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The Journal of Immunology, 2006, 176: 3342-3349.
Copyright © 2006 by The American Association of Immunologists

In Vitro Suppression of CD8+ T Cell Function by Friend Virus-Induced Regulatory T Cells1

Shelly J. Robertson, Ronald J. Messer, Aaron B. Carmody and Kim J. Hasenkrug2

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T cell (Treg)-mediated suppression of CD8+ T cells has been implicated in the establishment and maintenance of chronic viral infections, but little is known about the mechanism of suppression. In this study an in vitro assay was developed to investigate the suppression of CD8+ T cells by Friend retrovirus (FV)-induced Tregs. CD4+CD25+ T cells isolated from mice chronically infected with the FV suppressed the development of effector function in naive CD8+ T cells without affecting their ability to proliferate or up-regulate activation markers. In vitro restimulation was not required for suppression by FV-induced Tregs, correlating with their high activation state in vivo. Suppression was mediated by direct T cell-T cell interactions and occurred in the absence of APCs. Furthermore, suppression occurred irrespective of the TCR specificity of the CD8+ T cells. Most interestingly, FV-induced Tregs were able to suppress the function of CD8+ effector T cells that had been physiologically activated during acute FV infection. The ability to suppress the effector function of activated CTLs is likely a requisite role for Tregs in limiting immunopathology by CD8+ T cells during antiviral immune responses. Such activity may also have adverse consequences by allowing viruses to establish and maintain chronic infections if suppression of antiviral immune responses occurs before virus eradication.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During the last decade, a subset of CD4+ T cells has been identified that induces peripheral tolerance to self-Ags and prevents autoimmune diseases (1, 2, 3). These cells, termed "natural" regulatory T cells (Tregs),3 constitutively express the IL-2R {alpha}-chain (CD25) (4, 5), and their suppressive activity depends on expression of the transcriptional repressor Foxp3 (6, 7, 8, 9). It has been reported that there is a significant pool of natural Tregs that are continuously stimulated by self-Ags in vivo, thereby producing a basal level of T cell suppression to maintain self tolerance (10). In vitro assays developed to assess the suppressive activity of the natural Treg require in vitro activation or reactivation of the Treg to achieve suppression (11, 12). Thus, it appears that constant antigenic stimulation may be required for natural Treg suppression. Numerous studies have demonstrated that a predominant suppressive effect of natural Tregs in vitro is to inhibit the proliferative responses of CD4+ (12, 13) or CD8+ T cells (14, 15, 16).

More recently, Tregs have been implicated in the down-regulation of immune responses to foreign Ags (17, 18, 19, 20, 21). These induced Tregs are thought to minimize the immunopathological consequences of T cell responses to microbial infection (5, 19, 22). For example, in a mouse model of ocular infection with HSV, depletion of Tregs results in more severe immunopathological eye lesions (23). Not surprisingly, infectious organisms have evolved mechanisms to subvert the immunosuppressive properties of Tregs to establish and/or maintain chronic infections. For example, mice infected with Leishmania major undergo an expansion of natural Tregs at the site of infection that prevents eradication of the pathogen (20). Thus, pathogen-specific Tregs may arise from the pool of natural Tregs.

In studies of Friend retrovirus (FV) (24, 25, 26) infection of mice, it was found that immunosuppressive CD4+ T cells (17) facilitated virus persistence by inhibiting CD8+ T cell responses. Interestingly, inhibition was not due to diminished CD8+ T cell proliferation or activation, but rather to the inhibition of effector functions (27). In this study, we describe an in vitro assay used to investigate the molecular mechanisms of FV-induced Treg suppression of CD8+ T cell function. These experiments focus primarily on the subset of CD4+CD25+ T cells because it contains the most potent suppressive activity in vitro. Findings from this assay closely duplicate the known activity of FV-induced Tregs in vivo and provide new insights into the properties and mechanisms of action of these cells. This assay demonstrates suppression through cell-to-cell interactions between Tregs taken directly ex vivo and activated CD8+ T cells. Furthermore, because there is no requirement for in vitro activation of the Treg, the cellular responses probably reflect the physiological state more closely than assays that use artificial ligands such as anti-CD3 to stimulate suppressive activity.


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

Experiments were conducted using 12- to 24-wk-old female (C57BL/10 x A.BY) F1 mice bred at the Rocky Mountain Laboratories. The relevant FV resistance genotype of these mice is H-2b/b, Fv1b/b, Fv2r/s, and Rfv3r/s. FV-specific TCR transgenic (Tg) mice, which carry a transgene for a TCR that recognizes the Gag leader peptide of FV (28, 29), were also bred at Rocky Mountain Laboratories. For in vivo activation studies, (FV-specific TCR x B6.GFP) F1 mice were bred to obtain FV-specific TCR Tg CD8+ T cells that express the GFP (30). OT-1 TCR Tg mice that carry a TCR transgene specific for OVA residues 257–264 (31) were purchased from The Jackson Laboratory. Mice were treated in accordance with the regulations and guidelines of the Animal Care and Use Committee of the Rocky Mountain Laboratories and the National Institutes of Health.

Virus and virus infection

The FV stock used in these experiments was uncloned FV complex containing a B-tropic Friend murine leukemia helper virus and a polycythemia-inducing, spleen focus-forming virus (32). Mice were infected by i.v. injection of 0.5 ml of phosphate-buffered balanced salt solution containing 2% FBS and 1500 spleen focus-forming units of FV complex. Mice that had recovered from FV-induced splenomegaly were considered chronically infected at 8 wk postinfection (33). Virus levels stabilize at ~104 infectious centers per spleen by 6–8 wk postinfection (33).

