Impaired thymopoietic potential of immature CD3CD4+CD8 T cell precursors from SIV-infected rhesus monkeys

Kai Neben, Marc Heidbreder, Justus Müller1, Anke Marxer, Harald Petry2, Andrea Didier2, Anneliese Schimpl, Thomas Hünig and Thomas Kerkau

Institute of Virology and Immunobiology, University of Würzburg, Versbacher Strasse 7, 97078 Würzburg and
1 Institute of Pathology, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany
2 German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany

Correspondence to: T. Kerkau


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immature thymocyte subpopulations were examined for their capacity to differentiate in a newly developed xenogeneic monkey–mouse fetal thymus organ culture (FTOC) system. We provide evidence for impaired precursor function of CD3CD4+CD8 thymocytes after in vivo infection with SIVmac251 as indicated by a reduced cell number per FTOC and a lower percentage of thymocytes with more mature phenotypes. Addition of recombinant SIV glycoprotein 120 (rgp120) also resulted in a dose-dependent impairment of T cell maturation in FTOC. The data suggest that in patients infected with HIV, T cell maturation and thus replenishment of peripheral pools may be compromised as a result of intrathymic infection or circulating viral gp120.

Keywords: AIDS, immunodeficiency viruses, T cell development, thymus


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Infection with HIV initiates a series of complex events that culminates in profound immunosuppression due to functional abnormalities and quantitative depletion of CD4+ T lymphocytes (1). In addition to peripheral destruction of mature CD4+ T cells, an impaired replenishment by thymic emigrants may contribute to the inability of the immune system to maintain cellular homoeostasis in AIDS. Indeed, proviral DNA (2) and infectious virus (3) are found in the thymus in human HIV and in macaque SIV infection (4), and injection of HIV into human thymus fragments grafted into SCID mice severely impairs thymopoiesis and organ structure (5). A detrimental effect of HIV infection on T cell differentiation was also demonstrated in fetal thymus organ culture (FTOC) repopulated with human cord blood-derived precursor cells (6).

Within the thymus the CD4 molecule is not only present on mature CD3+CD4+CD8 cells, but also on the main population of immature thymocytes, the CD3–/loCD4+CD8+ cells (7) and on their CD3CD4+CD8 precursors (8).

Several in vitro studies demonstrated that multiple subsets of developing thymocytes are direct targets of HIV infection (911), allowing rapid virus replication with consequent cell death. Furthermore, ex vivo analyses of HIV-1 infection in the SCID human–mouse model suggested that immature human CD3CD4+CD8 and CD3–/loCD4+8+ thymocytes as well as their mature CD3+CD4+CD8 and CD3+CD4CD8+ progeny may harbor HIV-1 (12).

The disease associated with SIV infection in rhesus monkeys is very similar to human AIDS (13) except for a greatly shortened time course, which in most animals leads to full-blown disease within 1 year (14). SIV-infected animals develop lymphadenopathy and splenomegaly early after infection followed by generalized lymphoid depletion including thymic involution in the terminal phases of the disease. As in human AIDS, a hallmark of disease progression is a decrease in the number of peripheral helper CD4+ T cells and a concomitant decrease in the CD4:CD8 ratio (13,15).

In previous studies we analyzed early phenotypic and functional alterations in the thymus of SIV-infected rhesus monkeys (1618). We showed that the thymus is infected from the first week post-SIV inoculation and that proliferative responses of thymocytes from infected animals are impaired in spite of a very low frequency of SIV-infected cells (<= 1 in 104 thymocytes). Since, on the other hand, histological examination revealed a gradual loss of cortical epithelial cells, it remained unclear whether the functional defects observed in thymocytes from SIV-infected animals reflected an impaired thymopoietic potential of the thymocytes themselves or was solely due to the destruction of the thymic microenvironment. In order to dissociate these aspects, we developed a xenogeneic fetal thymus organ culture (FTOC) system in which the monkey-derived thymocytes but not the mouse stromal cells are potential targets for SIV infection.

Immature triple-negative CD3CD4CD8 and CD3CD4+CD8 thymocyte subpopulations were isolated from SIV-infected and control rhesus monkeys, and examined for their capacity to differentiate into CD4+CD8+ and mature CD3+CD4+CD8 or CD3+CD4CD8+ cells in mouse fetal thymic lobes.

