Expansion of Functional Endogenous Antigen-Specific CD4⁺CD25⁺ Regulatory T Cells from Nonobese Diabetic Mice

Mice

NOD mice were purchased from Taconic Farms; BDC2.5 TCR Tg⁺ mice were obtained from D. Mathis (Joslin Clinic, Harvard University, Boston, MA) (11); and glutamic acid decarboxylase (GAD)₂₈₆ TCR Tg⁺ mice (12), NOD.CD28^−/− mice (13), and NOD.TCRα^−/− mice (The Jackson Laboratory) were housed and bred under specific pathogen-free conditions at the University of California San Francisco Animal Barrier Facility in accordance with institutional guidelines.

Reagents

Anti-CD3ε (145-2C11), anti-CD28 (PV-1), anti-CD4 (GK1.5), and anti-glucocorticoid induced tumor necrosis factor receptor (GITR) (DTA-1; a gift from S. Sakaguchi, Kyoto University, Kyoto, Japan) were produced and purified in our laboratory. Anti-CD4 and anti-GITR were conjugated to FITC in our laboratory. R-PE-conjugated anti-CD25 (7D4) was purchased from Southern Biotechnology Associates. R-PE-conjugated anti-CTLA4 (UC10-4F10-11), biotinylated-anti-Vβ2 TCR (B20.6), anti-Vβ4 TCR (KT4), anti-Vβ12 TCR (MR11-1), and anti-ICOS (7E 17G9) were purchased from BD Pharmingen. Allophycocyanin-conjugated anti-CD62L (MEL-14), anti-CD4 (GK1.5), and streptavidin were purchased from eBioscience. p31-I-A^g7-mouse(m)IgG2a and hen egg lysozyme (HEL)-I-A^g7-mIgG2a were produced in our laboratory, as previously described (10). Peptides were produced by the University of California Biomolecular Resource Center. The 1040-31 peptide, specific for BDC2.5 TCR Tg⁺ T cells, consisted of amino acids YVRPLWVRME (9). The HEL_11–25 protein consisted of amino acids AMKRHGLDNYRGYSL. GAD_286–300 was a gift from E. Sercarz (Torrey Pines Institute for Molecular Studies, San Diego, CA).

Both latex and paramagnetic beads were used throughout the described studies with equal efficiency. For coating of latex beads, 1 × 10⁸ 5-μm white sulfate latex beads (Interfacial Dynamics) were coated with 30 μg of anti-CD28 and 3 μg of either anti-CD3 or p31-I-A^g7-mIgG2a. For coating of paramagnetic beads, 1 × 10⁸ Dynabeads M-450 Tosylactivated (Dynal Biotech) were coated with 50 μg of anti-CD28 and 5 μg of either anti-CD3 or p31-I-A^g7-mIgG2a, following the manufacturer’s instructions.

Cell sorting and in vitro expansion of T cells

CD4⁺ T cells were enriched from spleen and lymph nodes (inguinal, axillary, brachial, submandibular, pancreatic, and mesenteric) by negative selection using an AutoMACS (Miltenyi Biotec) or Stemsep beads (StemCell Technologies). Cells were stained and sorted, as previously described, on a Mo-Flo (DakoCytomation) into CD4⁺CD25⁺CD62L⁺ T_reg and CD4⁺CD25⁻CD62L⁺ T cell effector (T_eff) populations to >97% purity (6). Purified T cells at 10⁶/ml were stimulated in flat-bottom plates at a 1:1 bead-cell ratio with anti-CD3 and anti-CD28- or p31-I-A^g7mIgG2a and anti-CD28-coated beads in medium supplemented with 2000 IU/ml human rIL-2 (Chiron). Complete DMEM was made, as previously described (6). Cultures were expanded with IL-2-supplemented medium when needed. Beads were removed at the end of the culture period before further experimentation. To remove coated latex beads, cultures were incubated with biotinylated anti-mouse IgG2a (Southern Biotechnology Associates) and biotinylated anti-hamster Ig (Vector Laboratories), washed, incubated with streptavidin microbeads (Miltenyi Biotec), washed, and run over an MS column (Miltenyi Biotec). Cells in the flow through fraction were collected. Dynabeads were removed using a Dynal MPC-L magnet (Dynal Biotech).

