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The Journal of Immunology, 2007, 178, 5762-5768
Copyright © 2007 by The American Association of Immunologists, Inc.
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T Cell Activation and Proliferation Characteristic for HIV-Nef Transgenic Mice Is Lymphopenia Induced1

Paul G. F. Koenen, Frans M. Hofhuis, Mariëtte A. Oosterwegel and Kiki Tesselaar2

Department of Immunology, University Medical Center, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The HIV-Nef protein has been implicated in generating high viral loads and T cell activation. Transgenic (tg) mice with constitutive T cell-specific Nef expression show a dramatic reduction in T cell number and highly increased T cell turnover. Previous studies in Nef tg mice attributed this T cell activation to a direct effect of Nef at the cellular level. Given the strongly reduced peripheral T cell numbers, we examined whether this enhanced T cell division might instead be lymphopenia induced. Adoptively transferred naive wild-type T cells into lymphopenic Nef tg mice showed high T cell turnover and obtained the same effector/memory phenotype as the autologous Nef tg T cells, supporting the idea that the microenvironment determines the phenotype of the T cells present. Moreover, in bone marrow chimeras from mixtures of wild-type and Nef tg bone marrow, with a full T cell compartment containing a small proportion of Nef tg T cells, Nef tg T cells kept a naive phenotype. These results demonstrate that T cell activation in the Nef tg mice is lymphopenia induced rather than due to a direct T cell-activating effect of Nef.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human immunodeficiency virus-Nef is one of the key determinants in the pathogenesis of AIDS. HIV strains with a modified or deleted Nef gene were found in HIV-infected long-term nonprogressor patients who remain asymptomatic for >10 years and maintain low viral loads and stable CD4 T cell counts (1, 2). In analogy, rhesus monkeys infected with Nef-deleted SIV strains maintain low or undetectable levels of virus and show a strongly attenuated clinical course of infection (3, 4).

Additional evidence for the importance of Nef in development of AIDS comes from a transgenic (tg)3 mouse model in which constitutive expression of the whole HIV genome in CD4 T cells and in cells of the monocyte/macrophage lineage leads to severe AIDS-like pathology (5). Further dissection of this mouse model leads to the conclusion that Nef alone was the major disease determinant (5, 6). Yet, the mechanism of Nef causing this severe phenotype in mice is still not understood.

Nef is a small protein of 27 kDa that is expressed abundantly in the early stages of viral replication. It is posttranslationally modified by myristilation of the N terminus, which targets Nef to the plasma membrane (reviewed in Ref. 7). Nef enhances viral replication (3). The best documented functions of Nef in vitro are down-regulation of CD4 and MHC class I cell surface expression via the endocytosis machinery (8, 9) and interference with TCR signaling (reviewed in Ref. 7). Simmons et al. (10) have demonstrated that Nef expression in Jurkat T cells induces a very similar gene expression profile as TCR signaling. Other groups have shown that Nef leads to enhanced activation after stimulation of the TCR and CD28, resulting in increased IL-2 production (11, 12). Two recent studies showed an effect of Nef on the threshold of T cell activation either by influencing formation of the immunological synapse or by down-regulation of the TCR-CD3 complex (13, 14). Numerous proteins have been reported to interact with Nef, many of which are part of the TCR signaling cascade, which is compatible with the effects of Nef expression on T cell activation in cell lines (reviewed in Ref. 7). Yet, functional evidence for such effects of Nef in primary T cells is very limited.

To study the consequence of Nef expression in primary cells in vivo, various Nef tg murine models have been generated (6, 15, 16, 17). All, except one, which expresses Nef in CD4 T cells as well as in cells of the monocyte/macrophage lineage (6), express Nef specifically in the T cell compartment. In all of these tg mouse strains, depletion of the T cell compartment is observed. In addition, T cells derived from secondary lymphoid organs showed increased activation (15, 18, 19). Upon viral challenge with vesicular stomatitis virus and lymphocytic choriomeningitis virus, it appeared that Nef tg T cells were less capable of inducing virus-specific CTL responses and to clear the virus (15). In line with this finding, Nef tg T cells had decreased in vitro proliferative capacities compared with wild-type (wt) T cells (15, 18, 19).

Altogether, these models show that Nef expression in the T cell compartment leads to T cell activation, decreased functional capacity of T cells, and thymic atrophy, all features of the T cell compartment of HIV-infected humans. These studies suggest that Nef expression in peripheral T cells of tg mice leads directly to HIV-like hyperactivation of T cells and thereby implies a dominant role for Nef on peripheral T cell functioning during HIV infection.