In addition, the effect of high-level SIV infection on thymopoiesis was analyzed by in vitro infection of thymocytes from uninfected monkeys. Finally, we investigated whether recombinant SIV gp120, when added into the FTOC, could influence T cell maturation.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Rhesus monkeys (Macaca mulatta) used in this study were of Chinese origin and kept at the German Primate Center, Göttingen as previously described (19). At the time of infection, the monkeys were between 31 and 42 months of age. All monkeys were seronegative for type D-virus, foamyvirus, STLV-1 (simian T cell lymphotropic virus-1) and SIV. Eight animals were inoculated i.v. with 1 ml of cell-free stock virus of SIVmac251 MPBMC (20) containing 100 MID50 (monkey infectious doses infecting 50% of recipients). At weeks 1, 2, 3, 7, 14, 17, 19 and 28 after infection, individual animals were sacrificed, and lymphocytes from blood, spleen, thymus and lymph nodes were isolated. Four additional rhesus monkeys served as controls and were sacrificed simultaneously with four infected animals (1, 2, 14 and 28 weeks post-infection). Fragments of freshly prepared organs were dispersed in IMDM (Sigma, Deisenhofen, Germany) supplemented with 10% heat-inactivated FCS, 0.07% glutamine, 0.025% penicillin and 0.025% streptomycin (all from Gibco/BRL, Eggenstein, Germany).

Purification of monkey thymocyte subpopulations
CD3CD4CD8 and CD3CD4+CD8 thymocytes from monkey thymus were purified by first depleting CD8+ cells with magnetic anti human CD8 beads (RPA-T8; Miltenyi Biotec, Bergisch Gladbach, Germany). The remaining CD8 cells were stained with the following anti-human mAb which cross-react with macaque lymphocytes (21): biotinylated anti-CD3 (FN18; BioSource, Camarillo, CA), anti-CD4–phycoerythrin (PE; OKT4; Ortho Diagnostic Systems, Neckargemünd, Germany) and anti-CD8–FITC (RPA-T8; PharMingen, Hamburg, Germany). CD3CD4CD8 and CD3CD4+CD8 subpopulations were electronically sorted on a FACS Vantage cell sorter (Becton Dickinson, San Jose, CA). Subsets showed >99% purity on reanalysis.

FTOC
An adaptation of the method described previously (22) for human–mouse xenogeneic FTOC was employed. Day 14 fetal thymuses of BALB/c mice were maintained in lobe medium (IMDM, 10% FCS) on top of Nucleopore polycarbonate filters (Costar, Bodenheim, Germany), supported by polyurethane sponges (Groll, Reutlingen, Germany) in the presence of 1.35 mM 2'-deoxyguanosine (Sigma) for 5 days to eliminate endogenous thymocytes (23). Purified CD3CD4CD8 and CD3CD4+CD8 monkey thymocytes were introduced into 2'-deoxyguanosine-treated thymic lobes in hanging drops. One murine thymic lobe and 5x104 monkey thymocytes were added to each well of a Terasaki plate (Nunc, Wiesbaden, Germany) in a total volume of 25 µl. The plates were inverted and kept in a humidified atmosphere in 7.5% CO2. After 40 h, free thymocytes were removed by washing and the recolonized lobes were transferred to sponge-supported nucleopore filters without addition of 2'-deoxyguanosine. For long-time propagation (>1 week), cultures were fed twice weekly with fresh medium. At the end of the culture period, single-cell suspensions were prepared from individual lobes and viable cells were counted by phase microscopy using a hematocytometer. In some experiments, purified recombinant viral gp120 (1, 2.5 and 5 µg/ml) was included during the hanging-drop recolonization period.

Immunofluorescence and flow cytometry
Cells were suspended in 100 µl PBS, pH 7.2, containing 0.2% BSA and 0.02% sodium azide, and treated with saturating concentrations of the respective antibodies for 20 min at 4°C. In addition to those mentioned above for cell sorting, the following anti-human mAb, cross-reacting with rhesus monkey antigens, were used for immunophenotyping of macaque lymphocytes: anti-CD2 (39C1.5), anti-CD14 (RMO52), anti-CD16 (3G8), anti-CD20 (H299B1), anti-CD34 (Immu133), anti-CD56 (B159; Coulter-Immunotech Diagnostics, Hamburg, Germany), anti-CD4 (OKT4), anti-CD38 (OKT10; Ortho Diagnostic Systems), anti-CD28 (Leu-28; Becton Dickinson) and anti-MHC I (W6/32; Serotec (Camon, Wiesbaden, Germany).

Cells were washed, fixed with 3.5% formaldehyde to inactivate SIV and analyzed in a FACScan flow cytometer (Becton Dickinson). Appropriate negative controls were used to set the cut-off points in dot plots and to calculate the fraction of positive cells. In addition, anti-mouse CD3 (145-2C11), CD4 (H129.19) and CD8 (Ly-2) mAb (all from PharMingen) were used to discriminate between murine and monkey cells.