Abs, surface and intracellular staining, and flow cytometry

Abs used for sorting and cell analysis were purchased from BD Pharmingen except where noted. The Abs used in flow analysis were as follows: biotin-labeled anti-glucocorticoid-induced TNFR (GITR) (DTA-1 hybridoma) (34); FITC-, PE-Cy7-, and allophycocyanin-anti-CD4 (RM4-5); PE- and allophycocyanin-anti-CD25 (PC61); FITC-anti-CD43 (1B11); PE-anti-CD44 (IM7); biotin-anti-CD45RB (16A); FITC-CD62L (MEL-14); PE-anti-CD69 (HI-2F3); PE-anti-LAG3 (CD223) (C9B7W); FITC-anti-CD103 (M290); PE- and allophycocyanin-anti-CD8 (53-6.7); PE-anti-CD152 (UC10 4F10 11); biotin-anti-goat IgG (Vector Laboratories); goat anti-mouse granzyme B (R&D Systems); and PE-anti-Foxp3 (FJK-16s) (eBioscience). Spleen cell suspensions were incubated with fluorochrome-conjugated or biotinylated primary Abs. Samples labeled with biotinylated Abs were subsequently incubated with allophycocyanin-Cy7-streptavidin (Caltag Laboratories). Before analysis, cells were resuspended in PBS containing 2% FBS and propidium iodide (2 µg/ml). Dead cells (propidium iodidehigh) were excluded from all cell surface analyses. For intracellular CD152 staining, cells were first incubated with FITC-anti-CD4 and allophycocyanin-anti-CD25. The cells were then washed, fixed with 3.7% formaldehyde, permeabilized with 0.1% saponin in PBS, and incubated with PE-anti-CD152. Intracellular Foxp3 staining was performed using the PE-anti-mouse/rat Foxp3 staining kit (eBioscience) according to the supplier’s instructions after surface labeling with FITC-anti-CD4 and allophycocyanin-anti-CD25. Intracellular granzyme B staining was performed using a method described by R.A. Robins on the Sanquin Blood Supply Foundation (Amsterdam, The Netherlands) web site (<www.sanquinreagents.com>). Briefly, cultured cells were incubated with PE-anti-CD8, fixed overnight in 0.5% paraformaldehyde in PBS, and permeabilized with 0.1% saponin in PBS containing 0.1% sodium azide, 0.5% BSA, and 50 mM glucose. Cells were washed and incubated with goat anti-mouse granzyme B diluted in PBS/saponin/sodium azide containing 10% FBS. Bound Ab was then detected by sequential incubation with biotin-anti-goat IgG and allophycocyanin-streptavidin (BD Pharmingen). Data were acquired on either a FACSCalibur or FACSAria flow cytometer (BD Biosciences) and analyzed using FlowJo software, version 6.1.1 (Tree Star).

Enrichment of lymphocyte subsets

The MidiMACS separation system (Miltenyi Biotec) was used for the enrichment of T lymphocyte subsets as recommended by the manufacturer. Briefly, spleen cell suspensions were incubated sequentially with an anti-Fc{gamma}III/II receptor (2.4G2) to block FcR and then with biotin-conjugated anti-CD25 (PC61 5.3) (Caltag Laboratories) and streptavidin paramagnetic microbeads (MACS beads; Miltenyi Biotec). Labeled cells were purified by immunomagnetic separation as recommended by the manufacturer. CD4+CD25– T cells were subsequently purified from the CD25-depleted fraction using anti-CD4 MACS beads (Miltenyi Biotec). The purity of the cell fractions was monitored by flow cytometry and was >80%. All cell fractions contained <5% dead cells after the separation procedure. Analysis of CD69 and CD43 expression on cells prepurification and postpurification indicated that the procedure had not activated the cells.

In vitro suppression assay and CFSE labeling

FV-specific TCR Tg CD8+ T cells were purified using anti-CD8{alpha} MACS beads and the MidiMACS system. The CD8-depleted spleen cells from TCR Tg mice served as the APCs, which were incubated with DbGagL peptide (5 µg/ml) (28) in IMDM (Cambrex) containing 10% normal mouse serum for 60 min at 37°C, irradiated (3000 rad), and washed two times with IMDM. For cocultures, 3 x 105 each of TCR Tg CD8+ T cells and peptide-pulsed APC were cultured per well of a flat-bottom 96-well plate in IMDM containing 10% FBS, 2 mM L-glutamine, 50 µM 2-ME, and 100 U/ml each penicillin and streptomycin at 37°C with 5% CO2. Naive CD4+CD25– (3 x 105) T cells were also included in cocultures for optimal IFN-{gamma} expression by stimulated CD8+ T cells. Unless otherwise stated, CD4+CD25+ (6 x 105) T cells from either naive or chronically infected mice were added to each well for a 2:1 ratio of Treg to CD8+ T cells. In Transwell experiments, CD4+CD25+ T cells were added to the upper chamber of 6.5-mm Transwell plate (Corning Costar) equipped with 1.4-µm pore-sized membrane. Coculture supernatants were collected after 60 h and assayed for IFN-{gamma} content by ELISA. For assessment of proliferation, purified FV-specific TCR Tg CD8+ T cells were labeled with CFSE (Molecular Probes) before coculture as described previously (27, 35).

IFN-{gamma} ELISA

IFN-{gamma} ELISA plates were prepared by coating 96-well Immulon II plates (Thermo Lab Systems) with 2 µg/ml purified anti-mouse IFN-{gamma} capture Ab (R4-6A2) (BD Pharmingen) in sodium carbonate buffer (pH 9.6). Culture supernatants diluted in PBS containing 5% FBS were added to the wells and incubated for 2 h at room temperature. Plates were washed four times with PBS containing 0.5% Tween 20 and incubated for 2 h at room temperature with biotinylated anti-mouse IFN-{gamma} (XMG1.2) (BD Pharmingen). Bound Ab was detected with HRP-conjugated streptavidin (Calbiochem) and developed with tetramethylbenzidine substrate. All culture supernatants were assayed in duplicate, and concentrations of IFN-{gamma} were determined based on a standard curve using purified mouse IFN-{gamma} (BD Pharmingen).

Preactivation and purification of virus-specific CTLs

FV-specific GFP+CD8+ T cells were purified using anti-CD8{alpha} MACS beads. To activate the purified FV-specific GFP+CD8+ T cells, 8 x 106 cells in 0.5 ml of PBS solution containing 15 U/ml heparin were adoptively transferred by i.v. injection into mice infected with FV for 2 days. After 4 days, activated GFP+CD8+ T cells were reisolated from the spleens of adoptively transferred mice by first enriching for CD8+ T cells with anti-CD8{alpha} MACS beads. Enriched cells were then stained with PE-anti-CD44 and allophycocyanin-anti-CD8 and sorted for GFP-positive CD44+CD8+ cells on a FACSAria flow cytometer (BD Biosciences) to obtain purities of >95%. Purified cells were then cocultured with CD4+CD25+ T cells from either naive or chronically infected mice in the presence of DbGagL MHC class I tetramer (Beckman Coulter) (36).

Intracellular cytokine staining

For intracellular cytokine staining, cocultured cells were incubated at 37°C and 5% CO2 with brefeldin A (10 µg/ml) for the last 5 h. Cells were then washed, stained with FITC-anti-CD8, fixed in 3.7% formaldehyde, permeabilized with 0.1% saponin in PBS, and incubated with allophycocyanin-anti-IFN-{gamma}. Data was collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo, version 6.1.1 (Tree Star).

Statistical analysis

Statistical significance was determined using two-tailed unpaired t tests except for Fig. 2A, where a paired t test was used. Values of p < 0.05 were considered statistically significant.