During HIV infection, T cells are chronically activated and have high proliferation rates (20, 21, 22, 23). Different causes for this activation have been proposed, varying from the presence of HIV viral load, which continuously drives immune activation (20), to particular activating effects of HIV proteins including Nef (20, 21, 22, 23).

We studied whether the T cell activation seen in Nef tg mice is indeed due to direct effects of Nef expression on T cells, compatible with HIV-induced hyperactivation, or whether this activation is lymphopenia induced, secondary to Nef-mediated thymic atrophy and T cell depletion. Adoptive transfer and chimeric mouse models demonstrated that in Nef tg mice peripheral T cell turnover results from lymphopenia, which is caused by Nef-mediated thymic atrophy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The CD2 Nef tg mice have been described previously (17) and were a gift from E. Dzierzak (Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands). Experiments were performed with mice 6–12 wk of age. Transgenic mice were compared with non-tg littermates. C57BL/6.SJL (Ly5.1) and C57BL/6 (Ly5.2) mice were obtained from The Jackson Laboratory and were maintained as a breeding colony in our animal facility. Ly5.1/Ly5.2 mice were bred by crossing (C57BL/6.SJL Ly5.1 x C57BL/6-Ly5.2). All mice were kept under specific pathogen-free conditions and housed in accordance with institutional guidelines of American Association of Accreditation of Laboratory Animal Care, in the mouse facility of the Central Laboratory Animal Institute Utrecht University (Utrecht, The Netherlands). The Institutional Animal Ethics Committee approved all experiments.

Abs and reagents

(BrdU)-APC flow kit, annexin V, mAbs (CD4 (RM4-5), CD8 (53-6.7), CD16/32 (2.4G2), CD44 (IM7), CD45.1 (A20), CD45.2 (104), and CD62L (MEL-14)), and streptavidin, unlabeled or labeled with appropriate fluorochromes, were all purchased from BD Biosciences.

CFSE labeling and adoptive transfer

Single-cell suspensions were prepared from peripheral lymph nodes (PLN). Cells were labeled with CFSE (Molecular Probes) as described previously (24). Briefly, cells were washed twice in PBS and resuspended at 107 cells/ml in PBS. CFSE was added to a final concentration of 0.5 µM, and cells were incubated for 10 min at 37°C. Unbound CFSE was quenched by washing labeled cells twice with RPMI 1640 medium supplemented with 10% FBS (Integro). A total of 5 x 106 cells was resuspended in 200 µl of PBS and injected i.v. into the tail vein of the mice.

Bone marrow transplantations

Bone marrow was obtained by the passage of iced RPMI 1640 through the tibias and femurs. T cells were depleted by use of CD90.2 (Thy1–2)-labeled magnetic beads and LD columns (Miltenyi Biotec), following the manufacturer’s instructions. Recipient mice were irradiated with a single sublethal dose of 7.0 Gy from an x-ray source. Within 6 h, the recipient mice were injected i.v. with a total number of 5 x 106 bone marrow cells.

Measurement of BrdU incorporation in vivo

BrdU (1 mg/mouse; Sigma-Aldrich) was injected three times i.p. with a time interval of 4 h, and mice were sacrificed 24 h after the first injection. Single-cell suspensions of lymphoid organs were prepared and cells were labeled with the appropriate Abs. Next, cells were fixed, permeabilized, and stained for BrdU with a BrdU-APC flow kit according to the manufacturer’s instructions.

Flow cytometry

Thymus, spleen, and PLN were forced through cell strainers (Falcon; BD Biosciences) in the presence of RPMI 1640 with 10% FCS to obtain single-cell suspensions. Erythrocytes were lysed by incubation of the cells in 0.15 M NH4Cl, 0.01 M KHCO3, and 0.1 mM EDTA (pH 7.4) for 2 min on ice. Cells were preincubated with Fc-block (mAb to CD16/32, 2.4G2; BD Biosciences) and washed in staining buffer (PBS, 0.5% BSA and 0.01% sodium azide). Afterward, cells were incubated with Abs and/or annexin V. Stained cells were analyzed using a FACSCalibur or BD LSR II and analyzed by CellQuest or BD FACSDiva software (BD Biosciences).

