FoxP3 and Bcl-xL cooperatively promote regulatory T cell persistence and prevention of arthritis development

DAB/1J and C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). OT-II TCR-transgenic mice, expressing a TCR composed of variable (Vβ5 and Vα2) chains responsive to the I-A^b-restricted ovalbumin (OVA) peptide 323-339 (ISQAVHAAHAEINAGR), were maintained by breeding with C57BL/6J mice. All experiments were in compliance with the regulations of the Pennsylvania State University College of Medicine Animal Care Committee and were in accordance with guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care.

Anti-CD28 (37.51), mouse IL-2, and interferon-gamma (IFN-γ) were from BD Pharmingen (San Diego, CA, USA). Anti-actin (C2, sc-8432) for Western blot was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-Bcl-xL (#2762), peroxidase-conjugated anti-rabbit (#7054), and anti-mouse Ig (#7056) for Western blot were purchased from Cell Signaling Technology (Beverly, MA, USA). All FITC (fluorescein isothiocyanate)-, PE (phycoerythrin)-, Cy5 (cyanine 5)-, and APC (antigen-presenting cell)-conjugated antibodies were purchased from BioLegend (San Diego, CA, USA).

Naive CD4⁺ T cells from spleen and lymph nodes of OT-II mice were enriched by magnetic sorting with the CD4⁺ T Cell Isolation Kit (#130-090-860; Miltenyi Biotec Inc., Auburn, CA, USA). The cells were more than 90% CD4⁺, and more than 95% of these cells expressed a naive phenotype and the appropriate TCR as determined by flow cytometry at the Penn State Hershey flow cytometry core facility.

T cells were cultured in 24-well plates containing 1 mL of RPMI 1640 (Invitrogen Corporation, Carlsbad, CA, USA) with 10% fetal calf serum (HyClone, Logan, UT, USA). Naive CD4⁺ T cells were stimulated with plate-bounded anti-CD3 (2C11, 4 μg/mL) and soluble anti-CD28 (37.51, 4 μg/mL) antibodies.

cDNA corresponding to human Bc1xL was subcloned into the murine bicistronic retroviral expression vector Mig [18]. Mig-FoxP3 was a gift from Alexander Y Rudensky (Department of Immunology, University of Washington, Seattle, WA, USA) [19]. Retrovirus-mediated transduction was performed as described previously [16]. Briefly, T cells (1 × 10⁶) were stimulated with plate-bounded anti-CD3 and soluble anti-CD28 antibodies. After 2 days, the supernatant was replaced with 1 mL of viral supernatant containing 5 μg/mL Polybrene (Sigma-Aldrich, St. Louis, MO, USA), and the cells were sedimented by centrifugation for 1.5 hours at 32°C and incubated at 32°C for 8 hours. This step was repeated the following day. Viral supernatant was removed and replaced with fresh medium, and T cells were re-cultured. Expression of green fluorescent protein (GFP) in CD4⁺ T cells, indicative of retrovirus-mediated transduction, was determined by flow cytometry. GFP-expressing T cells were purified by cell sorting using a MoFlo high-performance cell sorter (Beckman Coulter, Fullerton, CA, USA).

Male DBA/1J mice (greater than 4 months of age) were injected at the base of the tail with 0.1 mL of emulsion containing 100 μg of bovine type II collagen (CII) (Chondrex, Redmond, WA, USA) in complete Freund's adjuvant (CFA) (Chondrex) using a 1-mL glass tuberculin syringe with a 26-guage needle. Mice were assessed for clinical arthritis in the paws, with each paw individually scored using a 4-point scale: 0, normal paw; 1, minimal swelling or redness; 2, redness and swelling involving the entire forepaw; 3, redness and swelling involving the entire limb; 4, joint deformity or ankylosis or both. Animals achieving a clinical score of 4 were euthanized.

Mice were sacrificed on day 60 after the challenge of CII. Hind foot paws were amputated, fixed in 10% formalin, and decalcified in Formical-4 (Decal Chemical Corporation, Tallman, NY, USA). The tissues were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (HE) or Safranin O-fast green as described before [20]. HE staining for bone erosions was scored using a semiquantitative scoring system from 0 to 4 (0 = no erosions, 4 = extended erosions and destruction of bone). Safranin O staining for the loss of proteoglycans was scored with a semiquantitative scoring system (0 to 3), where 0 represents no loss of proteoglycans and 3 indicates complete loss of staining for proteoglycans.

