymocytes was inhibited by this antibody in a dosedependent manner, whereas a normal goat IgG at the highest concentration (25 mg/ml), used as a control, did not modify the replication level (Fig. 5C). Anti-GM-CSF was also tested in heterologous cocultures and was found to similarly inhibit HIV replication (data not shown). This inhibition indicates that GM-CSF mainly provided by TEC is required to attain a high level of HIV-1 replication in thymocytes. We then tested whether GM-CSF could induce HIV-1 replication by itself, and we compared its inducing effect to that of combined treatment with TNF, IL-1, and IL-6. In this experiment, thymocytes were first thoroughly deprived of adherent cells, which could be a source of cytokines, by using nylon wool columns. They were then infected with HIV-1B-LAIp and cocultivated with TEC in a medium containing GM-CSF, at a saturating concentration, alone or in association with the three other cytokines IL-1, IL-6, and TNF. A threshold level of HIV-1 replication was observed with GM-CSF alone, whereas

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FIG. 4. The soluble factors TNF, IL-1, and IL-6 secreted in the mixed culture are involved in the activation of HIV-1 replication in thymocytes. (A) Thymocytes infected with the primary isolate HIV-1B-LAIp were cultured alone (control thymocytes) or with autologous TEC (control coculture). MAbs directed against IL-6 (10 mg/ml) or IL-1 ra (2.5 ng/ml) or an immune rabbit serum against TNF alpha (1/300 dilution) were added either separately or in a combination in the mixed culture at days 0, 4, and 7 postinfection. Medium containing MAb IgG1 (10 mg/ml) and normal rabbit serum (1/300 dilution) was used as a control (control antibodies). (B) Thymocytes infected with the primary isolate HIV-1B-LAIp were treated or not with recombinant human cytokines IL-6, IL-1b, and TNF either separately or in combinations of two or three together. Cytokines were used at the same concentration of 20 ng/ml and added at days 0, 4, and 7 of culture. Each bar represents the p24 level determined in supernatants harvested at day 10 (B) or 14 (A) postinfection. The results shown here are from one representative experiment of three carried out with three different thymuses.

this cytokine induced a very high synergistic effect when associated with IL-1b, IL-6, and TNF (Fig. 5D). The observed induction of HIV-1 replication was even higher than that observed in the coculture. No comparable induction was observed when M-CSF, also produced by TEC, was used instead of GM-CSF (Fig. 5D). IL-3 and SCF were also tested and were found to have no effect on HIV replication (data not shown). We conclude that IL-1b, IL-6, TNF, and GM-CSF synergize to promote HIV-1 replication in thymocytes and may account together for the high level of HIV-1 replication obtained in infected thymocytes in mixed culture with TEC. TNF–IL-1–IL-6 and GM-CSF constitute two independent requirements for HIV replication in thymocytes. We showed that treatment with anti-GM-CSF reduces HIV replication to the level obtained with the combination TNF–IL-1–IL-6 alone (compare bars 2 and 4 in Fig. 5B with bar 6 in Fig. 5C and bar 4 in Fig. 5D). This suggested that these two treatments did not act through the same mechanism to support HIV replication. Furthermore, the synergy observed between GM-CSF and TNF–IL-1–IL-6 in Fig. 5D strengthened this hypothesis. To further confirm this point, nylon-purified thymocytes were infected and then treated with the combination TNF–IL-1–IL-6 in the presence or absence of anti-GM-CSF. The results (Fig. 6) show that addition of this antibody at various concentrations did not dramatically impair the induction effect of TNF–IL-1– IL-6. This result confirms that the combination of TNF, IL-1, and IL-6 supports HIV replication by a mechanism distinct from that of GM-CSF.