Flow cytometry

For staining with peptide-I-A^g7 multimers, peptide-I-A^g7-mIgG2a was complexed with Alexa 488-conjugated protein A (Molecular Probes), as previously described (10), or complexed with biotinylated protein A (Sigma-Aldrich) and allophycocyanin-conjugated streptavidin (eBioscience) at a 3:1:0.25 molar ratio. Cells (10⁶) were stained with 1 μg of p31-I-A^g7-mIgG2a for 2 h on ice. Abs against TCR Vβ were added at the same time as the peptide-I-A^g7 multimers. Other Abs were added during the last 30 min of staining or when using peptide-I-A^g7/biotinylated protein A multimers, during the secondary staining along with allophycocyanin-conjugated streptavidin. Isotype-matched Abs were used for control staining for CTLA-4 and ICOS expression on expanded T_reg. Freshly isolated CD4⁺CD25⁻ cells were used as negative control staining for GITR analysis. Flow cytometric analysis was performed on a FACSCalibur flow cytometer (BD Biosciences).

In vitro suppression assays

The indicated numbers of expanded T_reg cultures were added to 50,000 freshly isolated CD4⁺ T cells with 50,000 NOD.TCRα-deficient spleen cells or NOD CD4⁺/CD8⁺-depleted spleen cells and either the indicated peptide at 0.1 μM or anti-CD3 at 1 μg/ml. Cultures were incubated for 72 h and pulsed with 1 μCi/well [³H]thymidine during the last 14 h.

CFSE assays

The p31-I-A^g7-expanded T_reg, either unsorted or FACS sorted based on p31-I-A^g7 multimer binding, were labeled with 2.5 μM CFSE and cultured with irradiated splenic APC and 1 μM of either HEL or p31 peptide in the presence of 100 IU of IL-2/ml. Cells were collected at 72 h and analyzed by flow cytometry.

Real-time PCR analysis

Total RNA was isolated from expanded T cells using QIAshredder and RNeasy Mini Kit spin columns (Qiagen). cDNA was synthesized from 50–600 ng of sample RNA using SuperScript III reverse transcriptase and oligo(dT)12-18 primer (Invitrogen Life Technologies). Real-time PCR contained 1.25–28.5 ng of cDNA. Primers and probes for Foxp3 and 18S rRNA were purchased as premixed reagents from Applied Biosystems. Real-time PCR was performed on an ABI PRISM 7900 HT Sequence Detection System using TaqMan Universal PCR Master Mix (Applied Biosystems). All samples and controls were run in duplicate, and average threshold cycle (Ct) values were used to normalize the signal levels in samples relative to an endogenous control (18S rRNA). The normalized sample values were then used to calculate the fold difference in expression of Foxp3 between expanded T_reg and T_eff. The expression ratios of T_reg to T_eff were calculated by the following formula: (T_reg/T_eff)_Foxp3 = 2ⁿ, n = (T_effCt_Foxp3 − T_effCt_18S) − (T_regCt_Foxp3 − T_regCt_18S).

Cytokine ELISA

To measure cytokine production, 1 × 10⁶ p31-I-A^g7-expanded T_reg were cultured with 5 × 10⁶ irradiated APC, 100 nM 1040-31 peptide, and 1 μg/ml anti-CD28. Culture supernatants were harvested at 48 h. Cytokine concentrations were determined by ELISA using Ab pairs purchased from BD Pharmingen.

Adoptive transfer

The indicated numbers of p31-I-A^g7-expanded T_reg cultures or expanded polyclonal T_reg cultures were transferred to NOD.CD28^−/− mice via retro-orbital injection. Nonfasting blood glucose levels in recipient mice were monitored using an Accu-Check glucometer (Roche Diagnostic Systems). Mice were considered diabetic after two readings above 250 mg/dL.

