Convincing evidence has accumulated for the existence of heterogeneous populations of CD4+ T regulatory (Treg) cells that maintain peripheral self-tolerance.1 One regulatory subset that has been well characterized expresses cell surface CD25, the interleukin (IL)-2 receptor α chain.2 These CD4+CD25+ Treg3 cells, comprising 5% to 10% of peripheral CD4+ T cells, are generated by self-antigen presentation by the thymic epithelium and are functionally mature when exported from the thymus into the periphery. When activated by their cognate antigen, they acquire the potential of suppressing the proliferation and cytokine production of self-reactivating CD4+ T helper (Th) and CD8+ T cells.3,4 It is now well established that in addition to maintaining peripheral self-tolerance, CD4+CD25+ Treg cells are responsive to pathogens in the peripheral lymphoid tissues and play a major role in modulating immune responses to infectious agents.5-9 Naturally occurring thymic-derived CD4+CD25+ Treg cells, and pathogen-induced Treg cells, are phenotypically defined by constitutive expression of cell surface glucocorticoid-induced tumor necrosis factor (TNF) receptor family-related gene (GITR, also known as TNFRSF18) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) as well as the transcription factor Foxp3.2 When activated for suppressor function, Treg cells also express biologically active tumor growth factor-β (TGFβ) on their surface (membrane TGFβ [mTGFβ]).10,11
Whether pathogen-induced Treg cells are derived from natural Treg cells generated in the thymus, are peripherally generated Treg cells, or both has yet to be fully elucidated. It is essential that there exists a mechanism for maintaining peripheral Treg cell homeostasis, however, because Treg cells comprise a small fraction (5% to 10%) of the total T-cell population in the blood and are anergic and cannot expand without T-cell help because of a deficiency in IL-2 production.12
The peripheral generation of Treg cells may be of particular importance in the case of feline immunodeficiency virus (FIV) and HIV infections, given that both viruses productively infect CD4+CD25+ T cells13-15 and that Treg cells are chronically activated in vivo in both infections.16 Because HIV/FIV-infected T cells are prone to lysis and activated T cells are highly susceptible to apoptosis, survival of Treg cells in HIV and FIV infections could be problematic. In HIV and FIV infections, however, the numbers of Treg cells in the circulation do not change appreciably, or may actually increase in the percentage of the total CD4+ T-cell population in long-term infections.16,17 Eggena et al17 observed increased numbers of Treg cells (expressed as percentage of total CD4+ T cells) in HIV-positive patients with low CD4+ cell counts, suggesting a slower decline in the CD4+CD25+ population or recruitment of Treg cells from the existing Treg or CD4+ Th pool with disease progression.
Data suggest that Treg cell homeostasis could be maintained in the periphery by expansion of the existing Treg population or by recruitment from the CD4+CD25− T-cell pool. A number of studies have demonstrated that Treg cells are activated and expand in numbers in response to several pathogens, including simian immunodeficiency virus (SIV)18,19 and HIV,17 and molecules such as lipopolysaccharide (LPS) and IL-2 have been shown to induce Treg proliferation.16,20 Chen et al21 reported that CD4+CD25− Th cells could be induced to attain phenotypic and functional Treg characteristics in vitro by stimulation with TGFβ in combination with T-cell receptor (TCR) engagement, suggesting an additional mechanism of Treg peripheral homeostasis independent of existing Treg cells or the thymus. Liang et al22 also reported that CD4+CD25− T cells converted into CD4+CD25+ Treg cells when injected into thymectomized congenic mice, suggesting an extrathymic mechanism of maintenance of Treg homeostasis. TGFβ-induced immunosuppressive Treg cells express the characteristic phenotype of natural Treg cells, including CD25, CTLA-4, GITR, mTGFβ, and Foxp3.