DISCUSSION Overwhelming evidence indicates that viral load and decline of CD41 T cells are the pathogenic correlates of HIV disease. Invasion of lymphoid tissues, and particularly of lymph nodes, by the virus was shown to be associated with the destruction of organ architecture. Since HIV-1 infection of the thymus is also associated with a loss of architecture together with a decline of precursor (CD41 CD81) and mature (CD41) T lymphocytes (44), it was essential to define the signals that regulate HIV-1 replication within this primary lymphoid organ. To investigate the possible physiological role of TEC-thymocyte interaction, which could influence HIV-1 replication in thymocytes, a mixed culture system of infected human thymocytes and autologous TEC was established at short time after surgical excision of the thymus (Table 1). We report here that TECthymocyte interaction is required for a high and sustained level of HIV-1 replication in thymocytes and that this virus production was qualitatively and quantitatively identical irrespective of the HIV-1 strain used to infect the thymocytes (Fig. 1). We also demonstrate that interaction with TEC is necessary and sufficient to promote a high level of HIV replication in thymocytes. This requirement is a general rule that applied to both autologous (8 donors) and heterologous (20 donors) coculture conditions, i.e., to a total of 28 thymuses from donors ranging from a few days to 2 years old. Furthermore, these data suggest that thymocytes which are permissive to a wide variety

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FIG. 5. GM-CSF, secreted mainly by TEC, synergizes with TNF–IL-1–IL-6 to enhance HIV-1 replication. (A) Thymocytes infected with the primary isolate HIV-1B-LAIp were cultured either alone (control thymocytes) or with autologous TEC (control coculture). A combined treatment with IL-6, IL-1b, and TNF, used at 20 ng/ml, was performed on isolated infected thymocytes cultivated either in a regular medium (control cytokines) or in a medium supplemented with 50% of a conditioned medium from a separate TEC culture (TEC CM). These cytokines were also added in a mixed culture of infected thymocytes with TEC treated with 2% PFA. (B) Thymocytes infected with the primary isolate HIV-1B-LAIp were left unstimulated (control thymocytes) or were stimulated with TNF, IL-1, and IL-6. These cytokine-stimulated thymocytes were cultured in regular medium (control cytokines) or in a medium supplemented with 50% of conditioned medium from a separate TEC culture (TEC CM). This TEC CM was either preincubated or not (no antibody) with goat polyclonal IgG against human GM-CSF (anti-GM-CSF) or with a normal goat IgG (control antibody) used at a concentration of 10 mg/ml. (C) Thymocytes infected with primary isolate HIV-1B-LAIp were cultured alone (control thymocytes) or with TEC treated or not with a goat IgG directed against GM-CSF used at different concentrations (0, 1, 2.5, 10, and 25 mg/ml) or with a normal goat IgG (control antibody) used as a control at 25 mg/ml. Goat IgGs were added at day 0. (D) Infected thymocytes were treated with recombinant GM-CSF or with a combination of IL-6, IL-1b, and TNF with or without GM-CSF or M-CSF. All recombinant cytokines were repeatedly added at the same concentration of 20 ng/ml at days 0, 4, and 7 postinfection. Each bar represents the p24 level determined in supernatants harvested at day 10 (A, B, and D) or 14 (C) postinfection. The results shown here are from one representative experiment of three carried out with three different thymuses.

of viruses in vitro might thus be a potentially frequent target of HIV-1 infection in vivo. TEC might also be infected by some HIV-1 strains (HIV1D-NDK and HIV-1E-4039) that we tested here (10). However, following infection of activated TEC, virus production was detectable only after a long time (17 days), suggesting that under our coculture conditions and within the period of time of the experiment (at most 14 days), viral replication occurs mainly in thymocytes. Virus production in thymocytes results from a specific interaction with TEC, excluding any other cell type that we tested, even cells with epithelial characteristics originating from other tissues (Fig. 2). Of course, we cannot entirely rule out a possible role of other thymic cells, such as macrophages and dendritic cells, which might remain at a very low density in the thymocyte population. However, from the degree of purity (98%) of the thymocyte population, it is clear that viral replication depends mostly on the presence of TEC. The explanation for this restricted response may be provided by the fact that thymocyte-TEC contact is required to promote optimal HIV-1 replication (Fig. 3A). Furthermore, we showed that chemical fixation of TEC abolished the induction of HIV-1 replication, whereas it was restored, albeit less efficiently, with conditioned medium from a noninfected mixed culture (Fig. 3B). This indicates that HIV-1 replication is mediated by soluble factors secreted during the cell-to-cell contact rather than by an inductive signal resulting from the interaction between cell surface ligands. This contact, creating a favorable cytokine microenvironment, might occur through interactions between