Figure 2
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  		FIGURE 2. Effects of CD4+CD25+ T cells from chronically infected mice on stimulated CD8+ T cell proliferation, activation, and effector function. A, CFSE-labeled FV-specific CD8+ T cells were stimulated with peptide-pulsed APCs and cocultured in the presence of CD4+CD25+ or CD4+CD25– T cells from either naive or chronically infected mice. After 60 h the CFSE content of CD8+ T cells was determined by flow cytometry. Representative CFSE histograms from CD8-gated cells are shown. Data from three separate experiments were analyzed by paired t test to determine the mean percentages of cells that had undergone more than two divisions. For cocultures with naive CD4+CD25+ T cells the mean was 43.2%, and for cocultures with CD4+CD25+ T cells from chronically infected mice the mean was 38.4%. This difference was not statistically significant (n = 11 mice per group; p = 0.1340). B, Stimulated FV-specific CD8+ T cells were cocultured in the presence of CD4+CD25+ T cells from naive mice or chronically infected mice at various ratios of CD4+ to CD8+ T cells as indicated. After 60 h the culture supernatants were collected and analyzed for IFN-{gamma} by ELISA. Cocultures containing CD4+CD25+ T cells from chronically infected mice at ratios of 2:1 and 1:1 produced significantly lower levels of IFN-{gamma} when compared with cocultures containing naive CD4+CD25+ T cells (*, p = 0.0007 and 0.0155, respectively). Values represent the mean ± SD of IFN-{gamma} (ng/ml) and were obtained from analysis of four mice per group. Similar results were obtained in three independent experiments. C, Histogram overlays of CD43 and CD69 activation markers on unstimulated CD8+ T cells (thin line); CD8+ T cells stimulated with peptide-pulsed APCs ( {permzspch020}) and stimulated CD8+ T cells c ocultured with CD4+CD25+ T cells from chronically infected mice (thick line) demonstrate very similar up-regulation of CD43 and CD69 following activation regardless of the presence of FV-induced Tregs. D, OVA-specific OT-I CD8+ T cells were stimulated with OVA peptide-pulsed APCs in cultures containing one of the following: 1) no additional cells as control (No Treg); 2) CD4+CD25+ from naive mice (CD25+/Naive); 3) CD4+CD25– T cells from chronically infected mice (CD25–/Chronic); or 4) CD4+CD25+ from chronically infected mice (CD25+/Chronic). A parallel in vitro assay using FV-specific CD8+ T cells and FV peptide-pulsed APCs was included for comparison. Data are from cells from four individual chronically infected mice, and bar graphs depict the mean IFN-{gamma} levels (ng/ml) ± SEM in supernatants after 60 h. Compared with the no Treg controls, CD4+CD25+ T cells from chronically infected mice suppressed IFN-{gamma} production by OT-1-specific CD8+ T cells ( {permzspch069}) (p = 0.00003) as well as FV-specific CD8+ T cells ( {permzspch142}) (p = 0.0003).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Chronic FV infection is associated with increased numbers of CD4+CD25+ T cells and changes in their phenotype

The IL-2R {alpha}-chain (CD25) is expressed on a subset of CD4+ T cells that exhibit suppressive activity, and this marker has commonly been used to identify natural Tregs (2). To determine whether chronic FV infection resulted in changes in this cell population, we assessed the proportion and absolute numbers of CD4+CD25+ T cells in infected mice compared with naive control mice. Flow cytometric analysis of splenocytes from FV-infected and naive mice revealed no significant difference in the percentage of lymphocytes coexpressing CD4 and CD25 (Fig. 1A) or in the percentage of CD4+ T cells that coexpressed CD25 (Fig. 1B). However, chronically infected mice had 2- to 3-fold higher numbers of CD4+CD25+ T cells compared with naive mice because of their enlarged spleens (Fig. 1A).


Figure 1
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  		FIGURE 1. FV infection is associated with increased numbers of phenotypically distinct CD4+CD25+ Tregs. A, Flow cytometric analysis of freshly isolated spleen cells stained for cell surface CD4 and CD25. The data represent the percentage of lymphocytes expressing both CD4 and CD25 ( {permzspch142}) and the absolute numbers of CD4+CD25+ T cells per spleen ( {permzspch069}) in naive and chronically infected mice. Chronically infected mice had significantly higher numbers of CD4+CD25+ T cells compared with naive mice (*, statistical significance; p = 0.0002). Data were compiled from analysis of four individual mice per group per experiment and are representative of three independent experiments. B, Flow cytometric analysis of freshly isolated spleen cells stained for cell surface CD4 and CD25. The data represent the percentage of CD4+ T cells coexpressing CD25. For naive mice, n = 15; for chronically infected mice, n = 16 (p = 0.1540). C, Histograms of the relative levels of expression of Foxp3, CD69, CD152, CD45RB, CD62L, GITR, LAG-3, and CD103 on CD4/CD25 double-positive cells from chronically infected mice (thick line) compared with naive mice (shaded) are shown. Differences in the mean fluorescence intensities (MFI) of GITR, LAG-3, CD69, and CD103 were statistically significant (p = 0.0162, 0.0015, 0.0052, and 0.0017, respectively). Data are representative of four or more individual mice per group.

 
Additional markers have been associated with Tregs and include Foxp3 (6, 7, 37), GITR (34, 38), CD152 (CTLA-4) (39), CD103 (40, 41), lymphocyte activation gene (LAG) 3 (42), CD62L (10, 43, 44), CD45RB (45), and CD69 (17). To determine whether expression of any of these proteins was altered on CD4+CD25+ T cells from chronically infected mice, each marker was assessed by flow cytometry. On CD4+CD25+ T cells, no significant differences were observed for Foxp3, CD62L, CD152, or CD45RB when comparing naive and chronically infected mice (Fig. 1C). However, there were slight but significant increases in the expression of GITR and LAG-3 and a striking increase in expression of CD69. Up-regulation of these surface markers has previously been associated with T cell activation (34, 38, 42, 46, 47). In addition, CD4+CD25+ T cells from chronically infected mice had markedly increased expression of CD103, an adhesion molecule that is up-regulated in Tregs responding to inflammatory stimuli. Thus, chronic FV infection was associated with increased activation and increased numbers of cells in the splenic CD25+CD4+ natural Treg subset, but no significant change in the proportion of CD4+ T cells expressing CD25 was found.

CD4+CD25+ and CD4+CD25– T cells from mice chronically infected with FV do not suppress Ag-induced CD8+ T cell proliferation

To investigate the means by which FV-induced Tregs suppress CD8+ T cells, an in vitro coculture assay was developed that examined the effects of Tregs on CD8+ T cell proliferation, activation, and effector function. CD4+CD25+ and CD4+CD25– T cells were purified from the spleens of both naive and chronically infected mice. With no in vitro stimulation, these CD4+ T cell subsets were then cocultured with FV-specific CD8+ T cells from mice carrying a TCR transgene specific for a FV epitope (DbGagL). The FV-specific CD8+ T cells were stimulated with peptide-pulsed APCs. To assess the effects on CD8+ T cell proliferation, these cells were labeled with the fluorescent dye CFSE before coculture. CFSE content was measured by flow cytometry following 60 h in culture. As shown in Fig. 2A, neither CD25+ nor CD25– subsets of CD4+ T cells from FV-infected mice affected the proliferation of stimulated CD8+ T cells.

