Cats and FIV infection
Specific pathogen-free cats were obtained from Liberty Labs (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. Cats were inoculated with the NCSU1 isolate of FIV, a pathogenic clade A virus, as described by Bucci et al. . FIV-infection was confirmed by immunoblot analysis and provirus detection by PCR using 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. Non-infected control cats ranged in age from 3 to 6 years and were housed separately from FIV-infected cats. Protocols were approved by the North Carolina State University Institutional Animal Care and Use Committee.
Sample collection and preparation
Whole blood (28 ml/cat) was collected by jugular venipuncture into EDTA Vacutainer tubes (Becton-Dickinson, Franklin Lakes, NJ). PBMC were isolated by Percoll density gradient centrifugation (Sigma-Aldrich, St. Louis, MO) as previously described  or by Ficoll-Histopaque-1077 density gradient centrifugation (Sigma-Aldrich, St-Louis, MO) following the manufacturer’s guidelines. Single-cell suspensions were prepared from popliteal or submandibular peripheral lymph nodes (PLN) obtained through surgical biopsies by gently and repeatedly injecting sterile PBS into the tissue using an 18G needle until the cells were released from the tissue. Cell counts and viability were determined by trypan blue dye exclusion and viability was always >90%.
Reagents and antibodies
Recombinant human IL2 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Dr. Maurice Gately, Hoffmann - La Roche Inc. LPS and Concanavalin A (ConA) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse IgG coated magnetic Dynabeads® M-450 were purchased from Dynal (Great Neck, NY). Streptavidin–PerCP was purchased from BD Biosciences PharMingen (San Diego, CA). Anti-TGFβ1 (MAB240) was purchased from R&D Systems (Minneapolis, MN) and conjugated to allophycocyanin (APC) or left unconjugated for blocking studies; PE-conjugated anti-TGFβ-RII (FAB241P), neutralizing anti-TGFβRII (AF-241-NA), and recombinant human TGFβ1 (240-B) were also purchased from R&D Systems. Mouse anti-feline CD25 (mAb 9 F23) was kindly provided by K. Ohno (University of Tokyo, Tokyo, Japan). Anti-CD21 was purchased from Serotec (Raleigh, NC). Mouse anti-feline CD4 (mAb 30A) and CD8 (mAb 3.357) were developed in our laboratory . PE conjugated rat anti-mouse FoxP3 (FJK-16 s) was purchased from eBioscience (San Diego, CA). FITC-conjugated anti-GARP IgG2b monoclonal antibody (LRRC32, Plato-1) was purchased from Enzo life Sciences (Ann Arbor, MI).
Flow cytometric analysis
For comparison of TGFβ and TGFβRII in chronic FIV-infected or uninfected cats, at least 5 × 105 PBMC were stained for surface expression using specific antibodies and three-color flow cytometry was performed on a FACSCaliber flow cytometer (BD Biosciences, Mountain View, CA). Lymphocytes were gated based on forward vs. side scatter, and 20,000 gated events were acquired and stored list-mode fashion for analysis using CellQuest software. For phenotyping studies, sorted and/or cultured cells were stained for surface expression of CD4, CD25, TGFβ, GARP and TGFβRII. For intracellular staining of FoxP3, cells were first stained for CD4 and CD25 expression, washed in PBS, incubated with 4% PFA for 10 minutes, and washed twice more. Cells were then incubated in 0.1% Triton x-100 for 30 minutes, washed with PBS + 4% FBS, resuspended in 100 uL of PBS and incubated with FoxP3-specific antibody at room temperature for 20 minutes. Cells were washed in PBS + 4% FBS and analyzed on the FACSCalibur flow cytometer, lymphocytes were gates on forward vs. side scatter and 20,000 gated events were acquired and stored list-mode fashion for analysis using CellQuest software. All gating was determined by isotype controls.
Purification of lymphocyte populations
CD4+CD25+ and CD4+CD25- cell populations were purified as previously described [17, 20]. Briefly, for FACS purification, PBMCs or LN cells were stained with anti-CD4, anti-CD25 and anti-CD8. CD4+CD25+, CD4+CD25- and CD4-CD8- (used as antigen presenting cells [APCs]) cell subsets were purified using a high-speed, high-purity fluorescence activated cell sorter (MoFlo, DakoCytomation). The purity of FACS sorted cell populations was always > 95%. CD4+CD25- T cells for use as target cells in proliferation assays were enriched using biomagnetic bead separation using goat anti-mouse IgG-coated beads as described by Bucci et al. . Briefly, CD21+ B cells, CD8+, and CD25+ T cells were depleted in successive steps using magnetic beads coated with anti-CD21, anti-CD8 and anti-CD25 antibody respectively. Purity of the magnetic bead enriched CD4+CD25- T cells was > 90%, as verified by flow cytometric analysis.