Quantitative DNA PCR
Quantification of the SIV provirus was achieved by amplifying a 328 bp fragment of the nef gene of SIVmac251/32H using the nef-specific primers AD1 and AD2. The sequence of the primers was: AD1 5'-AGCTATTTCCATGAGGCGGTCCAA-3' position 9066–9090 and AD2 5''-ATTGCTCTTAGGGGAACTTTTGGC-3' position 9366–9390. The competitor fragment was constructed by performing PCR on the 328 bp fragment using AD1 and {Delta}-nef (5'-ATTGCTCTTAGGGGAACTTTTGGCCTTTTTCTTAGCTGGGTTTCTC-3') as reverse primer. This PCR led to a 70 bp deletion in the wild-type fragment which is sufficient for discrimination on an agarose gel. The competitor was ligated into the pCR2.1 plasmid (Invitrogen, San Diego, CA).

PCR analyses were carried out in a 100 µl reaction volume containing 2.5 U Taq polymerase (Pharmacia Biotech, Freiburg, Germany) and 0.02 mM dNTPs (Boehringer, Mannheim, Germany) in a thermo-cycler (Biometra, Göttingen, Germany). The PCR was carried out for 30 cycles with the following profile: 30 s at 95°C, 30 s at 60°C and 1.15 min at 72°C, an increasing elongation time of 15 s every 10 cycles, an initial ramp time of 5 min at 94°C, and a final elongation time of 10 min at 72°C.

DNA extracted from at least 1x105 cells was used per reaction. If the proviral load was below the detection level of 1x103 copies after the first PCR, a second PCR was performed with 5 µl of the first PCR product, using the same reaction conditions. Detection of the PCR products was carried out by electrophoresis on 2% agarose gel stained with ethidium bromide.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Purification of CD3CD4CD8 and CD3CD4+CD8 thymocytes from rhesus monkeys
In order to establish a chimeric monkey–mouse FTOC model for the study of rhesus monkey T cell development, a protocol for the purification of immature thymocytes was developed. First, CD8+ cells were depleted by means of magnetic beads (Fig. 1Go), resulting in a strong reduction of CD4+CD8+ thymocytes and a corresponding disappearance of CD3lo cells. The earliest stages of thymocyte development, i.e. CD3CD4CD8 and CD3CD4+CD8 cells, were then isolated by staining the remaining CD8 cells with mAb directed against CD3, CD4 and CD8, followed by electronic cell sorting. Reanalyses showed >99% purity of both subsets. Both subpopulations were CD2, CD38 and MHC I positive, and weakly expressed CD34 (data not shown). In order to evaluate contamination of the two subsets by cells of non-T lineages, expression of CD20 (B cells), CD14 (monocytes) and CD16/56 (NK cells) was studied. Whereas both preparations contained only a small fraction of B cells (<0.4%) and monocytes (<0.1%), the CD3CD4CD8 population contained 5–7% NK cells (not shown). The virtual absence of monocytes is of particular importance because these cells are susceptible to infection by the SIV isolate employed.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 1. Purification of thymocyte subpopulations. Unseparated thymocytes (a and b) were depleted of CD8+ cells (c and d), and sorted by FACS Vantage into CD3CD4CD8 (e and f) and CD3CD4+CD8 subsets (g and h).

 
Repopulation of alymphoid embryonic mouse thymic lobes with monkey thymocytes
Purified monkey thymocytes were cultivated in hanging drops for 40 h with BALB/c mouse fetal thymic lobes previously depleted of thymocytes, and transferred to sponge-supported nucleopore filters for further incubation. Figure 2Go illustrates the maturation of monkey CD3CD4CD8 thymocytes in this xenogeneic FTOC system. From day 3 to 14, new thymocyte phenotypes appeared in the order predicted by the known maturational sequence of human T cell development (8), i.e. CD3CD4+CD8, CD3CD4+CD8+, CD3+CD4+CD8+, and finally CD3+CD4+CD8 and CD3+CD4CD8+ cells. The expression of monkey CD2 on all lymphoid cells recovered indicated that depletion of mouse thymocytes from the embryonic lobes with 2'-deoxyguanosine had been complete. This result was confirmed by the absence of cells reactive with antibodies to mouse CD3, CD4 and CD8 (data not shown). Phenotypic maturation was accompanied by dramatic numerical expansion. Thus, we usually recovered only a few thousand viable cells within the first days of FTOC, which increased to 3x104 and 3x105 cells per thymic lobe on days 6 and 10 respectively, before starting to decline to 2.2x105 and 8x104 cells after 14 and 18 days of culture respectively. Starting from ~5000 monkey cells in a lobe after the hanging-drop period (day 0), the cell number at day 14 represents a 60-fold increase. Figure 3Go shows the phenotype of monkey thymocytes after 18 days in FTOC, and documents that CD4 and CD8 single-positive thymocytes recovered at that time point expressed a high level of CD3, indicative of cells completing thymic maturation. The conclusion that monkey thymocyte maturation in mouse FTOC closely resembles the physiological in vivo setting was further supported by histological and electron microscopy examination which revealed normal thymic structure and interactions between thymocytes and thymic epithelial cells (not shown).