Blood samples were collected from the orbital sinus or by heart puncture on day 60 after primary immunization with CII. Total IgG was measured using the Easy-Titer IgG Assay Kit (Pierce, Rockford, IL, USA) in accordance with the recommendations of the manufacturer. The levels of anti-CII IgG in these sera were measured by enzyme-linked immunosorbent assay (ELISA) as described before [21]. Briefly, a 96-well microplate (Nunc, Roskilde, Denmark) was coated with 5 μg/mL CII at 4°C overnight followed by blocking with 0.2% bovine serum albumin and 0.05% Tween-20 in phosphate-buffered saline (PBS) at 4°C for 6 hours. Test samples were appropriately diluted with PBS containing 2% normal goat serum and added at 200 μL/well. After storage at 4°C overnight, wells were washed three times with PBS. Next, 100 μL of 1:1,000-diluted rabbit anti-mouse IgG1 or IgG2a conjugated with biotin and 100 μL of streptavvidin-peroxidase secondary antibody (Rockland Immunochemicals Inc., Gilbertsville, PA, USA) was added to each well and incubated at 4°C for 2 hours and 1 hour, respectively. After thorough washing, bound peroxidase activity was assayed by incubating samples at room temperature with 100 μL of TMB (3,3',5,5' tetramethylbenzidine) substrate (BioLegend). Absorbance at 405 nm was measured using a microplate reader.

T cells were cultured with CD3/CD28 and transduced on day 2/3 with retroviral vectors as described previously [16]. Cells were recultured for 3 more days. GFP⁺ CD4⁺ T cells were sorted by a MoFlo high-performance cell sorter, and 2.5 × 10⁶ cells were injected intravenously into the tail vein of male DBA/1J mice that had been induced to develop collagen-induced arthritis (CIA) 15 days prior by immunization with CII.

Cytokines were measured by ELISA [15]. T-cell survival in vitro was determined by trypan blue exclusion. Cell proliferation was measured by incorporation of 5-bromo-2-deoxyuridine (BrdU) (Invitrogen Corporation) overnight, staining with anti-BrdU, and analysis by flow cytometry. Alternatively, incorporation of [³H] thymidine (1 μCi/well; PerkinElmer Inc., Boston, MA, USA) during the last 12 hours of culture was measured.

Live CD4⁺ T cells were lysed in ice-cold RIPA lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, and 1 μg/mL leupeptin) for 30 minutes. Insoluble material was removed by centrifugation, and lysates were used for Western blotting. Protein content was determined by a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of protein (40 μg) were loaded onto 4% to 12% NuPage Bis-Tris precasting gels and separated by SDS-PAGE, transferred onto nitrocellulose membrane (Invitrogen Corporation), and immunoblotted. All blots were developed with the ECL (enhanced chemiluminescence) immunodetection system (Amersham Pharmacia Biotech, now part of GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Previously, we used the 2A peptide regions from picornavirus TaV (abbreviated herein as T2A) to generate multicistronic cassettes that linked Bcl-xL and survivin to make a single fragment encoding two proteins [18]. To generate reliable and versatile constructs to transduce primary CD4⁺ T cells that permit expression of FoxP3 and Bcl-xL genes, we used the T2A peptide to generate a retroviral vector with efficient translation of two cistrons (for example, FoxP3 and Bcl-xL) (Figure 1a). The human Bcl-xL gene and T2A were excised from Mig-Bcl-xL-2A-survivin [18] and subcloned into Mig-FoxP3. Thus, Bcl-xL and FoxP3 were linked with the 2A sequence in the Mig vector. The integrity of the Mig-Bcl-xL-2A-FoxP3 construct was confirmed by DNA sequencing (data not shown), and protein expression was verified by Western blot (Figure 1b). Both Bcl-xL expression and FoxP3 expression were induced by retroviral infection of CD4⁺ T cells following transduction with retroviral constructs expressing the individual genes or both genes (Figure 1b). By this method, approximately half of the cultured CD4⁺ T cells were induced to express FoxP3 following retrovirus-mediated transduction as visualized by flow cytometry (Figure 1c).

To determine whether induced co-expression of FoxP3 and Bcl-xL promotes the persistence of Tregs, we transduced naive CD4⁺ T cells with the GFP-IRES (GFP-internal ribosome entry site) retroviral vector containing FoxP3 and Bcl-xL (Mig-Bcl-xL-2A-FoxP3) (Figure 2a). After 2 to 3 days of transduction, the recovery of live T cells was monitored by GFP expression. On day 4 post-transduction, we found that the expansion of CD4⁺ T cells that had been transduced with both FoxP3 and Bcl-xL was greater than the expansion of cells transduced with FoxP3 alone (Figure 2b). Longer-term culture over 8 days showed that co-expression of FoxP3 and Bcl-xL enhanced the ability of CD4⁺ T cells to survive as compared with that of CD4⁺ T cells transduced with FoxP3 alone (Figure 2b). This result was not due to differences in FoxP3 expression between cells transduced with both FoxP3 and Bcl-xL versus FoxP3 alone, indicating that exogenous Bcl-xL did not affect FoxP3 expression (Figure 1b). Moreover, expression of CD25, a marker for Tregs, was significantly upregulated by day 4 in the CD4⁺ GFP⁺ T cells that were transduced with FoxP3 or with FoxP3 plus Bcl-xL as compared with cells in the vector control group, suggesting that these cells were characteristic of Tregs after gene transfection (Figure 2c). Similar profiles were observed in the expression of cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) (data not shown). Thus, co-expression of FoxP3 and Bcl-xL induced from a single retroviral vector produced a Treg phenotype in the resulting cells and provided increased long-term survival of these cells.