CD4+CD25+ T cells from mice chronically infected with FV suppress IFN-{gamma} production by both virus-specific and nonspecific CD8+ T cells

Production of IFN-{gamma} is an important mechanism by which activated CD8+ T cells mediate antiviral responses, including FV infections (48, 49, 50). As a measure of whether FV-induced Tregs influenced CD8+ T cell effector function, we next examined the production of IFN-{gamma} by CD8+ T cells in the cocultures. Stimulation of FV-specific CD8+ T cells with peptide-pulsed APCs induced IFN-{gamma} expression that was suppressed in a dose-dependent manner by CD4+CD25+ T cells from FV-infected mice, but not by the same subset of cells from naive mice (Fig. 2B). Suppression of CD8+ T cell IFN-{gamma} production was not associated with decreased expression of the early activation marker CD69 (46) or the activation-associated glycoform of CD43 (51) (Fig. 2C). Neither the CD25– subset of CD4+ T cells nor CD4– spleen cells from chronically infected mice suppressed IFN-{gamma} expression in vitro (data not shown). Moreover, because the vast majority of chronic virus is harbored in the CD4– subsets (52), suppression could not be attributed to virus infection within the cocultures. These results indicated that the immunosuppressive activity in mice chronically infected with FV was predominantly contained within the CD4+CD25+ T cell subset. Thus, subsequent studies were focused on the CD4+CD25+ T cell subset, which will be referred to as FV-induced Treg.

To determine whether FV-induced Tregs could suppress non-FV-specific CD8+ T cells in vitro, coculture experiments were conducted as described above, except that responses by OVA-specific (OT-1) TCR Tg CD8+ T cells were analyzed. As observed with FV-specific CD8+ T cells, CD4+CD25+ T cells from chronically infected but not from naive mice significantly reduced the production of IFN-{gamma} by stimulated OVA-specific CD8+ T cells, even at a 1:1 CD4 to CD8 ratio (Fig. 2D). Thus, in vitro suppression of IFN-{gamma} production by FV-induced Tregs was independent of the specificity of the CD8+ T cell.

FV-induced Tregs suppress granzyme B expression by CD8+ T cells

CD8+ T cell effector function was also measured by intracellular staining of granzyme B, an important molecule in the CD8+ T cell-mediated killing of FV-infected cells in vivo (53). Stimulation of FV-specific CD8+ T cells by peptide-pulsed APCs induced granzyme B production (Fig. 3A) and granzyme B levels were similar upon addition of CD4+CD25+ T cells from naive mice (Fig. 3B). In contrast, the overall granzyme B signal in CD8-gated cells was significantly lower in cocultures containing CD4+CD25+ T cells from chronically infected mice (Fig. 3B). Suppression of granzyme B expression was not observed with the CD25– subset of T cells from either naive or persistently infected mice (Fig. 3C). Thus, FV-induced Tregs suppressed expression of both IFN-{gamma} and granzyme B.


Figure 3
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  		FIGURE 3. Tregs inhibit granzyme B expression in stimulated TCR Tg CD8+ T cells. TCR Tg CD8+ T cells were stimulated in vitro with peptide-pulsed APCs and cocultured in the presence of CD4+CD25+ or CD4+CD25– T cells from either naive or chronically infected mice. After 60 h, cultured cells were stained for the expression of cell surface CD8 and intracellular granzyme B and analyzed by flow cytometry. Histograms represent granzyme B expression in CD8-gated T cells. A, The relative levels of granzyme B expression in TCR Tg CD8+ T cells cultured alone (dashed line; MFI = 5.0) or stimulated by coculture with peptide-pulsed APCs and naive CD4+CD25– T cells (solid line; MFI = 19.4) are shown. B, Stimulated TCR Tg CD8+ T cells were cocultured in the presence of CD4+CD25+ T cells from either naive (thick line; MFI = 12.2) or chronically infected mice (thin line; MFI = 7.5). The difference in MFI of granzyme B expression in CD8+ T cells was statistically significant (p = 0.014; n = 4 per group). C, Coculture with CD4+CD25– T cells from either naive (thick line; MFI = 11.5) or chronically infected mice (thin line; MFI = 12.0) had no effect on CD8+ T cell expression of granzyme B.

 
Suppressive activity of FV-induced Treg requires cell-to-cell contact

Mechanisms of Treg-mediated suppression have been proposed that include both soluble factors, such as IL-10 and TGF-beta, and cell-to-cell contact-dependent interactions (54, 55). To determine whether FV-induced Treg suppression of CD8+ T cells was mediated by soluble factors or required direct cell-to-cell contact, in vitro suppression assays were performed using a Transwell system in which the Tregs were physically separated from CD8+ T cell targets by a permeable membrane. Separation of Treg from stimulated CD8+ T cells completely abolished suppression (Fig. 4A). Furthermore, supernatants from standard cocultures did not transfer suppressive activity (Fig. 4B), nor was suppression in cocultures blocked by the addition of anti-TGFbeta and anti-IL-10R Abs (data not shown).


Figure 4
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  		FIGURE 4. FV-induced, Treg-mediated suppression requires direct cell-to-cell contact. FV-specific CD8+ T cells were cocultured with peptide-pulsed APCs and naive CD4+CD25– T cells. CD4+CD25+ T cells from either naive or chronically infected mice were added directly to the culture or to the top chamber of a Transwell culture system. A, Bar graphs depict the amount of IFN-{gamma} present in coculture supernatants after 60 h in culture. IFN-{gamma} production in cocultures that allowed cell contact and contained CD4+CD25+ T cells from chronically infected mice were significantly lower compared with those containing naive CD4+CD25+ T cells (p = 0.0027). B, Supernatants from cocultures containing CD4+CD25+ T cells from naive or chronically infected mice were transferred to cultures containing fresh FV-specific CD8+ T cells, peptide-pulsed APCs, and naive CD4+CD25– T cells. At 60 h, culture supernatants were collected and assayed for IFN-{gamma} by ELISA. Bar graphs depict the mean IFN-{gamma} (ng/ml) ± SD in supernatants.