different cell surface ligands. For instance, interaction involving CD2 on thymocytes and CD58/LFA3 on TEC (51) leads to the production of IL-1 (32), one of the cytokines involved in the induction of HIV-1 replication. Experiments are in progress to determine if any other adhesion pathway might participate in HIV-1 replication by inducing or increasing the other activating cytokines. A possible role of the interaction between the major histocompatibility molecules on the TEC surface and the CD3–T-cell receptor complex on thymocytes should also be investigated. Further studies, in particular with heterologous cell combinations, might be of help to elucidate the role of the specific cell-to-cell contact. A previous report also showed, in a mouse thymus reconstitution assay, that TNF, as well as IL-1, is present within the microenvironment (54). IL-1 and TNF, but also IL-6 and GMCSF, which were implicated in inducing or favoring HIV-1 replication, were thus good candidates for the active components of the conditioned medium on HIV-1 replication. The use of inhibitors of these four cytokines in the coculture (Fig. 4A and 5C), together with the demonstration of their capacity to induce HIV-1 replication in isolated thymocytes (Fig. 4B and 5D), clearly indicates that they were present within the microenvironment of the coculture and that their concomitant effect accounts for the productive and sustained replication observed in thymocyte-TEC mixed cultures. These data are in agreement with a previous report indicating that these cytokines used separately failed to induce a detectable level of HIV-1 replication in infected thymocytes (48), since we demonstrate here that the synergistic effect of these four cytokines

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FIG. 6. TNF–IL-1–IL-6 and GM-CSF are independent requirements for HIV replication in thymocytes. Thymocytes infected with the primary isolate HIV-1B-LAIp were cultured alone and left untreated (control thymocytes) or exposed to a combined treatment with IL-6, IL-1b, and TNF, used at 20ng/ml. These cytokine-stimulated thymocytes were treated or not with a goat IgG directed against GM-CSF used at different concentrations (0, 5, 10, and 25 mg/ml) or with a normal goat IgG (control antibody) used as a control at 25 mg/ml. Each bar represents the p24 level determined in the supernatants harvested at day 12 postinfection. The results shown here are from one representative experiments of three carried out with three different thymuses.

is required to obtain a high yield of HIV-1 replication. Convergent data (Fig. 5 and 6) indicate that TNF–IL-1–IL-6 and GM-CSF support HIV replication by two distinct mechanisms acting in synergy. This association of cytokines suggests at least two kinds of requirements in this process: one is the activation of the thymocytes through IL-1, IL-6, and TNF, and the other is the proliferation and/or protection of thymocytes against apoptosis through GM-CSF, as documented below. The effect of IL-1, IL-6, and TNF on HIV-1 replication might be an activation process occurring at the transcription level (4, 22, 25, 27, 37). Work is in progress in our laboratory to determine whether the TEC-thymocyte interaction activates HIV-1 transcription through IL-1- or TNF-induced activation of NF-kB. The potential role of IL-6 in HIV-1 transcription in thymocytes will also be investigated. GM-CSF, which acts synergistically with the other cytokines on HIV-1 replication, was not shown to act directly on HIV-1 transcription. However, its effect might be indirect by increasing IL-6 production by thymocytes, since it is one of the major inducers with IL-1 (36). The main role of GM-CSF might be to increase thymocyte survival by playing a protective role against apoptosis, as shown in various hematopoietic systems (4, 25, 27), and/or to induce proliferation of certain subpopulations of thymocytes, as shown for the CD42 CD82 subset in the murine thymus (43). IL-1 might also participate in a proliferative effect, as shown in the mouse model (32). Since immature thymocytes are permissive to the virus, GM-CSF might be responsible for an increasing expansion of infectable cells among the unfractionated population of thymocytes. This addresses the question of whether this interaction with TEC preferentially induces HIV-1 replication in certain subpopulations of thymocytes. Indeed, the subpopulations of thymocytes that actively repli-