Although natural CD4+CD25+ Treg cells and TGFβ-induced Treg cells express cell surface TGFβ when activated for suppressor function, the involvement of mTGFβ in their immunoregulatory function is controversial. Nakamura et al23 demonstrated that anti-CD3/allophycocyanin (APC)-stimulated Treg cells express mTGFβ and that treatment with anti-TGFβ neutralizing antibodies abrogated their contact-dependent suppressor function, suggesting that mTGFβ mediated suppression. In a follow-up study, Nakamura et al10 demonstrated that another TGFβ blocking molecule, recombinant latency-associated peptide of TGFβ (rLAP) reverses suppression by mouse CD4+CD25+ and human CD4+CD25hi Treg cells. Others have demonstrated that suppressor function of mouse and human CD4+CD25+ thymocytes is at least partially inhibited by neutralization of TGFβ.24,25 In contrast, others have reported that antibody neutralization of TGFβ failed to reverse CD4+CD25+ suppressor activity.3,26 In addition, CD4+CD25+ Treg cells from TGFβ-deficient mice could suppress CD4+CD25− T-cell proliferation, suggesting that TGFβ is not necessary for suppressor function in vitro.27 In the case of the CD4+CD25+ Treg phenotype induced by TGFβ/TCR stimulation of CD4+CD25− T cells, there is little information on the role of TGFβ in their suppressor function. Although Chen et al21 and Marie et al28 reported that TGFβ/TCR-converted Treg-like cells express mTGFβ and exhibit suppressor function, they did not determine the role of mTGFβ. To explore the mechanisms regulating Treg cell homeostasis and effector function and the effect that FIV infection has on these processes, we examined the role of mTGFβ and TGFβ receptor II (RII) in the Th-to-Treg conversion process and in their suppressor function. We report that conversion of CD4+CD25− Th cells to phenotypic and functional Treg-like cells requires stimulation by mitogen plus TGFβ. Further, we report that Th-to-Treg conversion can be inhibited by anti-TGFβ-RII antibodies. Finally, we show that CD4+CD25+ Treg cells freshly isolated from FIV-infected cats, in contrast to normal cats, are mTGFβ-positive and constitutively immunosuppressive and that suppressor function for CD4+ cells can be blocked by treatment of Treg cells with anti-TGFβ or Th target cells with anti-TGFβ-RII antibodies. These data suggest that recruitment of Treg cells from the Th cell pool and suppressor function by Treg cells are dependent on the TGFβ/TGFβ-R signaling pathway and that this pathway may be constitutively upregulated in asymptomatic chronically FIV-infected cats.
MATERIALS AND METHODS
Specific pathogen-free cats were obtained from Liberty Laboratories (Liberty Corners, NJ) or Cedar River Laboratory (Mason City, IA) and housed at the Laboratory Animal Resource Facility at the College of Veterinary Medicine, North Carolina State University (NCSU). All the protocols involving cats were reviewed and approved by the NCSU Institutional Animal Care and Use Committee. Cats were inoculated with the NCSU1 isolate of FIV, a pathogenic clade A virus,29 as described by Bucci et al.30 FIV infection was confirmed by immunoblot analysis, and provirus detection was confirmed by polymerase chain reaction (PCR) using antibodies and primers specific for the FIV-p24 GAG sequence. At the time samples were taken, cats had been infected with FIV for at least 5 years and were clinically asymptomatic. Uninfected control cats ranged in age from 3 to 6 years and were housed separately from FIV-infected cats.
Sample Collection and Preparation
Whole blood was collected by jugular venipuncture into ethylenediaminetetraacetic acid (EDTA) Vacutainer tubes (Becton-Dickinson, Franklin Lakes, NJ). Subsequently, peripheral blood mononuclear cells (PBMCs) were isolated by Percoll (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation, as previously described.31 Single-cell suspensions were prepared from peripheral lymph node (PLN) cells obtained from biopsies by gently and repeatedly injecting sterile phosphate-buffered saline (PBS) into the tissue using an 18-gauge needle until the cells were released from the tissue. Cell counts and viability were determined by trypan blue dye exclusion. Viability was always >90%.
Reagents and Antibodies
Recombinant human (rh) IL-2 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) from Dr. Maurice Gately (Hoffmann-La Roche, Nutley, NJ). LPS and Concanavalin A (ConA) were purchased from Sigma-Aldrich. Antimouse IgG-coated magnetic Dynabeads M-450 were purchased from Dynal (Great Neck, NY). Streptavidin-peridinin chlorophyll protein (PerCP) was purchased from BD Biosciences PharMingen (San Diego, CA). Anti-TGFβ (monoclonal antibody [mAb] 240) was purchased from R&D Systems (Minneapolis, MN) and conjugated to APC; phycoerythrin (PE)-conjugated anti-TGFβ-RII (FAB241P), goat anti-TGFβ-RII (AF-241-NA), and rhTGFβ (240-B) were also purchased from R&D Systems. Mouse antifeline CD25 (mAb 9F23) was kindly provided by K. Ohno (University of Tokyo, Tokyo, Japan). Anti-CD21 was purchased from P. Moore, (University of California, Davis, CA). Mouse antifeline CD4 (mAb 30A) and CD8 (mAb 3.357)32 were developed in our laboratory. Mouse (AB37355) and goat (AB37373) IgG isotype control antibodies were purchased from Abcam (Cambridge, MA).