 
FV-induced Treg inhibit effector function of activated CD8+ T cells in the absence of APCs

Because the in vitro suppression assay contained peptide-pulsed APCs to activate the CD8+ T cells, it was not known whether suppression was occurring via direct interactions between the Tregs and the CD8+ T cells, or whether it required the presence of APCs. Furthermore, although the in vitro assay described above revealed that FV-induced Treg prevented complete activation of naive CD8+ T cells, it did not address whether Treg could suppress previously activated CD8+ T cells. Suppression of activated cells might be critical if the function of the virus-induced Treg in vivo was to limit immunopathology during an ongoing immune response. To address both questions, we assessed the effects of FV-induced Tregs on in vivo-activated TCR Tg CD8+ T cells and modified the assay such that APC were no longer needed for in vitro activation. To obtain activated FV-specific CD8+ T cells, TCR Tg CD8+ T cells that were marked with a GFP transgene were adoptively transferred into mice acutely infected with FV, a context in which the cells proliferate and differentiate into effector cells normally (27). After 4 days, donor cells were repurified from host spleens by sorting for GFP-positive CD8+ T cells. Ninety-eight percent or more of the transferred CD8+ T cells were activated as evidenced by the high expression of CD44 (Fig. 5A). Activation of the CD8+ T cells was maintained in vitro by the addition of FV-specific MHC class I tetramers, thereby eliminating the need for APCs. Intracellular staining demonstrated that the majority of CD8+ T cells from cocultures containing naive CD4+CD25+ T cells expressed IFN-{gamma} (Fig. 5B). In contrast, CD8+ T cells cocultured in the presence of CD4+CD25+ T cells from chronically infected mice expressed significantly lower levels of IFN-{gamma} (Fig. 5B). Virtually none of the IFN-{gamma} produced in these cocultures came from the CD4+ T cells (Fig. 5C). Thus, FV-induced Treg cells directly suppressed IFN-{gamma} production by activated CD8+ T cells in the absence of APCs.


Figure 5
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  		FIGURE 5. FV-induced Tregs suppress in vivo-activated CD8+ T cells in the absence of APCs. A, The activation of adoptively transferred GFP+ FV-specific CD8+ T cells, as indicated by CD44 expression, was determined by flow cytometry. Histograms depict the relative expression of CD44 in the in vivo-activated CD8+ T cells (thick line; MFI = 11,413) compared with naive FV-specific CD8+ T cells before transfer (shaded; MFI = 44.2). In vivo-activated, GFP-positive, FV-specific CD8+ T cells (3 x 105) were stimulated with a specific tetramer and cocultured with CD4+CD25+ T cells (6 x 105) from either naive or chronically infected mice. After 48 h, cells were stained for intracellular IFN-{gamma} and analyzed by flow cytometry. B and C, Representative histograms of CD8-gated (B) or CD4-gated (C) T cells from cocultures containing CD4+CD25+ T cells from chronically infected (thick line) or naive mice (shaded) are shown. Data were confirmed by ELISA and showed significant suppression of IFN-{gamma} production from two independent experiments (p = 0.0128 and 0.014). The MFI values for IFN-{gamma} staining in CD8+ T cells (B) were 67.0 for naive and 18.5 for chronically infected mice. For CD4+ T cells (C) the MFI values were 6.0 for naive and 6.2 for chronically infected mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
One of the most significant findings from the current study is that FV-induced Tregs suppress the effector function of naturally expanded and activated CD8+ T cells. This ability is integral to the hypothesized function for Tregs in preventing CD8+ T cell-mediated immunopathology during the course of a viral infection (22, 23, 56). Most viral infections elicit potent CD8+ T cell responses that are characterized by a preset program of rapid proliferation coupled with development of effector functions that include secretion of IFN-{gamma} and formation of cytolytic granules containing granzyme B (57, 58). The expansion and differentiation of CD8+ T cells is not only essential for virus clearance, but the degree of expansion also correlates with the pool size of memory cells necessary for protection from future infections (58, 59). This expansion and differentiation process leads to circulation of large numbers of activated CTLs that have the potential to cause significant collateral damage due to cross-reactivity or bystander killing. Thus, in order for Treg to play a role in preventing immunopathological damage from these cells without disrupting immunity, they must have the capacity to suppress at a postproliferative stage of the process. Our data demonstrate that FV-induced Tregs indeed possess this capacity and operate by suppressing activated and differentiated CD8+ T cells at the level of effector function.

The beneficial aspect of protection from immunopathology is counterweighted by the ability of some pathogens to subvert the immunoregulatory system to assist in establishing and maintaining chronic infection. Since the initial description of Tregs in chronic FV infections of mice was reported (17), Tregs have also been shown to suppress immune responses in humans chronically infected with HIV (60, 61, 62, 63), the hepatitis C virus (18, 64), and EBV (65). It is notable that these are all viruses that cause chronic infections, and evidence suggests that immunosuppressive activity by Tregs before complete eradication of the infectious agent contributes to their evasion of host immune responses. Another adverse consequence of virus-induced Tregs could be generalized immunosuppression due to the nonspecific nature of Treg-mediated suppression (66, 67, 68). Consistent with this hypothesis, we show that FV-induced Treg suppress CD8+ T cells in vitro regardless of the TCR specificity of the CD8+ T cell. This result is in keeping with previous in vivo experiments showing that FV-induced Tregs suppressed CD8+ T cell-mediated rejection of both FV-induced tumors and tumors not expressing FV Ags (17). Indeed, HIV patients have an increased risk of developing certain malignancies, especially those associated with viruses (69, 70, 71). Of course, HIV patients are subject to other forms of virus-induced immunosuppression such as CD4+ T cell depletion, but virus-induced Treg cells may be a contributing factor in increased susceptibility to cancer.

The ability to inhibit CD8+ T cell effector function rather than proliferation distinguishes FV-induced Treg from natural Tregs, which generally inhibit at the level of proliferation in vitro. Another notable difference between natural Tregs and virus-induced Tregs is that natural Tregs typically require stimulation in vitro to exhibit suppressive activity (11, 12). In our in vitro assay, FV-induced Tregs demonstrated suppressive activity directly ex vivo. This finding is consistent with studies of the hepatitis C virus (64) and the feline immunodeficiency virus (72) showing that virus-induced Tregs suppress in vitro without additional stimulation. This may be related to the higher activation status of virus-induced Treg as demonstrated by increased expression of activation markers such as CD69 (46), GITR (34, 38, 47), and LAG-3 (42). Indeed, a small subset of natural Tregs that have an activated phenotype also suppresses T cell responses without in vitro activation (73). Interestingly, in addition to their classification as activation markers, CD69, GITR, and LAG-3 may also have direct roles in Treg function. CD69 is involved in production of the immunosuppressive molecule TGF-beta (74). GITR, which is constitutively expressed on natural Tregs, has been reported to protect them against activation-induced cell death (47, 75, 76). Recent studies have revealed that LAG-3 (42) may be directly involved in Treg-mediated suppression as evidenced by reduced regulatory activity in LAG3–/– mice and by Abs blocking LAG-3. Thus, the increases in expression of these molecules in CD4+CD25+ T cells from chronically infected mice may reflect higher immunosuppressive potential in subsets of Treg. At this point we can make no conclusion about the whether FV-induced Tregs are differentiated from natural Tregs or from a separate subset of CD4+ T cells.