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2. CD2, CD3, CD4 and CD8 phenotype of monkey thymocytes in chimeric organ culture. Purified CD3CD4CD8 thymocytes were cultured in hanging drops with 2'-deoxyguanosine-treated murine thymuses. After 40 h (day 0) lobes were placed on nucleopore filters after washing away unattached cells. As indicated, thymocytes were recovered, and stained for surface expression for CD3, CD4 and CD8 at different times showing phenotypic maturation. Murine and monkey thymocytes were discriminated by measuring expression of monkey CD2.

 


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3. CD3 expression of monkey thymocyte subpopulations after 18 days in FTOC. The dot-plot indicates expression of CD4 and CD8 in the recovered thymocyte subsets A–D. Histograms A–D illustrate the CD3 expression of the respective subpopulations.

 
Impaired thymopoietic potential of immature thymocytes from SIV-infected rhesus monkeys
In agreement with recent results obtained in mouse thymi repopulated with human cord blood derived T cells (6), we found that addition of infectious SIV led to destruction of developing macaque thymocytes in the xenogeneic FTOC system (data not shown). In view of the very low frequency of SIV-producing thymocytes observed in vivo (~1 in 104, 17), we next studied whether thymopoiesis of thymocytes derived from SIV-infected animals is also affected. A group of eight age-matched Chinese rhesus monkeys inoculated i.v. with cell-free SIVmac251 MPBMC was studied. Productive infection was confirmed by PCR and co-cultivation of peripheral blood lymphocytes with a susceptible indicator cell line (data not shown). CD4:CD8 ratios declined in infected animals after 2 weeks of infection in all organs examined. The animals appeared healthy and well nourished, and showed no signs of opportunistic infections, other overt disease or AIDS-associated malignancies at the time of sacrifice.

To test the impact of in vivo SIV infection on the thymopoietic potential of immature thymocyte subsets, CD3CD4CD8 and CD3CD4+CD8 cells were isolated from both uninfected and SIV-infected rhesus monkeys, and cultured in FTOC. At different time points (days 6, 10, 14, 18 and 22), thymic lobes were analyzed for cell numbers and phenotypes, and total numbers of the various subsets were calculated. Figure 4aGo shows that at 1 and 2 weeks post-infection, CD3CD4CD8 thymocytes from SIV-infected monkeys and uninfected controls developed into CD4/CD8 double-positive cells with comparable efficiency. At later stages of infection (14, 17, 19 and 28 weeks post-infection), however, the capacity of triple-negative thymocytes to differentiate in FTOC appeared to be slightly diminished (~2- to 3-fold) as compared to uninfected controls.




View larger version (78K):
[in this window]
[in a new window]
 
Fig. 4. Abundance and growth kinetics of CD4+CD8+ thymocytes in FTOC. Alymphoid murine embryonic thymic lobes were recolonized with purified CD3CD4CD8 (A) and CD3CD4+ CD8 (B) thymocytes from uninfected and SIV-infected monkeys. At the indicated days cells were recovered, counted, and stained for CD3, CD4 and CD8. By three-color flow cytometric analysis the percentage of double-positive cells was determined and multiplied with the absolute cell number per thymic lobe. The absolute number of CD4+CD8+ thymocytes is shown on a logarithmic scale.

 
In contrast to this rather mild impairment of progenitor function in the CD3CD4CD8 subset, a clear-cut effect was observed when CD3CD4+CD8 thymocytes isolated from SIV-infected animals were compared with control preparations (Fig. 4bGo). At all time points studied, SIV infection led to a pronounced reduction in thymopoietic potential which greatly exceeded that observed in CD3CD4CD8 cells isolated from the same animals. In the case of the animal sacrificed 2 weeks post-infection, for example, the number of CD4+CD8+ cells which developed from CD3CD4+CD8 thymocytes was up to 36-fold lower when compared to control cultures, while maturation and expansion of CD3CD4CD8 thymocytes was hardly affected. In addition, the generation of CD3high cells was determined in FTOC at day 18. Again, there was a marked negative effect of in vivo SIV infection when CD3CD4+CD8 and little effect if CD3CD4CD8 thymocytes were used to seed the lobes (not shown). Thus, within a few weeks after SIV infection, the potential of immature thymocytes to expand and differentiate is markedly impaired at the transitory CD3CD4+CD8 but appears relatively normal at the preceding CD3CD4CD8 stage.