Flow Cytometric Analysis
At least 5 × 105 PBMCs or PLN cells were stained for surface expression of CD25, CD4, TGFβ, and TGFβ-RII using fluorescein isothiocyanate (FITC)-conjugated anti-CD25, biotin-conjugated anti-CD4, and APC-conjugated anti-TGFβ mAbs and PE-conjugated anti-TGFβ-RII. T-cell preparations were stained in 5% fetal bovine serum (FBS)/PBS for 30 minutes on ice. When necessary, T cells also were washed and incubated with streptavidin PerCP secondary antibodies for 30 minutes on ice. Flow cytometry data were acquired using multicolor flow cytometry and analyzed (FACSCalibur and CellQuest software, respectively; BD Biosciences, Mountain View, CA). Lymphocytes were gated based on forward versus side scatter, and 20,000 gated events were acquired and stored in list-mode fashion for analysis using CellQuest software.
Purification of T Cells
For fluorescence-activated cell sorting (FACS) purification, lymph node cells were stained with anti-CD4 biotin/streptavidin PerCP and anti-CD25 FITC; CD4+CD25+ and CD4+CD25− T-cell subsets were purified using a MoFlo high-speed and high-purity fluorescence-activated cell sorter (DakoCytomation, Fort Collins, CO). The purity of FACS-purified CD4+CD25+ and CD4+CD25− cell populations was always >95%. CD4+CD25− T cells for use as target cells in proliferation assays were enriched using biomagnetic bead separation performed using goat antimouse IgG-coated beads as described by Bucci et al.30 Briefly, CD21+ B cells, CD8+ T cells, and CD25+ T cells were depleted in successive steps using magnetic beads coated with anti-CD21, anti-CD8, and anti-CD25 antibodies, respectively. The purity of the magnetic bead-enriched CD4+CD25− T cells was >90%, as verified by flow cytometric analysis.
Reverse Transcriptase Polymerase Chain Reaction Analysis
Preliminary to assessing the effect of ConA/TGFβ stimulation of CD4+CD25− cells on FoxP3 expression, it was necessary to develop and validate primers for feline Foxp3 messenger RNA (mRNA). To do this, total RNA was isolated from MYA-1 cells, a feline CD4+CD25+, CD3+CD8−, IL-2-dependent T-lymphoblastoid cell line established from PBMCs.33,34 After reverse transcription, a 385-base pair (bp) fragment from the resulting complementary DNA (cDNA) was amplified by PCR using primers designed from the published human FoxP3 sequence. Sequence analysis of a purified 385-bp feline product showed high homology to several published mammalian FoxP3 sequences. Total RNA was isolated from 2 × 106 to 5 × 106 CD4+CD25+ or CD4+CD25− T cells using the RNeasy Protect Mini Kit (Qiagen, Valencia, CA). Samples were treated on column with RNAse-free DNAse to eliminate contaminating DNA. Reverse transcription (RT) was carried out using the RT system kit from Promega according to the manufacturer's protocol, followed by PCR using HotStar Taq polymerase. Foxp3 message was detected using feline-specific primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was determined and used as a normalizing control. The PCR assay consisted of a 15-minute 95°C denaturing step followed by 35 cycles of 1 minute at 94°C, 45 seconds at 60°C, and 45 seconds at 72°C, with a final extension of 5 minutes at 72°C. Primers used were as follows: Foxp3 (5′-ATTTCATGCACCAGCTCTCAACGG-3′ and 5′-ACCATCTTCCTGGATGAGAAGGGCA-3′) and GAPDH (5′-CCTTCATTGACCTCAACTCCAT-3′ and 5′-GGTCATCCATGACCACTTCGG-3′).
Real-Time Polymerase Chain Reaction Analysis
Primers for target gene 1 (Foxp3: forward primer, GCC TGC CAC CTG GAA TCA AC; reverse primer, TCC CAA GCC CCA GCA CAC; probe, CAG TGC TGG CTC CCT GGA CAC CCA), target gene 2 (TGFβ: forward primer, GAG GTC ACC CGC GTG CTA ATG; reverse primer, TCT TCT CCG TGG AGC TGA AGC AAT), and reference gene (GAPDH: forward primer, GGA GAA GGC TGG GGC TCA C; reverse primer, GGT GCA GGA GGC ATT GCT GA; probe, CCC CTT CTG CTG ATG CCC CCA TGT TTG TGA TGG G) were generated (IDT, Coralville, IA) and optimized to an equal annealing temperature of 62°C. RT-PCR amplification products were separated by electrophoresis on a 2% agarose gel and analyzed with the VersaDoc Imaging System (BioRad, Hercules, CA). RT-PCR products resulted in a single product with the predicted length (Foxp3, 87 bp; TGFβ, 558 bp; and GAPDH, 142 bp), as documented by gel electrophoresis.