Statistical analysis

Results were analyzed using a Student’s t test (two-tailed). Differences between groups were considered statistically significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Strongly reduced T cell numbers and a highly activated T cell compartment in Nef tg mice

To determine the cause of peripheral T cell activation in vivo, we studied a Nef tg mouse model as described earlier by Dzierzak and coworkers (17, 18) (founder line F). In these mice, HIV-1BRU Nef gene expression is under control of the CD2 promoter and locus control region resulting in constitutive transgene expression in all T cells, starting during the earliest stages of T cell development. The most striking phenotype in these mice is a dramatic depletion of thymocytes (17). Total thymocyte cellularity of Nef tg mice was 10-fold reduced compared with wt littermates (Fig. 1A) as shown before (17). Nef expression disturbs thymocyte development, as evidenced by changes in thymic subset distribution, resulting in low numbers of single-positive thymocytes (17, 18) (data not shown).


Figure 1
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  		FIGURE 1. Nef tg mice have a strong reduced and highly activated T cell compartment. Thymocytes and T cells derived from Nef tg mice and wt littermates 6–8 wk old were counted and analyzed by flow cytometry. A, Absolute cell numbers of the thymus were determined. B, CD4 expression levels on thymocytes and T cells derived from lymph nodes were determined. C, Lymph nodes and spleens were recovered from tg mice and wt littermates of 6–8 wk old, and absolute CD4 and CD8 T cell numbers were determined. D, Spleen-derived CD4 and CD8 T cells were analyzed for differential expression of CD44 and CD62L by flow cytometry, and percentages of naive (N) and E/M T cells are indicated. Indicated are mean numbers ± SD of three mice (*, p < 0.005; **, p < 0.0005). Data shown are representative of three independent experiments.

 
As described before, Nef mRNA could be detected in thymocytes and peripheral T cells by RT-PCR (data not shown). Nef expression resulted in a reduced level of CD4 expression on both thymocytes (wt mean fluorescence intensity (MFI) of ~300 vs Nef tg ~135) and peripheral CD4 T cells (wt MFI of ~900 vs Nef tg ~650) (Fig. 1B), indicative for the presence of functional Nef protein. Analysis of peripheral lymphoid organs revealed a marked decrease in T cell numbers in both PLN and spleen (17) (Fig. 1C). Phenotypical analysis of tg peripheral T cells in previous studies using these Nef tg mice showed enhancement of T cell activation as evidenced by increased expression of the cell surface molecules CD25, CD69, and CD44 (17). Next to induction of these activation markers, we determined the composition of the peripheral T cell pool. Ag-inexperienced naive T cells can be distinguished from Ag-experienced effector and memory T cells on basis of their CD62L and CD44 expression levels. Although T cells expressing CD44mediumCD62Lhigh are defined as naive, CD44highCD62Llow and CD44highCD62Lhigh are defined as effector/memory (E/M/) cells (25, 26, 27).

Phenotypical analysis of spleen-derived T cells showed that in wt mice ~77% of all CD4 T cells and ~57% of all CD8 T cells were naive, compared with ~22% naive CD4 and ~12% naive CD8 T cells in Nef tg mice (Fig. 1D). Calculation of absolute T cell number showed a reduction of all T cell subsets in the Nef tg mice, which was most pronounced for naive T cells (Table I). Expression of activation markers was increased in Nef-expressing mice as was shown before.


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  		Table I. Shifted ratio of naive vs effector/memory T cells in Nef transgenic micea

 
Despite their severely decreased T cell numbers, Nef-expressing mice, housed in an open facility or under specific pathogen-free conditions, had a normal life span and showed no overt increase in infection or other physiological abnormalities when studied macroscopically. Taken together, in these tg mice Nef expression led to strong depletion of the thymus and to a depleted, activated E/M like peripheral T cell compartment.

Increased T cell turnover in Nef tg mice

Once we had observed low thymic cellularity and an activated peripheral T cell compartment, we examined the dynamics of the T cell pool. The proliferation of tg vs wt T cells was examined in vivo by measuring BrdU incorporation in DNA of dividing cells. Analysis of CD4 and CD8 T cells revealed a 6-fold increase in BrdU incorporation in both CD4 and CD8 T cells derived from the PLN of tg mice (as depicted in Fig. 2A), showing a 6-fold increase in cell proliferation compared with wt littermates. As a measure for cell death, we determined the fraction of annexin V-positive cells ex vivo by FACS analysis. A >7-fold increase in the percentage of death CD4 T cells vs a 3-fold increase among CD8 T cells derived from the PLN was measured in the tg mice (Fig. 2B). The fraction of dividing cells in the various T cell subsets did not differ between tg and wt mice. Increased overall turnover in T cells from tg mice was thus due to the increased proportion of E/M cells, which have a larger fraction of dividing cells compared with the naive T cell subset (data not shown) (26, 28).