Quantitation of proviral DNA in unseparated thymocytes
In order to determine the frequency of thymocytes that had been infected by SIV in vivo, an SIV-specific quantitative competitive PCR was performed as detailed in Methods with thymocytes of the four SIV-infected animals used as thymus donors for the experiments shown in Fig. 4Go. Only 0.2–5x102 SIV proviral copies/105 cells were found in the individual specimens, indicating a very low viral load in the thymus at the time of sacrifice.

Effect of gp120 on T cell maturation in FTOC
The very low (pro)viral input suggested that mechanisms distinct from direct virus-mediated cytolysis contribute to the impaired thymopoiesis of thymocytes from infected rhesus monkeys in FTOC. We therefore investigated the possibility that binding of circulating gp120 had influenced thymopoietic potential. For peripheral T cells it has been shown that binding of gp120 to CD4 is sufficient to induce anergy and/or apoptosis (24), and recent observations suggest that it may interfere with signals mediated by the common cytokine receptor {gamma} chain via JAK3, both of which are essential for IL-7-driven thymocyte expansion (25). As shown in Fig. 5Go, addition of gp120 in various concentrations (1, 2.5 and 5 µg/ml) resulted in a dose-dependent impairment of development of thymocytes from uninfected animals. Specifically, percentages and absolute numbers of developing CD4+CD8+ thymocytes were greatly reduced 10 days after addition of 5 µg/ml gp120, while the CD3CD4CD8 starting population was not affected. Thus, the results demonstrate that—at least in organ culture—gp120 is able to interfere with T cell maturation even in the absence of infectious virus.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. Effect of gp120 on T cell maturation in FTOC. CD3CD4CD8 thymocytes were isolated from three different uninfected rhesus monkey and SIV gp120 (5 µg/ml) was added during the hanging-drop recolonization period of the FTOC. Absolute numbers (A) and percentages (B) of developing thymocytes 10 days after addition of gp120 are shown.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have established a monkey–mouse xenogeneic FTOC system which allows the successful seeding and development of rhesus monkey thymocytes in fetal mouse thymus lobes, and have used this system to investigate possible effects of in vitro and in vivo SIV infection on the thymopoietic potential of defined immature monkey thymocytes.

The present monkey–mouse FTOC complements the recently developed human–mouse FTOC (22,26) as well as a co-culture system in which the differentiation of human (27,28) or monkey (29) CD34+ progenitor cells is supported by thymic stromal cells from rhesus monkeys. One major caveat about the use of mouse stroma in the xenogeneic FTOC is the potential lack of some species-specific molecular interactions between stroma and lymphocytes. On the other hand, we observed a normal thymic architecture and progression through well-established developmental stages. An advantage of the xenogeneic FTOC is that it allows us to investigate the effects of SIV infection on thymocyte development in the absence of virus-induced stromal alterations. The dramatic impairment of thymopoiesis as a result of SIV infection observed thus can be attributed to reduced progenitor cell function rather than to the destruction of the stroma which we have previously observed at later stages of SIV infection in situ (17).

In contrast to the almost complete abrogation of progenitor function in CD3CD4CD8 cells after in vitro infection with SIV (data not shown), such cells exhibited a near to normal repopulation potential when isolated from infected animals. This is in line with the very low viral load in the thymus (2–50 SIV proviral copies/104 cells) as detected by quantitative PCR in this study and previously reported by others (30). The difference in thymic repopulation potential of CD3CD4CD8 cells infected in vivo and in vitro is, therefore, likely to be due to the smaller number of cells infected in vivo, resulting in a viral load insufficient to cause appreciable damage in FTOC.

In contrast to CD3CD4CD8 thymocytes, the next intermediate on their differentiation pathway towards CD3+CD4+CD8+ cells, i.e. the CD3CD4+CD8 thymocytes, was clearly defective in thymopoietic potential when isolated from SIV-infected monkeys. This functional impairment is unlikely to be the direct consequence of infection. Thus, unless one assumes a dramatically higher viral load than that of the average thymocyte in this subset, indirect mechanisms are likely to affect these cells more strongly than their CD3CD4CD8 precursors. Indeed, a highly sensitive nested DNA PCR identified SIV DNA only in two out of four animals in the 105 cells of the CD3CD4+CD8 subset while CD3CD4CD8 cells were positive in all cases. This finding suggests that the viral load of CD3CD4+CD8 cells is not higher but may actually be lower than that of CD3CD4CD8 cells (data not shown). Possibly, those few CD3CD4CD8 cells which are infected do not differentiate into the downstream subsets and the short time spent in the transitory CD3CD4+CD8 compartment may not suffice to result in detectable infection.