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cate HIV-1 in vivo remain uncertain. Controversial reports showed that in thymuses from infected infants, viral RNA was detected either in the medullary (35) or in the cortical (38) area, suggesting, respectively, the specific infection of mature and immature thymocytes. In thymuses from simian immunodeficiency virus-infected macaques, virus particles were only detected in the cortical area (8). It will thus be of interest to define which thymocyte subpopulations preferentially replicate HIV-1 in the microenvironment of TEC. In conclusion, we identified here signals associated with the TEC-thymocyte interaction which are responsible for increasing the viral load within the thymus. We cannot exclude the possibility that other soluble factors might also be potentially active in this process, since other cytokines might have similar functions. We should also consider chemokines, which might down regulate the viral spread in thymocytes according to their interactions with the second receptors for HIV-1 (6, 14, 17). In addition to the regulatory signals involved in HIV replication, it is also of importance to clarify the pathways by which the viral load itself might be responsible, in turn, for thymocyte depletion and impairment in TEC function and survival, leading to a progressive destruction of the thymus in infants infected in utero. ACKNOWLEDGMENTS We thank M. Papiernik, A. Rein, and G. Chaouat for review of the manuscript. We are very grateful to Sonia Berrih-Aknin and to Claude Planche´ (Hospital Marie Lannelongue, Le Plessis-Robinson, France) for providing us with thymuses from infants undergoing cardiac surgery. We also thank Sonia Berrih-Aknin and Nathalie Moulion for helpful discussions. This work was supported by the Agence Nationale pour la Recherche sur le SIDA (ANRS). J. Braun was a fellow of the Medical Research Foundation through funding of SIDACTION, and M. Rothe and L. Cheˆne were fellows of the French Ministry of Education and Research (MENESR). M. Rothe was also supported by the Medical Research Foundation. REFERENCES 1. Aldrovandi, G. M., G. Feuer, L. Gao, B. Jamieson, M. Kristeva, I. S. Y. Chen, and J. A. Zack. 1993. The SCID-hu mouse as a model for HIV-1 infection. Nature 363:732–736. 2. Anderson, G., E. J. Jenkinson, N. C. Moore, and J. J. T. Owen. 1993. MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature 362:70–73. 3. Anderson, G., N. C. Moore, J. J. Owen, and E. J. Jenkinson. 1996. Cellular interactions in thymocyte development. Annu. Rev. Immunol. 14:73–99. 4. Bach, M. K., and J. R. Brashler. 1995. Evidence that granulocyte/macrophage-colony-stimulating factor and interferon-gamma maintain the viability of human peripheral blood monocytes in part by their suppression of IL-10 production. Int. Arch. Allergy Immunol. 107:90–92. 5. Bachmann, M. H., E. L. Delwart, E. G. Shpaer, P. Lingenfelter, R. Singal, and J. I. Mullins. 1994. Rapid genetic characterization of HIV type 1 strains from four World Health Organization-sponsored vaccine evaluation sites using a heteroduplex mobility assay. WHO Network for HIV Isolation and Characterization. AIDS Res. Hum. Retroviruses 10:1345–1353. 6. Baggiolini, M., B. Dewald, and B. Moser. 1997. Human chemokines—an update. Annu. Rev. Immunol. 15:675–705. 7. Barre´-Sinoussi, F., J.-C. Chermann, F. Rey, M.-T. Nugeyre, S. Chamaret, C. Axler-Blin, F. Vezinet-Brun, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983. Isolation of a T lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868–870. 8. Baskin, G. B., M. Murphey-Corb, L. N. Martin, B. Davison-Fairburn, F.-S. Hu, and D. Kuebler. 1991. Thymus in simian immunodeficiency virus-infected rhesus monkeys. Lab. Invest. 65:400–407. 9. Blanche, S., M. Tardieu, A. M. Duliege, C. Rouzioux, F. Le Deist, K. Fukunaga, M. Caniglia, C. Jacomet, A. Messiah, and C. Griscelli. 1990. Longitudinal study of 94 symptomatic infants with perinatally acquired human immunodeficiency virus infection. Evidence for a bimodal expression of clinical and biological symptoms. Am. J. Dis. Child. 144:1210–1215. 10. Braun, J., H. Valentin, M.-T. Nugeyre, H. Ohayon, P. Gounon, and F. Barre´-Sinoussi. 1996. Productive and persistent infection of human thymic epithelial cells in vitro with HIV-1. Virology 225:413–418.