For ICycler (BioRad) reactions, TaqMan Universal PCR Master Mix or SYBR Green Master Mix (Applied Biosystems, Foster City, CA) was used. Master Mix (23 μL) was added to each well of a 96-well plate, followed by 2 μL of cDNA. The plate was then placed in the ICycler and analyzed. The following ICycler experimental run protocol was used: 95°C for 15 minutes; 40 cycles of 95°C for 20 seconds, 62°C for 20 seconds, and 72°C for 30 seconds with a single fluorescence measurement; a melting curve program from 62°C to 95°C with a heating rate of 0.05°C per second and a continuous fluorescence measurement; and, finally, a cooling step to 10°C. Melting curve analysis performed by the ICycler for TGFβ resulted in a single product with a specific melting temperature.
Ten-fold serial dilutions of feline CD4+CD25+ Treg cell cDNA were used to generate a 5-point curve to obtain real-time PCR efficiencies calculated by the ICycler software. Transcripts showed high real-time PCR efficiency rates (Foxp3, 1.803; TGFβ, 1.840; and GAPDH, 1.922) with high correlation coefficients (r > 0.99). All experiments were carried out using 500 ng of transcribed total RNA. Fold increase in mRNA expression was calculated based on the following equation:
Equation (Uncited)Image Tools
where E is efficiency of the reaction and Ct is the threshold cycle.
In Vitro T-Cell Suppression Assay
Enriched CD4+CD25− target cells (106 cells/mL) were stimulated for 4 hours with 5 μg/mL of ConA, washed twice in RPMI 1640, and plated at 5 × 104 viable cells/well in 96-well U-bottom plates. CD4+ effector cells were then added to the wells at various effector:target (E:T) ratios ranging from 0.125:1 to 1:1. CD4+CD25+ suppressor cells were added as (1) freshly isolated untreated cells, (2) after treatment in 24-well plates at 1 × 106 cells/mL for 4 days with 10 μg/mL of LPS and 100 U/mL of rhIL-2, or (3) after conversion from CD4+CD25− T cells by culture in 24-well plates at 1 × 106 cells/mL for 4 days with 10 ng/mL of rhTGF-β1 and 5 μg/mL of ConA. The cells were then washed, counted, and added to the target cells. Assays were run in triplicate. Effector and target cells were cocultured for 72 hours and pulsed with 1 μCi of [3H]thymidine per well for the last 18 hours and harvested using a Filtermate Harvester (Packard Bioscience, Meriden, CT). [3H]thymidine incorporation was measured using a Top Count NXT Microplate scintillation counter (Packard Bioscience). Percent inhibition of proliferation was determined based on proliferation of stimulated CD4+CD25− target cells alone and calculated as follows:
Equation (Uncited)Image Tools
The Mann-Whitney U test (t test-like for nonparametric data) was used for pairwise comparison of surface molecule expression. Differences were considered to be significant at P < 0.01.
ConA Plus TGFβ (ConA/TGFβ) Stimulation Induces Immunosuppressor Function in Feline CD4+CD25− T Cells
Murine CD4+CD25− T cells can be converted to functional Treg cells by stimulation with anti-CD3 and anti-CD28 in the presence of TGFβ,21,35 suggesting a novel mechanism of peripheral Treg homeostasis. We were therefore interested to determine whether TGFβ also induces feline CD4+CD25− T cells to acquire the characteristic Treg suppressor activity and what effect FIV infection of cats has on this process. ConA/TGFβ-stimulated CD4+CD25− T cells isolated from PLNs of control cats suppressed the proliferative response of heterologous ConA-stimulated CD4+CD25− target cells in a dose-dependent manner (Fig. 1A). Immunosuppression by ConA/TGFβ-induced Treg-like cells was comparable to that of IL-2/LPS-activated natural CD4+CD25+ Treg cells obtained from control cats (see Fig. 1A). Because it is possible that expansion of the residual CD4+CD25+ T cells in the CD25-depleted CD4+CD25− population was responsible for the observed suppressor activity, we also tested the suppressor activity of autologous CD4+CD25− T cells that were cultured in medium supplemented with IL-2, which is known to activate CD4+CD25+ Treg suppressor function,36 including feline Treg cells.16 CD4+CD25+-depleted T cells stimulated only with IL-2 were unable to suppress the proliferation of ConA-stimulated CD4+CD25− T cells at any ratio tested (see Fig. 1A). Further, neither TGFβ alone nor ConA alone was able to induce suppressor function in the CD4+CD25− T-cell population (see Fig. 1A), verifying that TGFβ and mitogen stimulation are both required for induction of suppressor function by feline CD4+CD25− T cells.