The puzzling finding that in spite of a very low viral load, CD3CD4+CD8 cells displayed a marked functional impairment suggests that indirect effects rather than cytolysis by virus are likely to have damaged these cells already in vivo. Indirect mechanisms of interference with thymopoiesis by SIV infection are also in line with reports by others (12) who found a discrepancy between the frequencies of HIV-1-expressing and apoptotic cells in the thymus of HIV-infected SCID-hu mice. Moreover, it was found that T cell progenitors from the peripheral blood of HIV-infected patients display diminished differentiation potential even if virus replication is suppressed in vitro by anti-retroviral drugs (26). The possibility that binding of circulating viral gp120 had adversely affected the thymopoieteic potential of immature thymocytes in vivo is supported by the negative effects of gp120 added in vitro (Fig. 5Go). Accordingly, indirect gp120-mediated effects on T cell development are likely to play a prominent role in the physiological setting characterized by a strong peripheral and mild intrathymic infection. On the other hand, massive infection of thymocytes as achieved experimentally by direct intrathymic injection of HIV-1 in SCID-hu mice adversely affects the thymus by lytic infection of CD4+ cells and can be overcome by antiviral therapy (31).

In summary, our results indicate that thymopoiesis is disturbed in SIV-infected rhesus monkeys at the level of thymocyte progenitor function and that this impairment is unlikely to be due to the direct effects of infection of these immature thymocytes but rather caused indirectly by viral products. In line with our findings are recent reports (32,33) which provide strong evidence that the initial increase in blood CD4+ T cell counts after antiretroviral therapy is the result of redistribution of CD4+ T cells from other lymphoid compartments to peripheral blood and not due to production of new cells in the thymus. Moreover, normalization of CD4+ T cell numbers in blood and lymph node of these patients by 12–36 weeks after therapy was associated with only a limited renewal of CD4+ T cells (3335). It is unclear at present whether this impaired capacity to replenish the CD4 compartment results from a negative effect of shed gp120 on the clonogenic potential of mature T cells and/or on T lymphocyte precursors in the thymus as illustrated in this study. The xenogeneic monkey–mouse FTOC system presently described should facilitate the identification of the mechanism(s) by which infection with immunodeficiency viruses damages the progenitor function of thymocytes.


    Acknowledgments
 
This study was supported by grant BHBF 01 KI 9479 from the German Ministry of Research and Development, and by Fonds der Chemischen Industrie eV. We thank Tanja Vey for excellent technical assistance and Dr Christiane Stahl-Hennig for veterinary supervision of the animals. In addition, we gratefully acknowledge the generous contributions of Dr Thomas Hanke and Dr Richard Boyd.