Recombinant peptide-MHC can specifically expand rare Ag-specific T_reg

To selectively expand Ag-specific T_reg, we adapted the technique used to expand polyclonal T_reg by substituting the anti-CD3 mAb with a rMHC class II I-A^g7 presenting the BDC2.5 TCR mimotope peptide 1040-31 (p31) (10). The mimotope peptide was used because the endogenous BDC2.5 Ag is not yet identified (9). Initial experiments were conducted with BDC2.5 Tg⁺ T_reg to determine whether p31-I-A^g7- and anti-CD28-coated beads could expand low frequency Ag-specific cells from a polyclonal population and whether T_reg expanded in this manner retained suppression activity. Polyclonal CD4⁺CD25⁺ T_reg from NOD mice were seeded with low frequencies of sorted BDC2.5 TCR Tg⁺ T_reg at 0.1–0.001% of the total population and cultured with p31-I-A^g7- and anti-CD28-coated beads in the presence of IL-2. The p31-I-A^g7-coated beads were extremely efficient in expanding CD4⁺CD25⁺ BDC2.5 TCR Tg⁺ T_reg. Although cultures initially seeded at 0.01 and 0.001% BDC2.5 TCR Tg⁺ T_reg did not expand appreciably based on total cell number (Fig. 1⇓A), flow cytometric analysis using p31-I-A^g7 multimers revealed that BDC2.5 TCR Tg⁺ T_reg had expanded in all cultures. At the lowest seeding, BDC2.5 TCR Tg⁺ T_reg grew from 0.001 to 34.3% of the population (Fig. 1⇓B), reflecting greater than 12 cell divisions during the 10-day culture period and resulting in nearly a 5000-fold expansion of the Ag-specific cells.

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FIGURE 1.

Peptide-I-A^g7 beads can expand low frequency Ag-specific BDC2.5 TCR Tg⁺ T_reg in vitro. A, CD4⁺CD25⁺CD62L⁺ BDC2.5 TCR Tg⁺ T cells were seeded at the indicated frequencies into CD4⁺CD25⁺CD62L⁺ NOD cells and cultured with p31-I-A^g7- and anti-CD28-coated beads along with IL-2. The fold expansion of total cell numbers relative to the initial cell input of BDC2.5 and NOD cells is shown. Cultures were quantitated by viable cell counting. B, Cultures initially seeded at 100 or 0.001% BDC2.5 Tg⁺ T_reg were expanded for 10 days with p31-I-A^g7 beads, stained with either p31-I-A^g7 multimers or control HEL-I-A^g7 multimers, and analyzed by flow cytometry. C, Graded doses of BDC2.5 Tg⁺ T_reg expanded with p31-I-A^g7 and anti-CD28 beads were cultured with 5 × 10⁴ freshly isolated CD4⁺CD25⁻ BDC2.5 Tg⁺ responder cells and 5 × 10⁴ irradiated APC. Responder cells alone and T_reg alone are also shown. Cultures were stimulated with 100 nM BDC2.5 mimotope peptide p31 for 72 h. Proliferation was measured by [³H]thymidine incorporation.

Expanded CD4⁺CD25⁺ BDC2.5 T_reg were then tested for suppressive activity in in vitro cultures using freshly isolated CD4⁺CD25⁻ BDC2.5 Tg⁺ cells as responders and the p31 peptide as an Ag-specific stimulus. Expanded CD4⁺CD25⁺ BDC2.5 T_reg efficiently suppressed the response the BDC2.5 Tg⁺ responder cells in a dose-dependent manner (Fig. 1⇑C). Thus, the expansion procedure using recombinant peptide-MHC class II as an Ag-specific stimulus resulted in a large expansion of rare Ag-specific T_reg that retained suppressive function.

Rare Ag-specific T_reg can be expanded from wild-type NOD mice

This approach was applied next to the expansion of Ag-specific CD4⁺CD25⁺ T_reg from conventional NOD mice. Initial staining of freshly isolated T_reg from NOD mice with the p31-I-A^g7 multimer failed to detect positively staining cells above background levels, indicating that, if present, these Ag-specific cells were present at low frequency (data not shown). Sorted CD4⁺CD25⁺CD62L⁺ cells from 4- to 8-wk-old NOD mice were cultured with p31-I-A^g7 beads, as described in Materials and Methods. Over a 2-wk period, T_reg cultured in this manner generally expanded from 1- to 10-fold based on total cell number, with 4- to 5-fold expansion being the most typical. Expansion profiles of four representative cultures are shown in Fig. 2⇓A. The lower fold expansion was usually seen when lower numbers of purified T_reg (<0.5 × 10⁶) were seeded into the wells. The reason for the greater expansion observed in some cultures remains unknown. There was no correlation with the age of mice, as starting populations of CD4⁺CD25⁺CD62L⁺ cells taken from 4- or 8-wk-old mice demonstrated the same range of expansion of 1- to 10-fold. Flow cytometry analysis demonstrated that after expansion with the p31-I-A^g7 beads, up to 10% of CD4⁺CD25⁺ cells stained positive for the p31-I-A^g7 multimer (Fig. 2⇓B). Control cultures of CD4⁺CD25⁺ cells expanded with anti-CD3-coated beads did not stain positive for p31-I-A^g7 above background levels. In cultures of CD4⁺CD25⁻CD62L⁺ T_eff expanded with p31-I-A^g7-coated beads under the same conditions, 40–50% of the population stained positive for the p31-I-A^g7 multimer (Fig. 2⇓B). The p31-I-A^g7-expanded T_reg were also tested for specificity against peptides in proliferation assays. The p31-I-A^g7-expanded T_reg responded to p31 peptide, but not control HEL peptide when cultured with APC and IL-2 (data not shown).