Figure 2
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  		FIGURE 2. The T cell compartment of Nef tg mice has an increased cell turnover. The level of proliferation of peripheral T cells derived from PLN was determined by BrdU incorporation. Mice 6–8 wk old were injected three times with 1 mg of BrdU 1 day before analysis. A, The percentage of T cells that had incorporated BrdU is depicted. B, CD4 and CD8 T cells derived from PLN of tg mice and wt littermates 6–8 wk old were analyzed for the expression of annexin V by flow cytometry. Indicated are mean percentages of positive cells ± SD of three mice (*, p < 0.01; **, p < 0.001). Data shown are representative of three independent experiments.

 
In conclusion, increased T cell proliferation and apoptosis in Nef tg mice was observed and the majority of tg cells showed an activated E/M phenotype. This result resembled T cell characteristics observed in lymphopenic mice (27, 29, 30) and raised the question of whether proliferation and activation are driven by Nef directly or indirectly due to homeostatic effects.

Activation of normal T cells in Nef tg mice

We studied whether activation of the T cells was caused by an intrinsic Nef effect on peripheral T cells or was brought about by the strongly reduced thymic output and the concomitant T cell-depleted environment in Nef tg mice. Wild-type T cells, which could be distinguished from host cells by Ly5 allele expression, were adoptively transferred into Nef tg or wt mice of similar age. Before transfer, PLN-derived Ly5.1-expressing lymphocytes were labeled with CFSE, to monitor their proliferative behavior. The fate and phenotype of the transferred cells was determined in blood at day 3 and 6 after transfer and in spleen at day 10 after transfer (for overview experiment, see Fig. 3A). At day 3 after injection of the wt Ly5.1 T cells, no CFSE dilution was observed in either of the acceptor mice. Yet 6 days after injection proliferation was detectable in Nef tg mice (Fig. 3B). In wt recipients, proliferation was only detected at day 10 after transfer. At this time point, a fraction of ~85% of the transferred wt donor Ly5.1 CD4 T cells had not divided when transferred to wt mice, compared with ~54% when transferred to Nef tg mice (Fig. 3B). Of the transferred wt Ly5.1 CD8 T cells, ~87% had not divided in wt recipients vs ~63% in Nef tg recipients by day 10 (Fig. 3B). Based on the CFSE profiles, it seemed that the cell cycle progression of transferred wt Ly5.1 T cells was similar in wt and Nef tg mice; however, the number of cells entering the cell cycle seemed to be increased in Nef tg mice.


Figure 3
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  		FIGURE 3. Microenvironment in Nef tg mice leads to activation of T cells. PLN were isolated from 8-wk-old C57BL/6 Ly5.1 mice, lymph node suspensions were CFSE labeled, and 5 x 106 cells were transferred into 6- to 8-wk-old Nef tg and wt mice. A, Three, 6, and 10 days after transfer, blood and spleens were recovered and analyzed, respectively, and an overview of the experiment is shown. CFSE profiles of donor Ly5.1 CD4 and CD8 T cells derived from either Nef tg (tg + wt Ly5.1) or wt (wt + wt Ly5.1) mice were determined by flow cytometry in the blood at day 3 and 6 and in the spleen at day 10 posttransfer. B, Percentages of undivided cells are indicated. Splenocytes derived at day 10 posttransfer were incubated with mAbs directed against CD44 and CD62L. CFSE expression together with CD44 or CD62L on donor Ly5.1 T cells was determined, and percentages of undivided cells are indicated. C, The level of proliferation of donor Ly5.1 T cells derived from either wt or Nef tg spleens were determined by BrdU incorporation (for method, see Fig. 2). D, The proportion of Ly5.1 donor T cells that had incorporated BrdU is depicted. Data are representative of two mice per group and the experiment is performed three times.

 
Upon proliferation, CD44 was up-regulated and CD62L down-modulated, resulting in a higher proportion of CD44high and CD62Llow (Fig. 3C) wt donor T cells in the tg mice compared with wt littermates. In vivo BrdU incorporation was measured to quantify cell division. Higher division rates were observed for donor wt Ly5.1 T cells transferred into Nef tg mice than for cells transferred into wt mice (Fig. 3D). This result was in good agreement with the CFSE data. Thus, wt T cells transferred into T cell-depleted Nef tg animals become activated E/M cells, suggesting that the microenvironment in Nef tg mice is able to cause T cell activation irrespective of Nef expression in the peripheral T cells.