In this experiment, we also asked whether chronic FIV infection of cats affected the ability of CD4+CD25− T cells to attain regulatory function after ConA/TGFβ stimulation. This is an important question, because we previously reported that CD4+CD25− T cells harbor a latent FIV infection that becomes replication competent when stimulated with ConA.15 FACS-purified CD4+CD25− T cells from PLN cells from asymptomatic FIV-infected cats and treated as described for control cats (see Fig. 1A) were also capable of being converted to immunosuppressive Treg cells by ConA/TGFβ treatment but not by ConA or TGFβ alone (see Fig. 1B). As a positive control, we also demonstrated that nonstimulated CD4+CD25+ Treg cells from FIV-infected cats suppressed ConA-induced CD4+CD25− T-cell proliferation in a dose-dependent manner (see Fig. 1B). These experiments demonstrate that ConA/TGFβ stimulation of feline CD4+CD25− T cells from control or FIV-infected cats confers suppressor activity in this population comparative to that seen in in vitro activated CD4+CD25+ T cells from control cats or in vivo activated CD4+CD25+ T cells from FIV-infected cats. The ConA/TGFβ-converted cells from control and FIV-infected cats also displayed an anergic phenotype characteristic of Treg cells because they failed to proliferate in response to ConA stimulation, as opposed to CD4+CD25− T cells (data not shown).
ConA/TGFβ Stimulation of Feline CD4+CD25− T Cells Induces Foxp3 Expression
Recent studies in mice have shown that TGFβ coupled with TCR stimulation of CD4+CD25− T cells confers Treg-like function in this population through the induction of Foxp3.37 Therefore, we investigated the possibility that ConA/TGFβ-converted feline CD4+CD25− T cells also upregulated Foxp3 mRNA using PCR and real-time PCR. Feline primers developed from conserved sequences were used to assess Foxp3 expression in FACS-purified CD4+CD25+ Treg cells and CD4+CD25− Th cells from PBMCs of a normal cat. The feline primer set revealed that primary feline CD4+CD25+ T cells expressed high levels of Foxp3, whereas minimal expression was detected in CD4+CD25− Th cells (Fig. 2A), indicating that Foxp3 expression is preferentially confined to CD4+CD25+ Treg cells in cats. Sequence analysis of the feline Foxp3 product revealed 90.1% homology with human and macaque sequences. RT-PCR confirmed that CD4+CD25+ Treg cells expressed markedly higher levels of Foxp3 mRNA than CD4+CD25− T cells (see Fig. 2B).
Using the feline-specific Foxp3 primers, we next assessed Foxp3 expression in ConA/TGFβ-stimulated CD4+CD25− T cells from FIV-positive cats and found a marked upregulation of Foxp3 message (see Fig. 2C). A similar upregulation of Foxp3 was observed when CD4+CD25− T cells from control cats were used (data not shown). Foxp3 expression was minimally increased when cells were treated with ConA alone but not with TGFβ alone. Real-time PCR confirmed that ConA/TFG-β1 markedly upregulated Foxp3 mRNA expression in CD4+CD25− T cells (Fig. 2D). These data suggest that TGFβ plus mitogen activation is required for the induction of a Foxp3+ immunosuppressive Treg-like phenotype from CD4+ Th cells.