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FIGURE 2.

Ag-specific T_reg can be expanded from wild-type NOD mice. A, Sorted CD4⁺CD25⁺CD62L⁺ from wild-type NOD mice were cultured with p31-I-A^g7 and anti-CD28 beads in the presence of IL-2. The expansion of four representative cultures over a 2-wk period is shown. B, CD4⁺CD25⁺CD62L⁺ cells cultured with either p31-I-A^g7 beads or anti-CD3 (2C11) beads were stained with anti-CD4 and either p31- or HEL-I-A^g7 multimers to identify Ag-specific cells. Staining of CD4⁺CD25⁻CD62L⁺ T_eff cultured with p31-I-A^g7 beads is also shown. C, Cultures of p31-I-A^g7-expanded T_reg were left unsorted or sorted into p31-I-A^g7 multimer-positive and -negative populations using FACS. The resulting populations were CFSE labeled and stimulated with HEL or p31 peptide in the presence of irradiated APC and IL-2. Cultures were analyzed at 72 h by flow cytometry for CFSE intensity.

As shown in Fig. 2⇑B, in general, 90% of the p31-I-A^g7-expanded T_reg population failed to stain for the p31-I-A^g7 multimer when analyzed by flow cytometry. It was possible that the unstained population was comprised of p31-I-A^g7-reactive cells with TCRs with too low of an affinity to be detected by flow cytometry (14). Alternatively, this population could be comprised of cells that were expanding in an Ag-nonspecific manner. To distinguish between these two alternatives, p31-I-A^g7-expanded T_reg populations were sorted into p31-I-A^g7 multimer-positive and -negative populations using FACS. The resulting populations were then labeled with CFSE and stimulated with the polyclonal activator anti-CD3 mAb or with the p31 peptide or a control HEL peptide in the presence of APC and IL-2. All three populations, unsorted, p31-I-A^g7 multimer positive, and p31-I-A^g7 multimer negative, expanded equally well to anti-CD3 mAb when analyzed for CFSE dilution (data not shown). As shown in Fig. 2⇑C, >50% of the T_reg from the unsorted population entered into cell cycle when stimulated with the p31 peptide compared with only ∼14% when stimulated with the HEL peptide. The background proliferation with the HEL peptide probably reflected the residual presence of beads in the culture before cell sorting into p31-I-A^g7 multimer-positive and -negative populations. In the p31-I-A^g7 multimer-positive population, >65% of the cells entered into the cell cycle when stimulated with the p31 peptide. Importantly, >50% of the cells from the p31-I-A^g7 multimer-negative population entered into cell cycle when stimulated with the p31 peptide, demonstrating the presence of p31-reactive cells in this population. The expansion protocol using recombinant peptide-MHC class II was therefore very efficient in expanding Ag-specific T_reg with TCRs with a broad range of affinities for Ag.

Characterization of expanded Ag-specific T_reg

Expanded Ag-specific T_reg were examined for the expression of the T_reg lineage marker Foxp3 using quantitative real-time PCR. To ensure that p31-I-A^g7-reactive cells were analyzed, p31-I-A^g7-expanded T_reg and T_eff were sorted into p31-I-A^g7 multimer-positive and -negative populations by FACS before analysis. In a representative experiment shown in Fig. 3⇓A, expanded p31-I-A^g7 multimer⁺ T_reg expressed ∼3000-fold more Foxp3 relative to expanded p31-I-A^g7 multimer-positive T_eff. Upon challenge with Ag and APC, p31-I-A^g7-expanded T_reg expressed low levels of the proinflammatory cytokines IL-2, IL-4, and IFN-γ, and expressed high levels of the anti-inflammatory cytokine IL-10 (Fig. 3⇓B). Peptide-I-A^g7-expanded T_reg retained high levels of expression of CD62L and CD25 throughout the culture period (Fig. 4⇓A). In contrast, peptide-I-A^g7-expanded T_eff down-regulated CD62L and expressed relatively lower levels of CD25. Peptide-I-A^g7-expanded T_reg also expressed high levels of CTLA-4, GITR, and ICOS (Fig. 4⇓B). Peptide-I-A^g7-expanded T_eff expressed levels of CTLA-4 comparable to peptide-I-A^g7-expanded T_reg (data not shown). GITR was also up-regulated on the expanded T_eff as compared with naive T_eff, but remained lower than levels observed on expanded T_reg. ICOS expression was higher on expanded T_eff as compared with expanded T_reg (data not shown).