Normal phenotype of Nef tg T cells developing in a full T cell compartment

To dissect the effect of the T cell-depleted environment from the effect of expression of Nef on division and activation of Nef tg T cells, we examined the T cell activation status of tg cells in a nonlymphopenic environment. Therefore, we generated mixed bone marrow chimeras, where cotransfer of Nef tg and wt bone marrow resulted in Nef tg T cells in a normally developed T cell compartment of a mostly wt origin. Host and donor cells could be distinguished on the basis of Ly5 allele expression. Wild-type bone marrow-recipient mice of 10–12 wk old, which expressed both Ly5.1 and Ly5.2 on their hemopoietic cells, were sublethally irradiated and reconstituted with wt Ly5.1 bone marrow in combination with either Ly5.2 wt (wt/wt group) or Ly5.2 Nef tg (wt/tg group) bone marrow, in a ratio of 1:10 (Ly5.1:Ly5.2).

Blood analysis revealed that after 3 wk naive T cells numbers had increased, indicative of de novo thymic output. Six weeks after bone marrow transplantation, lymphocyte numbers in blood had stabilized and mice were sacrificed for ex vivo analysis of lymphoid organs (for overview experiment, see Fig. 4A).


Figure 4
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  		FIGURE 4. Nef tg T cells that developed in a full T cell compartment show a normal naive phenotype. Bone marrow was isolated from 6- to 10-wk-old C57BL/6 Ly5.1, Nef tg Ly5.2, and wt Ly5.2 littermates. After T cell depletion, 5 x 106 bone marrow cells were injected in 10- to 12-wk-old irradiated Ly5.1/Ly5.2 mice. One group of irradiated mice was injected with a mixture of Ly5.1 wt with Ly5.2 wt bone marrow (wt/wt group), and the other group of irradiated mice was injected with a mixture of wt Ly5.1 with Nef tg Ly5.2 bone marrow (wt/tg group), the ratio of bone marrow was 1:10 (Ly5.1:Ly5.2) for both groups. Six weeks after transfer, lymphoid organs were dissected and analyzed by flow cytometry, an overview of the experiment is shown (A). Single-cell suspensions derived from thymus and PLN were incubated with Abs directed against Ly5.1, Ly5.2, CD4, and CD8 and analyzed by flow cytometry. Absolute (B) and relative proportions (C) of the different Ly5 subsets in the thymus were determined for both the wt/wt and wt/tg group. D, The different Ly5 subsets present in the thymus were analyzed for differential expression of CD4 and CD8. E, Relative proportions of the Ly5.2 subset among CD4 and CD8 T cell derived from PLN were determined for both the wt/wt and wt/tg group. F, Ly5.1 wt and Ly5.2 tg lymphocytes derived from PLN of the wt/tg group were analyzed for CD4 expression levels by flow cytometry. T cells derived from PLN of mice from the wt/wt and wt/tg group were analyzed for differential expression of CD62L and CD44. Dot plots of one representative mouse from each group is shown, percentages of naive (CD44mediumCD62Lhigh) CD4 and CD8 T cells are depicted in the dot plots, and mean percentages of three mice are shown in bar charts (G). Data are representative of two experiments (one experiment with n = 3 per group and one experiment with n = 5 per group). No significant differences were found, using the Student’s t test. Indicated are mean percentages ± SD of three mice (*, p < 0.005; **, p < 0.0005).

 
Absolute cell numbers of the thymus derived from mice of the wt/wt group contained ~80 x 106 thymocytes, whereas thymi derived from mice of the wt/tg group contained ~50 x 106 thymocytes (Fig. 4B). Thymi derived from mice of the wt/wt group were composed of ~90% Ly5.2 thymocytes, due to the administered ratio of Ly5.1 and Ly5.2 bone marrow (1:10). The thymus of mice from the wt/tg group, however, contained ~15% thymocytes of the Ly5.2 lineage; the remaining 85% was predominantly from the Ly5.1 wt donor lineage (Fig. 4C). To substantiate functionality of the thymus, subset distribution within the thymus was determined by analyzing CD4 and CD8 expression profiles for the different Ly5 subsets in the thymus of mice from both wt/wt- and wt/tg-reconstituted groups. CD4 and CD8 single-positive thymocytes of all Ly5 subtypes were present (Fig. 4D), which indicated normal thymic development and an efflux of mature CD4 and CD8 T cells into the peripheral T cell compartment. Examination of total cellularity of the secondary lymphoid organs demonstrated rather normal T cell numbers among PLN and spleens of both the wt/wt and the wt/tg group (data not shown). The PLN-derived T cells from wt/wt-reconstituted mice consisted of ~50% of wt Ly5.2 T cells, whereas the wt/tg-reconstituted group contained only 1–2% of Nef tg Ly5.2 T cells in their PLN-derived T cells (Fig. 4E). This poor outgrowth of tg Ly5.2 T cells is compatible with the Nef-mediated block in T cell development.