ConA/TGFβ-Stimulated CD4+CD25− Th Cells and Activated CD4+CD25+ Treg cells From FIV-Infected Cats Express mTGFβ
Because surface TGFβ is also a marker associated with murine Treg cells activated in vitro,23 mTGFβ on ConA/TGFβ-converted CD4+CD25− T cells was evaluated by flow cytometry. Culturing of CD4+CD25− T cells in IL-2 or TGFβ alone for 48 hours caused no change in expression of cell surface CD25 or TGFβ (Fig. 3A). Treatment of CD4+CD25− T cells with ConA alone for 48 hours caused a marked upregulation of surface CD25 and a small increase in mTGFβ-positive cells (see Fig. 3A), whereas ConA/TGFβ stimulation significantly increased the number of mTGFβ-positive T cells, which were almost exclusively CD4+CD25+ T cells (see Fig. 3A). Because CD4+CD25+ Treg cells from FIV-infected cats are constitutively immunosuppressive, CD4+CD25+ Treg cells from normal and FIV-infected cats were also assessed for mTGFβ. Figure 3B clearly shows increased mTGFβ on Treg cells from an FIV-infected cat as compared to Treg cells from a normal cat. LPS/IL-2 stimulation of CD4+CD25+ Treg cells from control cats induced mTGFβ (data not shown), suggesting that mTGFβ is a characteristic of in vivo and in vitro activated Treg cells. The data in Figure 3A do not distinguish whether the cell surface TGFβ on ConA/TGFβ-stimulated CD4+CD25+ Th cells or CD4+CD25+ Treg cells from FIV-infected cats is endogenously produced TGFβ or the result of exogenous TGFβ binding to TGFβ-R on activated Th cells. To determine if TGFβ is transcriptionally upregulated in these cells, TGFβ mRNA was quantitated by real-time RT-PCR. The data in Figure 3C show that TGFβ mRNA is marginally upregulated in CD4+CD25− T cells stimulated with ConA/TGFβ (see Fig. 3C).
ConA-Stimulated CD4+CD25− T Cells Upregulate Cell Surface TGFβ-RII
If mTGFβ-positive Treg cells suppress proliferation of ConA-stimulated CD4+CD25− T cells by means of the TGFβ signaling pathway, it follows that ConA stimulation must upregulate TGFβ-R on CD4+CD25− T cells. To address this question, FACS-purified CD4+CD25− T cells were stimulated with ConA and assessed for mTGFβ-RII expression by flow cytometry. TGFβ-RII was upregulated on a large fraction of ConA-stimulated CD4+CD25− T cells as compared to freshly purified cells or cells cultured in medium supplemented only with rhIL-2 (Fig. 4). Interestingly, there seems to be 2 populations of TGFβ-RII-positive T cells, as indicated by forward scatter, suggesting that a fraction of CD4+CD25+ cells expressing TGFβ-RII are entering a blast-like or early proliferative state. Two-color flow cytometry revealed that most TGFβ-RII-positive CD4+ cells were, as expected, also CD25+ (data not shown). Real-time RT-PCR confirmed that ConA stimulation of CD4+CD25− T cells upregulated TGFβ-RII at the transcription level (data not shown).
Blockade of TGFβ-RII on CD4+CD25− Th Target Cells Abrogates Phenotypic and Functional Conversion by TGFβ
To address the role of TGFβ-RII in ConA/TGFβ conversion of CD4+CD25− T cells to a Treg phenotype more fully, FACS-purified naive CD4+CD25− T cells from normal cats were stimulated with ConA for 18 hours and then washed and incubated with goat anti-TGFβ-RII antibody or goat isotype control antibody for 30 minutes and washed again before addition of TGFβ to the culture medium. After 48 hours, mTGFβ was assayed by flow cytometry. TGFβ surface expression was completely blocked when anti-TGFβ-RII antibody was added to ConA-stimulated CD4+CD25− T cells before addition of TGFβ (Fig. 5). Cells were incubated in medium alone or with the isotype control antibody expressed mTGFβ (see Fig. 5). To assess the effect of TGFβ-RII blockade on TGFβ-induced Foxp3 expression in CD4+CD25− T cells, Th cells were treated as discussed previously, and after 48 hours, RNA was extracted and analyzed for Foxp3 mRNA by quantitative real-time RT-PCR. Pretreatment of ConA-stimulated CD4+CD25− cells with anti-TGFβ-RII antibodies markedly reduced TGFβ-induced Foxp3 mRNA expression (Fig. 6). In a second set of experiments, ConA-stimulated CD4+CD25− cells were treated with anti-TGFβ-RII and cultured with TGFβ for 48 hours before assessing suppressor function in the ConA-stimulated CD4+CD25− proliferation assay. Treatment of ConA-stimulated CD4+CD25− T cells with anti-TGFβ-RII antibodies also inhibited TGFβ-induced conversion to a suppressor phenotype (Table 1). Collectively, these data strongly suggest that mTGFβ mediates conversion of Th cells to Foxp3+ mTGFβ-positive immunosuppressive Treg cells through the TGFβ-RII signaling pathway.