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11. Calvelli, T. A., and A. Rubinstein. 1990. Pediatric HIV infection. Immunodef. Rev. 2:83–127. 12. Denning, S. M., J. Kurtzberg, P. T. Le, D. T. Tuck, K. H. Singer, and B. F. Haynes. 1988. Human thymic epithelial cells directly induce activation of autologous immature thymocytes. Proc. Natl. Acad. Sci. USA 85:3125–3129. 13. de Rossi, A., M.-L. Calabro, M. Panozzo, D. Bernardi, B. Caruzo, G. Tridente, and L. Chieco-Banchi. 1990. In vitro studies of HIV-1 infection in thymic lymphocytes: a putative role of the thymus in AIDS pathogenesis. AIDS Res. Hum. Retroviruses 6:287–297. 14. Dsouza, M. P., and V. A. Harden. 1996. Chemokines and HIV-1 second receptors—confluence of two fields generates optimism in AIDS research. Nat. Med. 2:1293–1300. 15. Duh, E. J., W. J. Maury, T. M. Folks, A. S. Fauci, and A. B. Rabson. 1989. Tumor necrosis factor a activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kB sites in the long terminal repeat. Proc. Natl. Acad. Sci. USA 86:5974–5978. 16. Ellrodt, A., F. Barre´-Sinoussi, P. Le Bras, M.-T. Nugeyre, L. Palazzo, F. Rey, F. Brun-Vezinet, C. Rouzioux, P. Segond, R. Caquet, L. Montagnier, and J.-C. Chermann. 1984. Isolation of a new human T-lymphotropic retrovirus (LAV) from a married couple of Zairians, one with AIDS, the other with prodromes. Lancet i:1383–1385. 17. Fauci, A. S. 1996. Host factors and the pathogenesis of HIV-induced disease. Nature 384:529–534. 18. Fogh, J., and G. Trempe. 1975. Human tumor cells in vitro. Plenum Press, New York, N.Y. 19. Folks, T. M., J. Justement, K. Kinter, C. A. Dinarello, and A. S. Fauci. 1987. Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line. Science 238:800–802. 20. Galy, A. H. M., C. A. Dinarello, T. S. Kupper, A. Kameda, and J. W. Hadden. 1990. Effects of cytokines on human thymic epithelial cells in culture. Recombinant IL1 stimulates thymic epithelial cells to produce IL6 and GMCSF. Cell. Immunol. 129:161–175. 21. Hays, E. F., C. H. Uittenbogaart, J. C. Brewer, L. W. Vollger, and J. A. Zack. 1992. In vitro studies of HIV-1 expression in thymocytes from infants and children. AIDS 6:265–272. 22. Hazan, U., D. Thomas, J. Alcami, F. Bachelerie, N. Israe¨l, H. Yssel, J.-L. Virelizier, and F. Arenzana-Seisdedos. 1990. Stimulation of a human T-cell clone with anti-CD3 or tumor necrosis factor induces NF-kB translocation but not human immunodeficiency virus 1 enhancer-dependent transcription. Proc. Natl. Acad. Sci. USA 87:7861–7865. 23. Ho, D. D., T. Moudgil, and M. Alam. 1989. Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. N. Engl. J. Med. 321:1621–1625. 24. Israe¨l, N., U. Hazan, J. Alcami, A. Munier, F. Arenzana-Seisdedos, F. Bachelerie, A. Israe¨l, and J.-L. Virelizier. 1989. Tumor necrosis factor stimulates transcription of HIV-1 in human T lymphocytes, independently and synergistically with mitogens. J. Immunol. 143:3956–3960. 25. Iversen, P. O., L. B. To, and A. F. Lopez. 1996. Apoptosis of hemopoietic cells by the human granulocyte-macrophage colony-stimulating factor mutant E21R. Proc. Natl. Acad. Sci. USA 93:2785–2789. 26. Jovaisas, E., M. A. Koch, A. Scha ¨fer, M. Stauber, and D. Lo ¨wenthal. 1985. LAV/HTLV-III in 20-week fetus. Lancet ii:1129 (Letter.) 27. Kitano, K., C. N. Abboud, D. H. Ryan, S. G. Quan, G. C. Baldwin, and D. W. Golde. 1991. Macrophage-active colony-stimulating factors enhance human immunodeficiency virus type 1 infection in bone marrow stem cells. Blood 77:1699–1705. 28. Kliman, H. J., J. E. Nestler, E. Sermasi, J. M. Sanger, and J. F. Strauss. 1986. Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118:1567–1582. 29. Koyanagi, Y., W. A. O’Brien, J. Q. Zhao, D. W. Golde, J. C. Gasson, and I. S. Y. Chen. 1988. Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science 241:1673–1675. 30. Le, P. T., S. Lazorick, L. P. Whichard, Y.-C. Yang, S. C. Clark, B. F. Haynes, and K. H. Singer. 1990. Human thymic epithelial cells produce IL-6, granulocyte-monocyte-CSF, and leukemia inhibitory factor. J. Immunol. 145: 3310–3315. 31. Le, P. T., D. T. Tuck, C. A. Dinarello, B. F. Haynes, and K. H. Singer. 1987. Human thymic epithelial cells produce interleukin 1. J. Immunol. 138:2520– 2526. 32. Le, P. T., L. W. Vollger, B. F. Haynes, and K. H. Singer. 1990. Ligand binding to the LFA-3 cell adhesion molecule induces IL-1 production by human thymic epithelial cells. J. Immunol. 144:4541–4547. 33. Mu ¨ller, J. G., V. Krenn, C. Schindler, S. Czub, C. Stahl-Hennig, C. Coulibaly, G. Hunsmann, C. Kneitz, T. Kerbau, A. Rethwilm, V. terMeulen, and H. K. Mu ¨ller-Hermelink. 1993. Alteration of thymus cortical epithelium and interdigitating dendritic cells but no increase of thymocyte cell death in the