To confirm Nef expression in peripheral T cells, the CD4 level on wt Ly5.1 and Nef tg Ly5.2 cells was measured in the wt/tg group. Wild-type cells expressed CD4 at MFI of ~124, whereas Nef tg cells expressed CD4 at MFI of ~73. This CD4 down-regulation is typical for a functional Nef protein being present in tg peripheral T cells (Fig. 4F). To study the activation state of the Nef tg T cells that developed in a full T cell compartment, the subset distribution of the different Ly5 populations in the wt/wt and wt/tg group were examined by measuring CD44 and CD62L expression by flow cytometry. As expected, no significant difference was found between the percentage of naive wt Ly5.1 and wt Ly5.2 T cells within the wt/wt group. This result was observed in both CD4 and CD8 T cells (Fig. 4G). Strikingly, the Ly5.2 Nef tg T cells had the same naive phenotype as the wt Ly5.1 T cells, which had developed together in the wt/tg group. This was observed for CD4 as well as for CD8 T cells (Fig. 4G). These data showed that when Nef tg T cells developed in a T cell-repleted normal environment, their behavior was similar to wt cells and they had a predominantly naive phenotype. We conclude that the T cell activation seen in Nef tg mice is mediated by lymphopenia-induced mechanisms rather than by an intrinsic effect of Nef expression on T cell activation and division.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we show that the cellularity of the thymus and, as a consequence, thymic output and T cell numbers in secondary lymphoid organs are dramatically reduced in Nef tg mice. Under these circumstances, the tg T cells are highly activated and show an increased turnover. By generating bone marrow chimeras, we were able to study tg T cells in a full T cell compartment. In contrast to the highly activated phenotype that was observed in Nef tg mice, Nef tg T cells in a full compartment showed an overall naive phenotype. This result strongly suggests that the T cell activation observed in Nef tg mice is a secondary effect of Nef-mediated abrogation of thymus output and that in this model, Nef does not lead to T cell activation directly. Our data closely resemble classical experiments in which T cells were adoptively transferred into lymphopenic mice leading to homeostatic proliferation and an E/M like phenotype (25, 28, 30). Our results thus are reminiscent of lymphopenia-induced mechanisms.

Based on the in vitro interference of Nef with TCR signaling, many of the in vivo effects of Nef in tg mice models have been ascribed to effects on T cell activation (7, 10). Studies by Jolicoeur and coworkers (19) in CD4C/HIV-Nef tg mice have shown similar thymic depletion and increased peripheral T cell activation and proliferation as we report here. To test whether this increased activation was TCR dependent, these investigators crossed their Nef tg mice with TCR tg mice (AD10). Also in these double-tg mice CD4 T cells had an activated E/M phenotype, suggesting an intrinsic Ag-independent effect of Nef on T cell activation (19). However, it was shown that under lymphopenic conditions TCR tg CD4 T cells can proliferate in the absence of their cognate Ag, resulting in the acquisition of an E/M-like phenotype (27), thereby questioning the former interpretation of their results.

Correlation between the expression levels of activation markers and the amount of Nef protein expressed in CD4 T cells in Nef tg mice further suggests an in vivo role for Nef in T cell activation (19). In these Nef tg mice, different CD4 populations, designated as CD4high and CD4low, with respectively low and high Nef expression have been described previously (19). In agreement with a direct effect of Nef on T cell activation, these two subpopulations also differed in their activation status, i.e., the more Nef protein expressed, the lower the CD4 expression, the more the cells appeared to be activated. However, when we evaluated the activation status of low and high CD4-expressing T cells derived from wt mice, expression of activation markers was also found to be unevenly distributed. Even in these wt mice, the CD4low-expressing T cells contained a higher fraction of cells expressing activation markers (our unpublished data). Apparently, this inverse correlation of CD4 and activation marker expression levels is a general phenomenon independent of Nef effects on T cell activation.