Anti-TGFβ or Anti-TGFβ-RII Treatment Diminishes CD4+CD25+ Treg Suppressor Function
Although expression of mTGFβ is a constant feature of activated natural Treg cells, the role of TGFβ in mediating immunosuppression has been controversial. Nakamura et al10,23 reported that mTGFβ mediates suppressor function, although others27,38 have suggested that it is not involved. To address this question, CD4+CD25+ Treg cells from FIV-infected cats were treated with anti-TGFβ or ConA-stimulated CD4+CD25− target cells were treated with anti-TGFβ-RII antibodies before coculture and evaluation of suppressor function. Suppressor function by natural CD4+CD25+ Treg cells from a chronically FIV-infected cat was diminished when pretreated with anti-TGFβ (see Table 1). In addition, suppressor function of in vivo FIV-activated CD4+CD25+ Treg cells was completely abolished by anti-TGFβ-RII treatment of CD4+CD25− target cells or by treatment of both suppressor and target cells (see Table 1), suggesting that suppressor function of natural CD4+CD25+ Treg cells is mediated by the TGFβ/TGFβ-R signaling pathway that is constitutively upregulated in Treg cells from FIV-infected cats. There is currently no information on the role of cell surface TGFβ on the suppressor function of Th to Treg-converted cells. To address this question, we converted FACS-purified CD4+CD25− T cells from PLN of normal cats to CD25+ mTGFβ-positive Treg-like suppressor cells as described previously. The converted cells were then incubated with anti-TGFβ for 30 minutes and washed before coculturing in the antiproliferation suppressor assay. As shown in Table 1, pretreatment of ConA/TGFβ-converted CD4+CD25− T cells with anti-TGFβ antibodies completely abrogated suppressor activity. Together, these experiments suggest that TGFβ/TGFβ-RII signaling plays a critical role in the recruitment of Treg cells from the Th cell pool and in mediating their suppressor function.
CD4+CD25+ Treg cells have emerged as a distinct lineage of T cells thought to function in maintenance of peripheral self-tolerance and to modulate immune responses to pathogens.39-45 Although the immunoregulatory property of these cells is no longer questioned, where and how they are generated and the scope and mechanism of their immunoregulatory function remain areas of active investigation. Although early studies indicated that the thymus is the sole reservoir of Treg cells,2 recent evidence suggests that Treg cell homeostasis may also be regulated in the periphery, particularly under conditions of chronic immune stimulation such as chronic infectious disease.46 CD4+CD25+ Treg cells express a number of Toll-like receptors (TLRs), including TLR4, and can be activated with LPS.20 Also, IL-2 has been shown to induce a proliferative response by peripheral Treg cells in vitro and to increase their immunosuppressor function.47,48 Others have reported that peripheral Treg cells can be recruited from the CD4+CD25− T-cell pool by TCR engagement in the presence of TGFβ. Conversion of CD4+CD25− T cells to CD4+CD25+Foxp3+ T-suppressor cells by anti-CD3 and anti-CD28 antibody treatment in the presence of exogenous TGFβ has been reported in rodents and humans.21,35 Here, we report that ConA stimulation followed by exogenous TGFβ converts feline CD4+CD25− T cells to a CD4+CD25+Foxp3+ mTGFβ-positive Treg-like phenotype.
Similar to the observations of Chen et al21 and Fantini et al,35 the ConA/TGFβ-converted CD4+CD25− T cells are anergic and possess potent immunosuppressor activity against ConA-stimulated CD4+CD25− T cells. We also investigated the dual role of ConA and TGFβ in the Th-to-Treg conversion process. ConA-stimulated CD4+CD25− T cells rapidly upregulated TGFβ-RII on their cell surface, rendering them susceptible to conversion to a Treg-like phenotype by TGFβ. In addition, we observed that pretreatment of ConA-stimulated CD4+CD25− T cells with anti-TGFβ-RII antibodies abrogated TGFβ-induced conversion to a Treg-like phenotype, including reduction in Foxp3 mRNA, mTGFβ expression, and suppressor function, suggesting a role for TGFβ/TGFβ-RII signaling in the conversion process. Chen et al21 also reported that TGFβ and TCR costimulation of CD4+CD25− T cells results in a Foxp3+ immunosuppressive Treg-like phenotype that expressed TGFβ on their surface. Whether the mTGFβ on ConA/TGFβ-induced Treg cells in our study or the TCR/TGFβ-induced Treg cells in the study by Chen et al21 is attributable to transcriptional upregulation of TGFβ in the converted Treg-like cell or the result of membrane-bound exogenous TGFβ is not known; however, our data suggest either is possible.