34. 35.

36. 37.

38. 39.

40.

41.

42.

43.

44. 45. 46. 47. 48. 49.

50. 51.

52.

53. 54.

5861

early course of simian immunodeficiency virus infection. Am. J. Pathol. 143:699–713. Osborn, L., S. Kunkel, and G. J. Nabel. 1989. Tumor necrosis factor a and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kB. Proc. Natl. Acad. Sci. USA 86:2336–2340. Papiernik, M., Y. Brossard, N. Mulliez, J. Roume, C. Brechot, F. Barin, A. Goudeau, J.-F. Bach, C. Griscelli, R. Henrion, and R. Vazeux. 1992. Thymic abnormalities in fetuses aborted from human immunodeficiency virus type 1 seropositive women. Pedriatrics 89:297–301. Papiernik, M., A. Herbelin, E. Schneider, and M. Dy. 1992. Characterization of thymic cell subpopulations involved in IL-1- or GM-CSF-induced IL-6 production. Eur. Cytokine Network 3:89–95. Poli, G., P. Bressler, A. Kinter, E. Duh, W. C. Timmer, A. Rabson, J. S. Justement, S. Stanley, and A. S. Fauci. 1990. Interleukin 6 induces human immunodeficiency virus expression in infected monocytic cells alone and in synergy with tumor necrosis factor a by transcriptional and posttranscriptional mechanisms. J. Exp. Med. 172:151–158. Rosenzweig, M., D. P. Clark, and G. N. Gaulton. 1993. Selective thymocyte depletion in neonatal HIV-1 thymic infection. AIDS 7:1601–1605. Saksela, K., C. Stevens, P. Rubinstein, and D. Baltimore. 1994. Human immunodeficiency virus type 1 mRNA expression in peripheral blood cells predicts disease progression independently of the numbers of CD41 lymphocytes. Proc. Natl. Acad. Sci. USA 91:1104–1108. Schnittman, S. M., S. M. Denning, J. J. Greenhouse, J. S. Justement, M. Baseler, J. Kurtzberg, B. F. Haynes, and A. S. Fauci. 1990. Evidence for susceptibility of intrathymic T-cell precursors and their progeny carrying T-cell antigen receptor phenotypes TCRab1 and TCRgd1 to human immunodeficiency virus infection: a mechanism for CD41 (T4) lymphocyte depletion. Proc. Natl. Acad. Sci. USA 87:7727–7731. Singer, K. H., R. M. Scearce, D. T. Tuck, L. P. Whichard, S. M. Denning, and B. F. Haynes. 1989. Removal of fibroblasts from human epithelial cell cultures with use of a complement fixing monoclonal antibody reactive with human fibroblasts and monocytes/macrophages. J. Invest. Dermatol. 92:166– 170. Stanley, S. K., J. M. McCune, H. Kaneshima, J. S. Justement, M. Sullivan, E. Boone, M. Baseler, J. Adelsberger, M. Bonyhadi, J. Orenstein, C. H. Fox, and A. S. Fauci. 