Next to TCR-dependent effects of Nef, in vitro studies have shown effects of Nef on CD28-mediated costimulation (11, 12). Crossing of our Nef tg mice on a B7-deficient background did not lead to any changes in the observed phenotype or in vitro characteristics of Nef tg T cells (our unpublished data), which questions the effect of Nef on CD28-mediated costimulation. Recently, it was proposed that two different mechanisms drive proliferation in a lymphopenic environment: homeostatic proliferation, which is slow, and IL-7 dependent and endogenous proliferation, which is fast and depends on competition of TCRs for stimulatory self-peptide/MHC interactions. It has been reported that CD28-mediated signals are not required for homeostatic proliferation but are necessary for endogenous proliferation. Although in our mice absence of CD28-mediated costimulation did not result in any change in the observed phenotype, this effect would point to homeostatic proliferation as the driving mechanism in our model (31).

Taken together, our data show that Nef expression in the T cell lineage of tg mice does not directly activate primary peripheral T cells. Instead, Nef expression severely affects T cell development in the thymus, resulting in lymphopenia and causing lymphopenia-induced division and differentiation of the remaining Nef tg T cells.


   Acknowledgments
 
We acknowledge F. Miedema, L. Meyaard, and J. Borghans for critically reading the manuscript. We thank A. Martens for helpful discussions and technical assistance concerning the bone marrow transfer-experiments. We thank E. Dzierzak for providing the CD2 Nef tg mice and R. Arens (Department of Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands) for supplying C57BL/6 Ly5.1-derived lymphocytes.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by Grant Number 6011 from Aids Fonds Netherlands. Back

2 Address correspondence and reprint requests to Dr. Kiki Tesselaar, Department of Immunology, University Medical Center, Lundlaan 6, Room KC02.085.2, 3584 EA Utrecht, The Netherlands. E-mail address: k.tesselaar{at}umcutrecht.nl Back

3 Abbreviations used in this paper: tg, transgenic; wt, wild type; PLN, peripheral lymph node; MFI, mean fluorescence intensity; E/M, effector/memory. Back