Although it is clear from our studies that ConA upregulates TGFβ-RII on CD4+CD25− T cells and that the exogenous TGFβ is capable of binding to these receptors and converting the CD4+CD25− T cells to a Treg phenotype, our data also show that TGFβ is transcriptionally upregulated in ConA/TGFβ-stimulated CD4+CD25− T cells. Fantini et al35 also reported that murine CD4+CD25− T cells stimulated with TGFβ plus anti-CD3/anti-CD28 expressed higher levels of TGFβ mRNA than cells cultured in the absence of TGFβ. Nakamura et al23 reported that anti-CD3-stimulated CD4+CD25+ Treg cells upregulated TGFβ on their surface, suggesting an active induction process. We also reported herein that in vivo activated CD4+CD25+ Treg cells from FIV-infected cats constitutively express higher levels of mTGFβ than Treg cells from control cats. Whether the cell surface TGFβ was of endogenous or exogenous origin was not determined. Although our data would support a role for exogenous TGFβ binding to TGFβ-R on activated Treg cells, we cannot rule out the possibility that the mTGFβ is an autocrine response.
This study also demonstrated that ConA/TGFβ-converted CD4+CD25− cells are phenotypically and functionally comparable to the in vivo activated Treg cells from chronically FIV-infected cats. Both constitutively express mTGFβ and mediate immunosuppressor function in the absence of additional in vitro stimulation. Treatment of converted Treg cells or natural CD4+CD25+ Treg cells from FIV-infected cats with anti-TGFβ reduced their suppressor activity against ConA-stimulated CD4+CD25− cells, suggesting a role for mTGFβ. This observation is consistent with observations by Nakamura et al,23 who also showed that activated CD4+CD25+ Treg cells express TGFβ on their surface and that treatment with anti-TGFβ antibodies inhibited their suppressor function. That TGFβ/TGFβ-RII signaling mediates Treg suppressor function in the feline model is confirmed by our observation that ConA treatment of CD4+CD25− T cells upregulates TGFβ-RII on their surface and that treatment of these target cells with anti-TGFβ-RII antibodies before addition of CD4+CD25+ Treg cells abrogates suppressor function. Others, however, have reported that TGFβ plays no role in the suppressor function of CD4+CD25+ Treg cells.3,26 Marie et al28 also presented data in a TGFβ-deficient mouse model to support the conclusion that TGFβ is not an effector molecule of suppression but is a mediator of signaling in Treg cells required to maintain Foxp3 expression and suppressor function. In another model, Walker et al49 reported that the suppressor activity of regulatory CD4+CD25+ generated from anti-CD3/anti-CD28-activated CD4+CD25− T cells was contact dependent and cytokine independent. They used transwell assays to draw this conclusion, however, and failed to consider the possibility of mTGFβ-mediated suppression.
The studies reported herein support the suggestion that ConA/TGFβ-induced Th-to-Treg-like conversion in vitro may be relevant to mechanisms maintaining Treg cell homeostasis and function in vivo, particularly in the case of chronic immune stimulation. In HIV and FIV infections, the numbers of Treg cells in the circulation do not change appreciably or may actually increase in the percentage of the total CD4+ T-cell population throughout the course of the infection.16,17 A role for TGFβ in recruitment or expansion of Treg cells in lentivirus infection was recently suggested in SIV primate models for AIDS. Kornfeld et al18 reported an early induction of TGFβ and Foxp3 expression that correlated with increased levels of CD4+CD25+ and CD8+CD25+ T cells in acutely SIV-infected African green monkeys. Estes et al19 also reported an increase in CD4+CD25+Foxp3+ T cells and TGFβ-positive T cells in acutely SIV-infected macaques. The origin of these CD4+CD25+ T cells (eg, expansion of Treg cells or recruitment from the CD4+ Th pool) was not determined in either of these studies. Studies in the FIV model for human AIDS are currently underway to address this question.
In summary, our data suggest that TGFβ/TGFβ-RII signaling and upregulation of Foxp3 may play a dual role in maintaining Treg peripheral homeostasis and suppressor function. Further, our data suggest that the TGFβ/TGFβ-RII signaling pathway is constitutively activated in CD4+CD25+ Treg cells from chronically FIV-infected cats and may play a role in the T-cell immunodeficiency that is the hallmark of this infection.
The authors thank Drs. Jonathan Fogle and Angela Mexas for assistance with collection of blood and lymph node cells and Deborah Anderson for excellent technical assistance.
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