CD103 ({alpha}Ebeta7), an epithelial cadherin-specific integrin whose expression on a subset of CD4+CD25+ T cells has been correlated with potent immunosuppressive activity and homing to sites of inflammation (40) (41), was also found to be up-regulated on Tregs from chronically infected mice. It is presently unclear why splenic Tregs from chronically infected mice would have increased expression of an integrin specific for E-cadherin, because the spleen does not normally contain epithelial cells. However, exposure of Tregs to the immunosuppressive cytokine TGFbeta up-regulates CD103 expression (77). Thus, increased expression of CD103 may simply reflect prior exposure to TGFbeta rather than cellular adhesion in the spleen.

In conclusion, the results from the current in vitro study are consistent with the salient features of in vivo Treg function in mice with chronic FV infections. For example, both in vivo and in vitro FV-induced Tregs inhibit virus-specific CD8+ T cell effector functions without inhibiting proliferative responses or the up-regulation of activation markers (27). The in vitro assay also duplicates the finding that FV-induced Treg suppression of CD8+ T cells occurs without regard to the specificity of the CD8+ T cells (17). Although little is known about the mechanisms of virus-induced Treg suppression in vivo, the in vitro assay has enabled us to determine that direct cell-to-cell interactions between Treg and CD8+ T cells mediate suppression with no involvement of APCs. This finding indicates that distinct cell surface interactions mediate inhibitory signaling events. Thus, this assay provides us with an opportunity to discover the molecules involved in Treg-mediated suppression of CD8+ T cells and investigate their function. Such information is critical for the development of therapeutics to modulate Treg function by blocking the receptor-ligand interactions or the downstream signaling pathways.


   Acknowledgments
 
We thank Robert Heinzen, Jeff Shannon, Brandon Walter, and Sonja Best for critical review of the manuscript, and Anita Mora and Gary Hettrick for assistance with graphics.


   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 research was funded by the National Institute of Allergy and Infectious Diseases Division of Intramural Research, National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Kim J. Hasenkrug, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South Fourth Street, Hamilton, MT 59840. E-mail: KHasenkrug{at}nih.gov Back

3 Abbreviations used in this paper: Treg, regulatory T cell; FV, Friend retrovirus; GITR, glucocorticoid-induced TNFR; LAG, lymphocyte activation gene; MFI, mean fluorescence intensity; Tg, transgenic. Back