1993. Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse. J. Exp. Med. 178:1151–1163. Stewart, A. A., J. S. Cairns, D. J. Tweardy, and S. A. McCarthy. 1994. Granulocyte-macrophage colony-stimulating factor augmentation of T-cell receptor-dependent and T-cell receptor-independent thymocyte proliferation. Blood 83:713–723. Su, L., H. Kaneshima, M. Bonyhadi, S. Salimi, D. Kraft, L. Rabin, and J. M. McCune. 1995. HIV-1-induced thymocyte depletion is associated with indirect cytopathicity and infection of progenitor cells in vivo. Immunity 2:25–36. Tanaka, K. E., W. C. Hatch, Y. Kress, R. Soeiro, T. Calvelli, W. K. Rashbaum, A. Rubinstein, and W. D. Lyman. 1992. HIV-1 infection of human fetal thymocytes. J. Acquired Immune Defic. Syndr. 5:94–101. Tremblay, M., K. Numazaki, H. Goldman, and M. Wainberg. 1990. Infection of human thymic lymphocytes by HIV-1. J. Acquired Immune Defic. Syndr. 3:356–360. Uittenbogaart, C. H., D. J. Anisman, B. D. Jamieson, S. Kitchen, I. Schmid, J. A. Zack, and E. F. Hays. 1996. Differential tropism of HIV-1 isolates for distinct thymocyte subsets in vitro. AIDS 10:9–16. Uittenbogaart, C. H., D. J. Anisman, J. A. Zack, A. Economides, I. Schmid, and E. F. Hays. 1995. Effects of cytokines on HIV-1 production by thymocytes. Thymus 23:155–175. Valentin, H., M.-T. Nugeyre, F. Vuillier, L. Boumsell, M. Schmid, F. Barre´Sinoussi, and R. Pereira. 1994. Two subpopulations of human triple-negative thymic cells are susceptible to infection by human immunodeficiency virus type 1 in vitro. J. Virol. 68:3041–3050. van Ewijk, W., E. W. Shores, and A. Singer. 1994. Crosstalk in the mouse thymus. Immunol. Today 15:214–217. Vollger, L. W., D. T. Tuck, T. A. Springer, B. F. Haynes, and K. H. Singer. 1987. Thymocyte binding to human thymic epithelial cells is inhibited by monoclonal antibodies to CD-2 and LFA-3 antigens. J. Immunol. 138:358– 363. Withersward, E. S., R. G. Amado, P. S. Koka, B. D. Jamieson, A. H. Kaplan, I. Chen, and J. A. Zack. 1997. Transient renewal of thymopoiesis in HIVinfected human thymic implants following antiviral therapy. Nat. Med. 3:1102–1109. Wolf, S. S., and A. Cohen. 1992. Expression of cytokines and their receptors by human thymocytes and thymic stromal cells. Immunology 77:362–368. Zuniga-Pflu ¨cker, J. C., D. Jiang, and M. J. Lenardo. 1995. Requirement for TNF-a and IL-1a in fetal thymocyte commitment and differentiation. Science 268:1906–1909.