    Abbreviations
 
FTOCfetal thymus organ culture

    Notes
 
The first two authors contributed equally to this work

Transmitting editor: H. R. MacDonald

Received 15 February 1999, accepted 31 May 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Fauci, A. S. 1988. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science 239:617.[ISI][Medline]
  2. Courgnaud, V., Laure, F., Brossard, A., Bignozzi, C., Goudeau, A., Barin, F. and Brechot, C. 1991. Frequent and early in utero HIV-1 infection. AIDS Res. Hum. Retroviruses 7:337.[ISI][Medline]
  3. Tanaka, K. E., Hatch, W. C., Kress, Y., Soeiro, R., Calvelli, T., Rashbaum, W. K., Rubinstein, A. and Lyman, W. C. 1992. HIV-1 infection of human fetal thymocytes. J. Acquir. Immune Defic. Syndr. 5:94.[Medline]
  4. Hirsch, V. M., Zack, P. M. and Johnson, P. R. 1990. Molecular characterization of SIV in tissues from experimentally infected macaques. J. Med. Primatol. 19:287.[ISI][Medline]
  5. Aldrovandi, G. M., Feuer, G., Gao, L., Jamieson, B., Kristeva, M., Chen, I. S. Y. and Zack, J. A. 1993. The Scid-hu mouse as a model for HIV-1 infection. Nature 363:732.[ISI][Medline]
  6. Greiner, D. L., Shultz, L. D., Deluca, D., Leif, J. H., Christianson, S. W. and Hesselton, R. M. 1996. HIV-1 infectivity of human T cells in a human/murine chimeric fetal thymic organ culture system. In Vivo 10:33.[ISI][Medline]
  7. Lanier, L. L., Allison, J. P. and Phillips, J. H. 1986. Correlation of cell surface antigen expression by multi-color flow cytometric analysis: implications for differentiation. J. Immunol. 137:2501.[Abstract/Free Full Text]
  8. Kraft, D. L., Weissman, I. L. and Waller, E. L. 1993. Differentiation of CD3CD4CD8 human fetal thymocytes in vivo: characterization of a CD3CD4+CD8 intermediate. J. Exp. Med. 178:265.[Abstract]
  9. Schuurman, H. J., Krone, W. J., Broekhuizen, R., van Baarlen, J., van Veen, P., Golstein, A. L., Huber, J. and Goudsmit, J. 1989. The thymus in acquired immunodeficiency syndrome. Comparison with other types of immunodeficiency diseases, and presence of components of human immunodeficiency virus type 1. Am. J. Pathol. 134:1329.[Abstract]
  10. Schnittman, S. M., Denning, S. M., Greenhouse, J. J., Justement, J. S., Baseler, M., Kurtzberg, J., Haynes, B. F. and Fauci, A. S. 1990. Evidence for susceptibility of intrathymic T cell precursors and their progeny carring T cell antigen receptor phenotypes TCR{alpha}ß+ and TCR{gamma}{delta}+ to human immunodeficiency virus infection: a mechanism for CD4+ (T4) lymphocyte depletion. Proc. Natl Acad. Sci. USA 87:7727.[Abstract]
  11. Tremblay, M., Numazaki, K., Goldman, H. and Wainberg, M. A. 1990. Infection of human thymic lymphocytes by HIV-1. J. Acquir. Immune. Defic. Syndr. 3:356.[Medline]
  12. Su, L., Kaneshima, H., Bonyhadi, M., Salimi, S., Kraft, D., Rabin, L. and McCune, J. M. 1995. HIV-1-induced thymocyte depletion is associated with indirect cytopathicity and infection of progenitor cells in vivo. Immunity 2:25.[ISI][Medline]
  13. Letvin, N. L., Daniel, M. D., Sehgal, P. K., Desrosiers, R. C., Hunt, R. D., Waldron, L. M., MacKey, J. J., Schmidt, D. K., Chalifoux, L. V. and King, N. W. 1985. Induction of AIDS-like disease in macaque monkeys with T cell tropic retrovirus STLV-III. Science 230:71.[ISI][Medline]
  14. Benveniste, R. E., Morton, W. R., Clark, E. A., Tsai, C. C., Ochs, H. D., Ward, J. M., Kuller, L., Knott, W. B., Hill, R. W., Gale, M. J. and Thouless, M. E. 1988. Inoculation of baboons and macaques with simian immunodeficiency virus/Mne, a primate lentivirus closely related to human immunodeficiency virus type 2. J. Virol. 62:2091.[ISI][Medline]
  15. Fahey, J. L., Prince, H., Weaver, M., Groopman, J., Visscher, B., Schwartz, K. and Detels, R. 1984. Quantitative changes in T helper or T suppressor/cytotoxic lymphocyte subsets that distinguish acquired immunodeficiency syndrome from other immune subset disorders. Am. J. Med. 76:95.[ISI][Medline]
  16. Kneitz, C., Kerkau, T., Müller, J., Coulibaly, C., Stahl-Hennig, C., Hunsmann, G., Hünig, T. and Schimpl, A. 1993. Early phenotypic and functional alterations in lymphocytes from simian immunodeficiency virus infected macaques. Vet. Immunol. Immunopathol. 36:239.[ISI][Medline]
  17. Müller, J. G., Krenn, V., Schindler, C., Czub, S., Stahl-Hennig, C., Coulibaly, C., Hunsmann, G., Kneitz, C., Kerkau, T., Rethwilm, A., ter Meulen, V. and Müller-Hermelink, H. K. 1993. Alterations of thymus cortical epithelium and interdigitating dendritic cells but no increase of thymocyte cell death in the early course of simian immunodeficiency virus infection. Am. J. Pathol. 143:699.[Abstract]