Received for publication October 13, 2005. Accepted for publication December 30, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]
  2. Sakaguchi, S.. 2005. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and nonself. Nat. Immunol. 6: 345-352. [Medline]
  3. Shevach, E. M., R. S. McHugh, A. M. Thornton, C. Piccirillo, K. Natarajan, D. H. Margulies. 2001. Control of autoimmunity by regulatory T cells. Adv. Exp. Med. Biol. 490: 21-32. [Medline]
  4. Sakaguchi, S.. 2004. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22: 531-562. [Medline]
  5. Bluestone, J. A., A. K. Abbas. 2003. Natural versus adaptive regulatory T cells. Nat. Rev. Immunol. 3: 253-257. [Medline]
  6. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057-1061. [Abstract/Free Full Text]
  7. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: 330-336. [Medline]
  8. Chen, W., W. Jin, N. Hardegen, K. J. Lei, L. Li, N. Marinos, G. McGrady, S. M. Wahl. 2003. Conversion of peripheral CD4+CD25– naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J. Exp. Med. 198: 1875-1886. [Abstract/Free Full Text]
  9. Walker, M. R., D. J. Kasprowicz, V. H. Gersuk, A. Benard, M. Van Landeghen, J. H. Buckner, S. F. Ziegler. 2003. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25– T cells. J. Clin. Invest. 112: 1437-1443. [Medline]
  10. Fisson, S., G. Darrasse-Jeze, E. Litvinova, F. Septier, D. Klatzmann, R. Liblau, B. L. Salomon. 2003. Continuous activation of autoreactive CD4+CD25+ regulatory T cells in the steady state. J. Exp. Med. 198: 737-746. [Abstract/Free Full Text]
  11. Shevach, E. M.. 2002. CD4+CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2: 389-400. [Medline]
  12. von Boehmer, H.. 2005. Mechanisms of suppression by suppressor T cells. Nat. Immunol. 6: 338-344. [Medline]
  13. Ermann, J., V. Szanya, G. S. Ford, V. Paragas, C. G. Fathman, K. Lejon. 2001. CD4+CD25+ T cells facilitate the induction of T cell anergy. J. Immunol. 167: 4271-4275. [Abstract/Free Full Text]
  14. Piccirillo, C. A., E. M. Shevach. 2001. Cutting edge: control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells. J. Immunol. 167: 1137-1140. [Abstract/Free Full Text]
  15. Chai, J. G., J. Y. Tsang, R. Lechler, E. Simpson, J. Dyson, D. Scott. 2002. CD4+CD25+ T cells as immunoregulatory T cells in vitro. Eur. J. Immunol. 32: 2365-2375. [Medline]
  16. Camara, N. O., F. Sebille, R. I. Lechler. 2003. Human CD4+CD25+ regulatory cells have marked and sustained effects on CD8+ T cell activation. Eur. J. Immunol. 33: 3473-3483. [Medline]
  17. Iwashiro, M., R. J. Messer, K. E. Peterson, I. M. Stromnes, T. Sugie, K. J. Hasenkrug. 2001. Immunosuppression by CD4+ regulatory T cells induced by chronic retroviral infection. Proc. Natl. Acad. Sci. USA 98: 9226-9230. [Abstract/Free Full Text]
  18. MacDonald, A. J., M. Duffy, M. T. Brady, S. McKiernan, W. Hall, J. Hegarty, M. Curry, K. H. Mills. 2002. CD4 T helper type 1 and regulatory T cells induced against the same epitopes on the core protein in hepatitis C virus-infected persons. J. Infect. Dis. 185: 720-727. [Medline]
  19. McGuirk, P., C. McCann, K. H. Mills. 2002. Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195: 221-231. [Abstract/Free Full Text]
  20. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, D. L. Sacks. 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420: 502-507. [Medline]
  21. Suvas, S., U. Kumaraguru, C. D. Pack, S. Lee, B. T. Rouse. 2003. CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses. J. Exp. Med. 198: 889-901. [Abstract/Free Full Text]
  22. Rouse, B. T., S. Suvas. 2004. Regulatory cells and infectious agents: detentes cordiale and contraire. J. Immunol. 173: 2211-2215. [Abstract/Free Full Text]
  23. Suvas, S., A. K. Azkur, B. S. Kim, U. Kumaraguru, B. T. Rouse. 2004. CD4+CD25+ regulatory T cells control the severity of viral immunoinflammatory lesions. J. Immunol. 172: 4123-4132. [Abstract/Free Full Text]
  24. Friend, C.. 1957. Cell-free transmission in adult Swiss mice of a disease having the character of a leukemia. J. Exp. Med. 105: 307-318. [Abstract]
  25. Kabat, D.. 1989. Molecular biology of Friend viral erythroleukemia. Curr. Top. Microbiol. Immunol. 148: 1-42. [Medline]
  26. Chesebro, B., M. Miyazawa, W. J. Britt. 1990. Host genetic control of spontaneous and induced immunity to Friend murine retrovirus infection. Annu. Rev. Immunol. 8: 477-499. [Medline]
  27. Dittmer, U., H. He, R. J. Messer, S. Schimmer, A. R. Olbrich, C. Ohlen, P. D. Greenberg, I. M. Stromnes, M. Iwashiro, S. Sakaguchi, et al 2004. Functional impairment of CD8+ T cells by regulatory T cells during persistent retroviral infection. Immunity 20: 293-303. [Medline]
  28. Chen, W., H. Qin, B. Chesebro, M. A. Cheever. 1996. Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors. J. Virol. 70: 7773-7782. [Abstract]
  29. Ohlen, C., M. Kalos, L. E. Cheng, A. C. Shur, D. J. Hong, B. D. Carson, N. C. Kokot, C. G. Lerner, B. D. Sather, E. S. Huseby, P. D. Greenberg. 2002. CD8+ T cell tolerance to a tumor-associated antigen is maintained at the level of expansion rather than effector function. J. Exp. Med. 195: 1407-1418. [Abstract/Free Full Text]
  30. Okabe, M., M. Ikawa, K. Kominami, T. Nakanishi, Y. Nishimune. 1997. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407: 313-319. [Medline]
  31. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76: 17-27. [Medline]
  32. Lilly, F., R. A. Steeves. 1973. B-tropic Friend virus: a host-range pseudotype of spleen focus-forming virus (SFFV). Virology 55: 363-370. [Medline]
  33. Hasenkrug, K. J., D. M. Brooks, M. N. Robertson, R. V. Srinivas, B. Chesebro. 1998. Immunoprotective determinants in Friend murine leukemia virus envelop protein. Virology 248: 66-73. [Medline]
  34. Shimizu, J., S. Yamazaki, T. Takahashi, Y. Ishida, S. Sakaguchi. 2002. Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3: 135-142. [Medline]
  35. Murali-Krishna, K., L. L. Lau, S. Sambhara, F. Lemonnier, J. Altman, R. Ahmed. 1999. Persistence of memory CD8 T cells in MHC class I-deficient mice. Science 286: 1377-1381. [Abstract/Free Full Text]
  36. Schepers, K., M. Toebes, G. Sotthewes, F. A. Vyth-Dreese, T. A. Dellemijn, C. J. Melief, F. Ossendorp, T. N. Schumacher. 2002. Differential kinetics of antigen-specific CD4+ and CD8+ T cell responses in the regression of retrovirus-induced sarcomas. J. Immunol. 169: 3191-3199. [Abstract/Free Full Text]
  37. Khattri, R., T. Cox, S. A. Yasayko, F. Ramsdell. 2003. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4: 337-342. [Medline]
  38. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach, M. Collins, M. C. Byrne. 2002. CD4+CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16: 311-323. [Medline]
  39. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192: 295-302. [Abstract/Free Full Text]
  40. Huehn, J., K. Siegmund, J. C. Lehmann, C. Siewert, U. Haubold, M. Feuerer, G. F. Debes, J. Lauber, O. Frey, G. K. Przybylski, et al 2004. Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells. J. Exp. Med. 199: 303-313. [Abstract/Free Full Text]
  41. Lehmann, J., J. Huehn, M. de la Rosa, F. Maszyna, U. Kretschmer, V. Krenn, M. Brunner, A. Scheffold, A. Hamann. 2002. Expression of the integrin {alpha}Ebeta7 identifies unique subsets of CD25+ as well as CD25– regulatory T cells. Proc. Natl. Acad. Sci. USA 99: 13031-13036. [Abstract/Free Full Text]
  42. Huang, C. T., C. J. Workman, D. Flies, X. Pan, A. L. Marson, G. Zhou, E. L. Hipkiss, S. Ravi, J. Kowalski, H. I. Levitsky, et al 2004. Role of LAG-3 in regulatory T cells. Immunity 21: 503-513. [Medline]