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FIGURE 3.

Expanded Ag-specific T_reg express Foxp3 and IL-10. A, p31-I-A^g7-expanded cells were sorted into p31-I-A^g7 multimer-positive and -negative populations by FACS. Levels of Foxp3 mRNA expression were determined for the indicated populations by real-time PCR analysis. The expression levels of Foxp3 in p31-I-A^g7-expanded CD4⁺CD25⁺ T_reg relative to p31-I-A^g7-expanded CD4⁺CD25⁻ T_eff are shown. B, p31-I-A^g7-expanded T_reg were stimulated with p31 peptide in the presence of irradiated APC. Secreted cytokine levels were determined 48 h after stimulation by ELISA.

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FIGURE 4.

Surface phenotype of expanded Ag-specific T_reg. A, Cultures of p31-I-A^g7-expanded T_reg or T_eff were stained for expression of CD25 and CD62L. B, Surface staining of p31-I-A^g7-expanded T_reg. Light lines indicate control staining. Heavy lines indicate staining of p31-I-A^g7-expanded T_reg populations. C, Cultures of p31-I-A^g7-expanded T_reg and T_eff were costained with p31-I-A^g7 multimers and the indicated TCR Vβ Abs. Numbers shown represent the percentage of p31-I-A^g7 multimer-positive cells staining positive for the indicated TCR Vβ.

We then examined the Vβ repertoire of the p31-I-A^g7-expanded T cells. The BDC2.5 TCR expresses a TCRβ derived from the Vβ4 family (11). However, when p31-I-A^g7-expanded T_reg and T_eff were costained with p31-I-A^g7 multimers and different TCR Vβ reagents, neither population was monoclonal. Although there was a significant number of Vβ4⁺ p31-I-A^g7 multimer⁺ T cells in the T_reg culture, other TCR Vβ were also present in significant numbers (Fig. 4⇑C). For instance, Vβ2 and Vβ12 accounted for 10.2 and 13.7% of the p31-I-A^g7 multimer⁺ T_reg, respectively, in this representative culture. TCR Vβ4⁺ T cells were generally present in the p31-I-A^g7 multimer⁺ T_eff population, but at a lower percentage. Together, these results suggest that broad repertoires of T_reg and T_eff reactive against the islet peptide mimic are resident in conventional NOD mice, and that the islet peptide mimic-reactive T_reg repertoire is not identical with the islet peptide mimic-reactive T_eff repertoire.

Suppression by Ag-specific T_reg in vitro

Previous studies have shown that CD4⁺CD25⁺ T_reg can suppress proliferation of CD4⁺ effectors in vitro and that the suppressive effect is dependent on stimulation of CD4⁺CD25⁺ T_reg through their TCR. Therefore, expanded p31-I-A^g7 T_reg were examined for suppressive activity and specificity in vitro. Expanded p31-I-A^g7 T_reg effectively suppressed the proliferation of Ag-specific CD4⁺ BDC2.5 TCR Tg⁺ cells in a dose-specific manner when cultures were stimulated with either anti-CD3 or 1040-31 peptide (Fig. 5⇓A). In contrast, p31-I-A^g7-expanded CD4⁺CD25⁺ T_eff failed to suppress freshly isolated BDC2.5 CD4⁺ T cells and resulted in augmentation of proliferation (data not shown). The p31-I-A^g7-expanded T_reg, but not the T_eff, were anergic to stimulation (both p31 peptide and anti-CD3) in the absence of CD28 costimulation or addition of IL-2 consistent with reports for freshly isolated T_reg (Fig. 5⇓B and data not shown). To further characterize the Ag specificity of the expanded T_reg, expanded polyclonal T_reg and p31-I-A^g7-expanded T_reg were assessed for the ability to suppress BDC2.5 TCR Tg⁺ or GAD₂₈₆ TCR Tg⁺ CD4⁺ cells (12) through either polyclonal T cell activation via anti-CD3 or through Ag-specific T cell activation. Both polyclonal T_reg and p31-I-A^g7-expanded T_reg suppressed BDC2.5 TCR Tg⁺ responders when stimulated with anti-CD3, whereas only the p31-I-A^g7-expanded T_reg suppressed cultures stimulated with the BDC2.5 1040-31 peptide (Fig. 5⇓B). Similarly, both polyclonal T_reg and p31-I-A^g7-expanded T_reg suppressed the response of GAD₂₈₆ TCR Tg⁺ CD4⁺ T cells when stimulated with anti-CD3 (Fig. 5⇓C, bottom). However, neither the polyclonal T_reg population nor the p31-I-A^g7-expanded T_reg population suppressed GAD₂₈₆ TCR Tg⁺ responders when stimulated with the GAD_286–300 peptide (Fig. 5⇓C, top). Most significantly, p31-I-A^g7-expanded T_reg were capable of suppressing GAD₂₈₆ TCR Tg⁺ responders when the culture was stimulated with both the GAD_286–300 and the 1040-31 peptide (Fig. 5⇓D). Collectively, these data demonstrate that the suppressive activity of the p31-I-A^g7-expanded T_reg is dependent on Ag-specific stimulation through the TCR, although, once stimulated with cognate Ag, p31-I-A^g7-expanded T_reg are capable of exerting bystander suppression.