Received for publication June 28, 2006. Accepted for publication February 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kirchhoff, F., T. C. Greenough, D. B. Brettler, J. L. Sullivan, R. C. Desrosiers. 1995. Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N. Engl. J. Med. 332: 228-232. [Free Full Text]
  2. Deacon, N. J., A. Tsykin, A. Solomon, K. Smith, M. Ludford-Menting, D. J. Hooker, D. A. McPhee, A. L. Greenway, A. Ellett, C. Chatfield, et al 1995. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270: 988-991. [Abstract/Free Full Text]
  3. Kestler, H. W., III, D. J. Ringler, K. Mori, D. L. Panicali, P. K. Sehgal, M. D. Daniel, R. C. Desrosiers. 1991. Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65: 651-662. [Medline]
  4. Alexander, L., P. O. Illyinskii, S. M. Lang, R. E. Means, J. Lifson, K. Mansfield, R. C. Desrosiers. 2003. Determinants of increased replicative capacity of serially passaged simian immunodeficiency virus with nef deleted in rhesus monkeys. J. Virol. 77: 6823-6835. [Abstract/Free Full Text]
  5. Hanna, Z., D. G. Kay, M. Cool, S. Jothy, N. Rebai, P. Jolicoeur. 1998. Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease. J. Virol. 72: 121-132. [Abstract/Free Full Text]
  6. Hanna, Z., D. G. Kay, N. Rebai, A. Guimond, S. Jothy, P. Jolicoeur. 1998. Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95: 163-175. [Medline]
  7. Fackler, O. T., A. S. Baur. 2002. Live and let die: Nef functions beyond HIV replication. Immunity 16: 493-497. [Medline]
  8. Garcia, J. V., A. D. Miller. 1991. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 350: 508-511. [Medline]
  9. Schwartz, O., V. Marechal, S. Le Gall, F. Lemonnier, J. M. Heard. 1996. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat. Med. 2: 338-342. [Medline]
  10. Simmons, A., V. Aluvihare, A. McMichael. 2001. Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity 14: 763-777. [Medline]
  11. Schrager, J. A., J. W. Marsh. 1999. HIV-1 Nef increases T cell activation in a stimulus-dependent manner. Proc. Natl. Acad. Sci. USA 96: 8167-8172. [Abstract/Free Full Text]
  12. Wang, J. K., E. Kiyokawa, E. Verdin, D. Trono. 2000. The Nef protein of HIV-1 associates with rafts and primes T cells for activation. Proc. Natl. Acad. Sci. USA 97: 394-399. [Abstract/Free Full Text]
  13. Schindler, M., J. Munch, O. Kutsch, H. Li, M. L. Santiago, F. Bibollet-Ruche, M. C. Muller-Trutwin, F. J. Novembre, M. Peeters, V. Courgnaud, et al 2006. Nef-mediated suppression of T cell activation was lost in a lentiviral lineage that gave rise to HIV-1. Cell 125: 1055-1067. [Medline]
  14. Thoulouze, M. I., N. Sol-Foulon, F. Blanchet, A. Dautry-Varsat, O. Schwartz, A. Alcover. 2006. Human immunodeficiency virus type-1 infection impairs the formation of the immunological synapse. Immunity 24: 547-561. [Medline]
  15. Lindemann, D., R. Wilhelm, P. Renard, A. Althage, R. Zinkernagel, J. Mous. 1994. Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF/3'-long terminal repeat transgene. J. Exp. Med. 179: 797-807. [Abstract/Free Full Text]
  16. Skowronski, J., D. Parks, R. Mariani. 1993. Altered T cell activation and development in transgenic mice expressing the HIV-1 Nef gene. EMBO J. 12: 703-713. [Medline]
  17. Brady, H. J., D. J. Pennington, C. G. Miles, E. A. Dzierzak. 1993. CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration. EMBO J. 12: 4923-4932. [Medline]
  18. Pennington, S. A., J. Hugh, M. Brady, G. Colin, G. Miles, E. A. Dzierzak, D. J. Abraham. 1997. HIV-Nef severely impairs thymocyte development and peripheral T-cell function by a CD4-independent mechanism. Genes Funct. 1: 321-335. [Medline]
  19. Weng, X., E. Priceputu, P. Chrobak, J. Poudrier, D. G. Kay, Z. Hanna, T. W. Mak, P. Jolicoeur. 2004. CD4+ T cells from CD4C/HIVNef transgenic mice show enhanced activation in vivo with impaired proliferation in vitro but are dispensable for the development of a severe AIDS-like organ disease. J. Virol. 78: 5244-5257. [Abstract/Free Full Text]
  20. Hazenberg, M. D., J. W. Stuart, S. A. Otto, J. C. Borleffs, C. A. Boucher, R. J. de Boer, F. Miedema, D. Hamann. 2000. T-cell division in human immunodeficiency virus (HIV)-1 infection is mainly due to immune activation: a longitudinal analysis in patients before and during highly active antiretroviral therapy (HAART). Blood 95: 249-255. [Abstract/Free Full Text]
  21. McCune, J. M.. 2001. The dynamics of CD4+ T-cell depletion in HIV disease. Nature 410: 974-979. [Medline]
  22. Mohri, H., A. S. Perelson, K. Tung, R. M. Ribeiro, B. Ramratnam, M. Markowitz, R. Kost, A. Hurley, L. Weinberger, D. Cesar, et al 2001. Increased turnover of T lymphocytes in HIV-1 infection and its reduction by antiretroviral therapy. J. Exp. Med. 194: 1277-1287. [Abstract/Free Full Text]
  23. Kovacs, J. A., R. A. Lempicki, I. A. Sidorov, J. W. Adelsberger, B. Herpin, J. A. Metcalf, I. Sereti, M. A. Polis, R. T. Davey, J. Tavel, et al 2001. Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV. J. Exp. Med. 194: 1731-1741. [Abstract/Free Full Text]
  24. Lyons, A. B., C. R. Parish. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171: 131-137. [Medline]
  25. Goldrath, A. W., M. J. Bevan. 1999. Selecting and maintaining a diverse T-cell repertoire. Nature 402: 255-262. [Medline]
  26. Surh, C. D., J. Sprent. 2002. Regulation of naive and memory T-cell homeostasis. Microbes Infect. 4: 51-56. [Medline]
  27. Tanchot, C., A. Le Campion, S. Leaument, N. Dautigny, B. Lucas. 2001. Naive CD4+ lymphocytes convert to anergic or memory-like cells in T cell-deprived recipients. Eur. J. Immunol. 31: 2256-2265. [Medline]
  28. Tough, D. F., J. Sprent. 1994. Turnover of naive- and memory-phenotype T cells. J. Exp. Med. 179: 1127-1135. [Abstract/Free Full Text]
  29. Goldrath, A. W., L. Y. Bogatzki, M. J. Bevan. 2000. Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation. J. Exp. Med. 192: 557-564. [Abstract/Free Full Text]
  30. Cho, B. K., V. P. Rao, Q. Ge, H. N. Eisen, J. Chen. 2000. Homeostasis-stimulated proliferation drives naive T cells to differentiate directly into memory T cells. J. Exp. Med. 192: 549-556. [Abstract/Free Full Text]
  31. Min, B., W. E. Paul. 2005. Endogenous proliferation: burst-like CD4 T cell proliferation in lymphopenic settings. Semin. Immunol. 17: 201-207. [Medline]




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