To investigate whether co-expression of FoxP3 and Bcl-xL promotes suppressive activity of Tregs, CD4⁺ T cells expressing FoxP3 and Bcl-xL from primary cultures were sorted based on GFP expression, and equal numbers of cells were re-stimulated with anti-CD3 plus anti-CD28 antibodies. Greater numbers of cells were recovered over time from Tregs derived from expression of both FoxP3 and Bcl-xL as compared with cells transduced with FoxP3 alone (Figure 3a). To evaluate Treg function, Tregs isolated from CD4⁺ T cells expressing both FoxP3 and Bcl-xL or FoxP3 alone were co-cultured with naive CD4⁺ T cells (Tregs/T cells = 1:1) in the presence of anti-CD3 and anti-CD28 antibodies for various periods of time. The results show that production of IL-2 and IFN-γ over the 48-hour period was significantly decreased in the presence of Tregs derived from expression of FoxP3 alone or FoxP3 plus Bcl-xL (Figure 3b). However, the proliferation in CD4⁺ GFP^- T cells was significantly suppressed by Tregs co-expressing FoxP3 and Bcl-xL as compared with cells expressing FoxP3 alone on day 4 but not day 2 (Figure 3c, d). These data indicate that the transduction of both FoxP3 and Bcl-xL sustains suppressive activity of CD4⁺ T cell-derived Tregs by promoting survival advantage.

We reported previously that induced expression of Bcl-xL significantly sustained T-cell survival [15, 22]. In the present study, we examined whether co-expression of Bcl-xL with FoxP3 is capable of increasing the persistence of Tregs in vivo. CD4⁺ T cells from OT-II TCR transgenic mice were induced to differentiate into Tregs by transduction of FoxP3 alone or FoxP3 and Bcl-xL. On day 6 post-transduction, GFP⁺ Tregs were sorted and adoptively transferred into syngeneic recipient mice. These mice were subsequently challenged with OVA protein. We found that the effect of Bcl-xL and FoxP3 was cooperative and long-lasting as we measured greater numbers of Tregs expressing both genes than cells derived by FoxP3 transduction alone on days 7 and 14 after Ag challenge (Figure 4). Thus, Tregs were maintained above background levels beyond the time of normal T-cell contraction (Figure 4). Overall, these data strongly support the conclusion that the combined activities of FoxP3 and Bcl-xL promote both the differentiation and long-term survival of Tregs.

To demonstrate that the gene transduction of FoxP3 and Bcl-xL sustains the Treg response in a physiologically and clinically relevant setting, GFP-sorted Tregs derived from DBA/1J mice were adoptively transferred into male DBA/1J mice (>4 months of age). Fifteen days prior to adoptive transfer (day 0), CIA was induced in the recipient DBA/1J mice by one intradermal immunization in the base of the tail with 100 μg of CII in CFA, containing 5 mg/mL killed Mycobacterium tuberculosis (H37Ra). Arthritis incidence (Figure 5a) and clinical score (Figure 5b) were assessed in the paws from three independent experiments (Additional files 1 and 2). Mice receiving FoxP3-plus-Bcl-xL-transduced Tregs had a significantly decreased incidence of arthritis compared with mice receiving Tregs transduced with FoxP3 alone (32% versus 70% on day 60; Figure 5a). Most importantly, mice receiving FoxP3-plus-Bcl-xL-transduced Tregs had a lower clinical score than those receiving Tregs transduced with FoxP3 alone (a score of 1.2 versus 2.8 on day 60; Figure 5b).

We also examined whether disease severity correlates with antibody production and histological observations of the joints. We found that mice receiving FoxP3-plus-Bcl-xL-transduced Tregs had similar levels of total IgG and anti-CII IgG1 in the serum as mice that received Tregs transduced with FoxP3 alone (Figure 6a, b). However, anti-CII IgG2a was significantly decreased following adoptive transfer of FoxP3-plus-Bcl-xL-transduced Tregs (Figure 6c). These results are similar to observations reported in a recent study that describes decreased levels of IgG2a following adoptive transfer of Tregs [23]. Because anti-CII IgG2a was the major component of anti-CII IgG on day 60, total anti-CII IgG in this case was significantly reduced by the treatment of adoptive transfer of FoxP3-plus-Bcl-xL-transduced Tregs. These results are similar to observations previously reported [24, 25]. The preventive effects of Treg transfer on CIA were also confirmed by histological analyses. In the control mice, there were significant signs of arthritic pathology as indicted by destruction of cartilage with fibrosis and leukocyte infiltration around bones and in the joint space on day 60 (Figure 7a). In contrast, the treatment of adoptive cell transfer of Tregs resulted in a marked suppression of these symptoms that are characteristic of CIA. In addition, there were only a few foci of leukocyte infiltration and less destruction of bones and joints in mice that received FoxP3-plus-Bcl-xL-transduced Tregs (Figure 7). Collectively, these findings show that the expression of FoxP3 and Bcl-xL by retrovirus-mediated transduction can cooperatively promote the persistence of Tregs and that these Tregs can effectively prevent the development of CIA.