  18. Müller, J. G., Krenn, V., Schindler, C., Stahl-Hennig, C., Coulibaly, C., Hunsmann, G. and Müller-Hermelink, H. K. 1993. The thymic epithelial reticulum and interdigitating cells in SIV-induced thymus atrophy and its comparison with other forms of thymus involution. Res. Virol. 144:93.[ISI][Medline]
  19. Stahl-Hennig, C., Herchenröder, O., Nick, S., Evers, M., Stille-Siegener, M., Jentsch, K. D., Kirchhoff, F., Tolle, T., Gatesman, T. J., Lüke, W. and Hunsmann, G. 1990. Experimental infection of macaques with HIV-2ben, a novel HIV-2 isolate. AIDS 4:611.[ISI][Medline]
  20. Stahl-Hennig, C., Voss, G., Dittmer, U., Coulibaly, C., Petry, H., Makoschey, B., Cranage, M. P., Aubertin, A. M., Lüke, W. and Hunsmann, G. 1993. Protection of monkeys by a split vaccine against SIVmac depends upon biological properties of the challenge virus. AIDS 7:787.[ISI][Medline]
  21. Sopper, S., Stahl-Hennig, C., Demuth, M., Johnston, I. C., Doerries, R. and ter Meulen, V. 1997. Lymphocyte subsets and expression of differentiation markers in blood and lymphoid organs of rhesus monkeys. Cytometry 29:351.[ISI][Medline]
  22. Merkenschlager, M. and Fisher, A. G. 1992. Selective manipulation of the human T cell receptor repertoire expressed by thymocytes in organ culture. Proc. Natl Acad. Sci. USA 89:4255.[Abstract]
  23. Cohen, A., Lee, J. W. W., Dosch, H. M. and Gelfand, E. W. 1980. The expression of deoxyguanosine toxicity in T lymphocytes at different stages of maturation. J. Immunol. 125:1578.[Abstract/Free Full Text]
  24. Chirmule, N. and Pahwa, S. 1996. Envelope glycoproteins of Human Immunodeficiency Virus Type 1: profound influences on immune functions. Microbiol. Rev. 60:386.[Abstract]
  25. Selliah, N. and Finkel, T. H. 1998. JAK3 activation and rescue of T cells from HIV gp120-induced unresponsiveness. J. Immunol. 160:5697.[Abstract/Free Full Text]
  26. Clark, D. R., Ampel, N. M., Hallett, C. A., Yedavalli, V. R., Ahmad, N. and DeLuca, D. 1997. Peripheral blood from human immunodeficiency virus type 1-infected patients display diminished T cell generation capacity. J. Infect. Dis. 176:649.[ISI][Medline]
  27. Plum, J., DeSmedt, M., Defresne, M. P., Leclercq, G. and Vande-kerckhove, B. 1994. Human CD34+ fetal liver stem cells differentiate to T cells in a mouse thymic microenvironment. Blood 84:1587.[Abstract/Free Full Text]
  28. Yeoman, H., Gress, R. E., Bare, C. V., Leary, A. G., Boyse, E. A., Bard, J., Shultz, L. D., Harris, D. T. and DeLuca, D. 1993. Human bone marrow and umbilical cord blood cells generate CD4+ and CD8+ single positive T cells in murine fetal thymus organ culture. Proc. Natl Acad. Sci. USA 90:10778.[Abstract]
  29. Rosenzweig, M., Marks, D. F., Zhu, H., Hempel, D., Mansfield, K. G., Sehgal, P. K., Kalams, S., Scadden, D. T. and Johnson, R. P. 1996. In vitro T lymphopoiesis of Human and Rhesus CD34+ progenitor cells. Blood. 87:4040.[Abstract/Free Full Text]
  30. Baskin, G. B., Murphey-Corb, M., Martin, L. N., Davison-Fairburn, B., Hu, F. S. and Kuebler, D. 1991. Thymus in simian immunodeficiency virus-infected rhesus monkeys. Lab. Invest. 65:400.[ISI][Medline]
  31. Withers-Ward, E. S., Amado, R. G., Koka, P. S., Jamieson, B. D., Kaplan, A. H., Chen, I. S. Y. and Zack, J. A. 1997. Transient renewal of thymopoiesis in HIV-infected human thymic implants following antiretroviral therapy. Nat. Med. 3:1102.[ISI][Medline]
  32. Pakker, N. G., Notermans, D. W., de Boer, R. J., Roos, M. T. L., de Wolf, F., Hill, A., Leonard, J. M., Danner, S. A., Miedema, F. and Schellekens, T. A. 1998. Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection: A composite of redistribution and proliferation. Nat. Med. 4:208.[ISI][Medline]
  33. Fleury, S., de Boer, R. J., Rizzarde, G. P., Wolthers, K. C., Otto, S. A., Welbon, C. C., Graziosi, C., Knabenhans, C., Soudeyns, H., Bart, P.-A., Gallant, S., Corpataux, J.-M., Gillet, M., Meylan, P., Schnyder, P., Meuwly, J.-Y., Spreen, W., Glauser, M. P., Miedema, F. and Pantaleo, G. 1998. Limited CD4+ T cell renewal in early HIV-infection: effect of highly active antiretroviral therapy. Nat. Med. 4:794.[ISI][Medline]
  34. Hellerstein, M., Hanley, M. B., Cesar, D., Siler, S., Papageorgopoulos, C., Wieder, E., Schmidt, D., Neese, R., Macallan, D. and McCune, J. M. 1999. Directly measured kinetics of circulating T lymphocytes in normal and HIV-infected humans. Nat. Med. 5:83.[ISI][Medline]
  35. Douek, D. C., McFarland, R. D., Keiser, P. H., Gage, E. A., Massey, J. M., Haynes, B. F., Polis, M. A., Haase, A. T., Feinberg, M. B., Sullivan, J. L., Jamieson, B. D., Zack, J. A., Picker, L. J. and Koup, R. A 1998. Change in thymic function with age and during the treatment of HIV infection. Nature 396:690.[ISI][Medline]