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FIGURE 5.

p31-I-A^g7-expanded T_reg suppress in an Ag-specific manner in vitro. A, 5 × 10⁴ freshly isolated BDC2.5 CD4⁺CD25⁻ T cells were cultured with varying amounts of p31-I-A^g7-expanded T_reg and 5 × 10⁴ irradiated APC. Cultures were stimulated with either 100 nM 1040-31 peptide or 1 μg/ml anti-CD3. Proliferation was determined, as described in Fig. 1⇑C. B and C, 5 × 10⁴ p31-I-A^g7 or 2C11-expanded T_reg were cultured with freshly isolated BDC2.5 TCR Tg⁺ CD4⁺CD25⁻ responders (B) or GAD₂₈₆ TCR Tg⁺ CD4⁺CD25⁻ responders (C), 5 × 10⁴ irradiated APC and the 1040-31 peptide (B) or GAD_286–300 peptide (C), or anti-CD3. Proliferation was determined, as described above. D, p31-I-A^g7-expanded T_reg were cultured with GAD₂₈₆ TCR Tg⁺CD4⁺CD25⁻ responder cells, as described in C, and stimulated with a combination of the 1040-31 and GAD_286–300 peptides.

Expanded Ag-specific T_reg prevent autoimmune diabetes

We then tested the ability of small numbers of p31-I-A^g7-expanded T_reg to suppress diabetes in CD28^−/− NOD mice. CD28^−/− NOD mice have normal numbers of T_eff and Th1 responses and undergo an accelerated form of autoimmune diabetes due to a deficiency in T_reg (4, 15). Previous studies have shown that delay or prevention of diabetes in this model required the transfer of high numbers of polyclonal T_reg (8–20 × 10⁶) (4). In sharp contrast, the transfer of as few as 1.8 × 10⁶ p31-I-A^g7-expanded T_reg into 5- to 7-wk-old mice prevented the development of diabetes in the majority of mice for at least 15 wk of age (Fig. 6⇓). The in vivo administered p31-I-A^g7-expanded T_reg cultures were an admixture of ∼10% high avidity cells, as determined by the ability to bind p31-I-A^g7 multimers, and lower avidity cells that were not detected by the p31-I-A^g7 multimers when analyzed by flow cytometry, but were capable of responding to p31 peptide presented by APC (see Fig. 2⇑, B and C). Thus, the Ag-specific p31-I-A^g7-expanded cells were highly efficient in protecting the onset of diabetes induced by a fully functional polyclonal T cell response. Suppression of autoimmunity by the p31-I-A^g7-expanded T_reg appeared to be organ specific. Although animals that received the p31-I-A^g7-expanded T_reg were protected from diabetes, the mice continued to exhibit other autoimmune syndromes based on continued lymphocytic infiltration in the salivary and thyroid glands (data not shown).

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FIGURE 6.

Expanded Ag-specific T_reg prevent diabetes in vivo. Five- to 7-wk-old prediabetic CD28-deficient NOD mice were injected with 1.8–2 × 10⁶ p31-I-A^g7-expanded T_reg (n = 11), 2 × 10⁶ (n = 6), or 8 × 10⁶ (n = 2) 2C11-expanded T_reg or left untreated (n = 6). Diabetes was determined by monitoring blood glucose levels.