Treg cell-induced conversion assay
Converter cells consisted of purified CD4+CD25+ T cells from either control cat or FIV-infected cat PLN. In the case of control cats, the converter cells were activated by culturing (4 × 106 per well, 24-well plate) for 4 days in the presence of LPS (10 μg/mL) plus IL2 (100 U/mL) then washed twice and labeled with Vybrant DiD (Molecular Probes) fluorescent dye. In the case of FIV-infected cats, as the CD4+CD25+ T cells are constitutively activated, the converter cells were freshly isolated and labeled with Vibrant DiD (see Figure 2). Converter cell control groups consisted of CD4+CD25- or CD4+CD25+ cells from control cats incubated in IL2 (100 U/ml) alone for 4 days then labeled with DiD. The positive control for conversion of CD4+CD25- Th was a 5d treatment of sorted cells with 5 μg/mL ConA and 10 ng/mL soluble TGFβ which has been described in previous conversion experiments . The target cells for testing conversion cell immunosuppressive function consisted of autologous unlabeled CD4+CD25- T cells freshly purified from peripheral blood and stimulated with 5 μg/mL ConA and 100 U/ml IL2 for 4 h. The target cells were washed and added to the converter cell cultures at a converter to target cell ratio of 1:2. Cultures were kept at 37°C for 5 days in RPMI 1640 supplemented with 10% heat-inactivated FBS, 50 μM 2-ME, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10U/ml IL2. After 5 days, DiD positive cells were FACS depleted and the remaining cells were used in suppression assays, cell surface molecule analysis, and mRNA analysis. The purity of DiD-depleted cells was >98% as verified by flow cytometric analysis. For conversion blocking experiments, 100 μg/ml anti–TGFβ or 100 μg/ml anti-GARP was added to the converter cell population, or 100 μg/ml anti–TGFβ−RII was added to the target cell population just prior to co-culture. Experimental design is outlined in Figure 2.
In vitro T cell IL2 suppression assay
Peripheral lymph node (PLN) cells from FIV-negative cats were FACS purified into CD4+CD25- target cells and CD4-CD8- APCs, combined at a 1:1 ratio, stimulated for 4 h with 5 μg/ml ConA, washed twice in RPMI 1640, and plated at 2 × 106 cells/mL in 12-well plates. DiD negative cells from the conversion assays described above were added as suppressor cells at a suppressor to target cell ratio of 1:2. Effector cell controls for the suppressor assay consisted of CD4+CD25+ cells from control cats that had been stimulated for 4 days with 10 μg/ml LPS and 100 U/ml rhIL2 (positive control) or non-stimulated CD4+CD25- cells from control cats (negative control) as suppressor cells. Controls for IL2 production consisted of ConA stimulated CD4+CD25- cells plus APCs without effector cells (positive control) and non-stimulated CD4+CD25- cells plus APCs without effector cells (negative control). After 24 hours, 100 uL of the supernatant from each well was analyzed in triplicate by IL2 ELISA using the Feline IL2 DuoSet (DY1890 R&D Systems, Minneapolis, MN) as per manufacturer’s protocol.
In vitro T cell proliferation suppression assay
target cells (106
cells/ml) were stimulated for 4 h with 5 μg/ml Con A, washed twice in RPMI 1640, and plated at 5 × 104
viable cells/well in 96-well U bottom plates. DiD negative cells from the conversion assays described above were added as suppressor cells at suppressor to target cell ratios ranging from 0.125:1 to 1:1. Controls for the suppressor assay consisted of CD4+
cells from control cats that had been stimulated for 4 days with 10 μg/ml LPS and 100 U/ml rhIL2 (positive control) or non-stimulated CD4+
cells from control cats (negative control) as suppressor cells. Effector and target cells were co-cultured at 37°C for 72 hrs, pulsed with 1 μCi of [3
H]TdR/well for the last 18 h and harvested using a Filtermate Harvester (Packard Bioscience, Meriden, CT). [3
H]thymidine incorporation was measured using a Top Count NXT Microplate scintillation counter (Packard Bioscience). Percent inhibition of proliferation was determined based on proliferation of CD4+
target cells alone and calculated as follows:
For suppressor activity blocking experiments, the suppressor cells were pretreated with 100 μg/ml anti-TGFβ for 30 minutes then washed, counted, and added to the target cells. Assays were run in triplicate.
Reverse transcription PCR analysis of FoxP3 and GARP
FoxP3 mRNA was detected by RT-PCR using feline specific primers for FoxP3 or GARP as described previously (Miller et al., 2013, Petty et al., 2008). GAPDH mRNA expression was used as a normalizing control. Briefly, total RNA was isolated from 1 × 106 CD4+CD25+ or CD4+CD25- T cells using RNeasy Protect Mini Kit (Qiagen, Valencia, CA). Reverse transcription was carried out using a reverse transcription system kit from Promega as per the manufacturer’s protocol, followed by PCR using HotStar Taq polymerase (Qiagen, Valencia CA). PCR products were resolved on agarose/ethidium bromide gels and visualized using GelRed (Biotium, Hayward CA).
The Mann–Whitney U test (t test for nonparametric data) was used for pair-wise comparison of parameters. Differences were considered to be significant at p < 0.05.