  43. Walker, L. S., A. Chodos, M. Eggena, H. Dooms, A. K. Abbas. 2003. Antigen-dependent proliferation of CD4+CD25+ regulatory T cells in vivo. J. Exp. Med. 198: 249-258. [Abstract/Free Full Text]
  44. Yamazaki, S., T. Iyoda, K. Tarbell, K. Olson, K. Velinzon, K. Inaba, R. M. Steinman. 2003. Direct expansion of functional CD25+CD4+ regulatory T cells by antigen-processing dendritic cells. J. Exp. Med. 198: 235-247. [Abstract/Free Full Text]
  45. Powrie, F., M. W. Leach, S. Mauze, S. Menon, L. B. Caddle, R. L. Coffman. 1994. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1: 553-562. [Medline]
  46. Hara, T., L. K. Jung, J. M. Bjorndahl, S. M. Fu. 1986. Human T cell activation. III: rapid induction of a phosphorylated 28 kD/32 kD disulfide-linked early activation antigen (EA 1) by 12-o-tetradecanoyl phorbol-13-acetate, mitogens, and antigens. J. Exp. Med. 164: 1988-2005. [Abstract/Free Full Text]
  47. Ronchetti, S., G. Nocentini, C. Riccardi, P. P. Pandolfi. 2002. Role of GITR in activation response of T lymphocytes. Blood 100: 350-352. [Abstract/Free Full Text]
  48. Iwashiro, M., K. Peterson, R. J. Messer, I. M. Stromnes, K. J. Hasenkrug. 2001. CD4+ T cells and {gamma} interferon in the long-term control of persistent friend retrovirus infection. J. Virol. 75: 52-60. [Abstract/Free Full Text]
  49. Peterson, K. E., M. Iwashiro, K. J. Hasenkrug, B. Chesebro. 2000. Major histocompatibility complex class I gene controls the generation of {gamma} interferon-producing CD4+ and CD8+ T cells important for recovery from Friend retrovirus-induced leukemia. J. Virol. 74: 5363-5367. [Abstract/Free Full Text]
  50. Stromnes, I. M., U. Dittmer, T. N. Schumacher, K. Schepers, R. J. Messer, L. H. Evans, K. E. Peterson, B. Race, K. J. Hasenkrug. 2002. Temporal effects of {gamma} interferon deficiency on the course of Friend retrovirus infection in mice. J. Virol. 76: 2225-2232. [Abstract/Free Full Text]
  51. Harrington, L. E., M. Galvan, L. G. Baum, J. D. Altman, R. Ahmed. 2000. Differentiating between memory and effector CD8 T cells by altered expression of cell surface O-glycans. J. Exp. Med. 191: 1241-1246. [Abstract/Free Full Text]
  52. Hasenkrug, K. J., D. M. Brooks, U. Dittmer. 1998. Critical role for CD4+ T cells in controlling retrovirus replication and spread in persistently infected mice. J. Virol. 72: 6559-6564. [Abstract/Free Full Text]
  53. Zelinskyy, G., S. Balkow, S. Schimmer, K. Schepers, M. M. Simon, U. Dittmer. 2004. Independent roles of perforin, granzymes, and Fas in the control of Friend retrovirus infection. Virology 330: 365-374. [Medline]
  54. Mills, K. H.. 2004. Regulatory T cells: friend or foe in immunity to infection?. Nat. Rev. Immunol. 4: 841-855. [Medline]
  55. O’Garra, A., P. Vieira. 2004. Regulatory T cells and mechanisms of immune system control. Nat. Med. 10: 801-805. [Medline]
  56. Mills, K. H., P. McGuirk. 2004. Antigen-specific regulatory T cells–their induction and role in infection. Semin. Immunol. 16: 107-117. [Medline]
  57. Kaech, S. M., R. Ahmed. 2001. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat. Immunol. 2: 415-422. [Medline]
  58. Wherry, E. J., R. Ahmed. 2004. Memory CD8 T-cell differentiation during viral infection. J. Virol. 78: 5535-5545. [Free Full Text]
  59. Homann, D., L. Teyton, M. B. Oldstone. 2001. Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory. Nat. Med. 7: 913-919. [Medline]
  60. Aandahl, E. M., J. Michaelsson, W. J. Moretto, F. M. Hecht, D. F. Nixon. 2004. Human CD4+CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens. J. Virol. 78: 2454-2459. [Medline]
  61. Andersson, J., A. Boasso, J. Nilsson, R. Zhang, N. J. Shire, S. Lindback, G. M. Shearer, C. A. Chougnet. 2005. Cutting edge: the prevalence of regulatory T cells in lymphoid tissue is correlated with viral load in HIV-infected patients. J. Immunol. 174: 3143-3147. [Abstract/Free Full Text]
  62. Kinter, A. L., M. Hennessey, A. Bell, S. Kern, Y. Lin, M. Daucher, M. Planta, M. McGlaughlin, R. Jackson, S. F. Ziegler, A. S. Fauci. 2004. CD25+CD4+ regulatory T cells from the peripheral blood of asymptomatic HIV-infected individuals regulate CD4+ and CD8+ HIV-specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status. J. Exp. Med. 200: 331-343. [Abstract/Free Full Text]
  63. Weiss, L., V. Donkova-Petrini, L. Caccavelli, M. Balbo, C. Carbonneil, Y. Levy. 2004. Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells which suppress HIV-specific CD4 T-cell responses in HIV-infected patients. Blood 104: 3249-3256. [Abstract/Free Full Text]
  64. Boettler, T., H. C. Spangenberg, C. Neumann-Haefelin, E. Panther, S. Urbani, C. Ferrari, H. E. Blum, F. von Weizsacker, R. Thimme. 2005. T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection. J. Virol. 79: 7860-7867. [Abstract/Free Full Text]
  65. Marshall, N. A., M. A. Vickers, R. N. Barker. 2003. Regulatory T cells secreting IL-10 dominate the immune response to EBV latent membrane protein 1. J. Immunol. 170: 6183-6189. [Abstract/Free Full Text]
  66. von Herrath, M. G., L. C. Harrison. 2003. Antigen-induced regulatory T cells in autoimmunity. Nat. Rev. Immunol. 3: 223-232. [Medline]
  67. Bach, J. F.. 2003. Regulatory T cells under scrutiny. Nat Rev Immunol. 3: 189-198. [Medline]
  68. Miller, A., O. Lider, H. L. Weiner. 1991. Antigen-driven bystander suppression after oral administration of antigens. J. Exp. Med. 174: 791-798. [Abstract/Free Full Text]
  69. Dal Maso, L., S. Franceschi. 2003. Epidemiology of non-Hodgkin lymphomas and other haemolymphopoietic neoplasms in people with AIDS. Lancet Oncol. 4: 110-119. [Medline]
  70. de Sanjose, S., J. Palefsky. 2002. Cervical and anal HPV infections in HIV positive women and men. Virus Res. 89: 201-211. [Medline]
  71. Scadden, D. T.. 2003. AIDS-related malignancies. Annu. Rev. Med. 54: 285-303. [Medline]
  72. Vahlenkamp, T. W., M. B. Tompkins, W. A. Tompkins. 2004. Feline immunodeficiency virus infection phenotypically and functionally activates immunosuppressive CD4+CD25+ T regulatory cells. J. Immunol. 172: 4752-4761. [Abstract/Free Full Text]
  73. Nolte-’t Hoen, E. N., J. P. Wagenaar-Hilbers, E. P. Boot, C. H. Lin, G. J. Arkesteijn, W. van Eden, L. S. Taams, M. H. Wauben. 2004. Identification of a CD4+CD25+ T cell subset committed in vivo to suppress antigen-specific T cell responses without additional stimulation. Eur. J. Immunol. 34: 3016-3027. [Medline]
  74. Sancho, D., M. Gomez, F. Viedma, E. Esplugues, M. Gordon-Alonso, M. A. Garcia-Lopez, H. de la Fuente, A. C. Martinez, P. Lauzurica, F. Sanchez-Madrid. 2003. CD69 down-regulates autoimmune reactivity through active transforming growth factor-beta production in collagen-induced arthritis. J. Clin. Invest. 112: 872-882. [Medline]
  75. Nocentini, G., L. Giunchi, S. Ronchetti, L. T. Krausz, A. Bartoli, R. Moraca, G. Migliorati, C. Riccardi. 1997. A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis. Proc. Natl. Acad. Sci. USA 94: 6216-6221. [Abstract/Free Full Text]
  76. Muriglan, S. J., T. Ramirez-Montagut, O. Alpdogan, T. W. Van Huystee, J. M. Eng, V. M. Hubbard, A. A. Kochman, K. H. Tjoe, C. Riccardi, P. P. Pandolfi, et al 2004. GITR activation induces an opposite effect on alloreactive CD4+ and CD8+ T cells in graft-versus-host disease. J. Exp. Med. 200: 149-157. [Abstract/Free Full Text]
  77. Robinson, P. W., S. J. Green, C. Carter, J. Coadwell, P. J. Kilshaw. 2001. Studies on transcriptional regulation of the mucosal T-cell integrin {alpha}Ebeta7 (CD103). Immunology 103: 146-154. [Medline]



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