Materials and Methods

Mice and Diabetes Detection. TNF/CD80 (31) and CD154^-/- C57BL/6 mice (N11) (33) mice have been described elsewhere. For TNF- repression, mice were fed a 2.3 g/kg doxycycline diet (Bio-Serv). All mice were maintained under specific pathogen-free conditions.

Diabetes was monitored by testing of urinary glucose using Diastyx (Bayer) and confirmed by blood glucose measurement using One-Touch strips (Lifescan). Mice with blood glucose >250 mg/dl on 2 consecutive days were deemed diabetic.

Cell Purification and in Vitro Assays. CD4⁺CD25⁺ T cells were isolated by using a MoFlo Cell Sorter (Dako Cytomation). CD8⁺ T cells were purified by negative selection using rat anti-mouse CD4 (RM4-4), Mac-1 (M1/70), B220 (RA3-6B2), and I-A^b (25-9-3) Abs (BD Pharmingen) followed by incubation with goat anti-rat IgG Biomag beads (Qiagen, Valencia, CA) according to the manufacturer's instructions.

Proliferation assays were carried out in triplicate by combining 2 x 10⁴ CD8⁺ T cells and 2 x 10⁴ T_R cells, as indicated in 96-well, round-bottomed microtiter plates (Corning). Cells were cultured in RPMI medium 1640 (Sigma) supplemented with 10% FCS, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, and 100 units/ml each of penicillin and streptomycin (Invitrogen). Cells were stimulated with 5 x 10⁵ irradiated splenocyte APCs and 10 µg/ml anti-CD3 for 72 h. During the last 6 h of culture, cells were pulsed with 1 µCi of [³H]thymidine. (Amersham Pharmacia; 1 Ci = 37 GBq) and proliferation was quantified as [³H] incorporation.

Flow Cytometry. The following fluorochrome-conjugated Abs were used for flow cytometry: anti-CD4 (GK1.5), anti-CD25 (PC61), and anti-CD8 (53.6) (BD Pharmingen). Hamster antimouse CD154 (39H5), Armenian hamster Ig isotype control, and anti-hamster FITC were obtained from (Serotec). Stained cells were acquired on a FACSCalibur and analyzed by using CELLQUEST software (BD Biosciences).

In Vivo Assays. For in vivo CD154 blockade, mice received i.p. injections of 250 µg of anti-CD154 Ab (MR1) or Armenian hamster isotype control Ab. For in vivo stimulation of CD40, mice were injected i.p. with 250 µg of CD40 agonistic Ab (FGK45) or rat IgG isotype control Ab. For adoptive transfer, purified CD8⁺ T cells or CD4⁺CD25⁺ T_R cells were washed twice in sterile tissue culture-grade PBS (Invitrogen) before injection into the lateral tail vein.

Real-Time PCR. CD4⁺CD25⁺, CD4⁺CD25^-, and CD8⁺ T cells were isolated by cell sorting. RNA was prepared by using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized with random primers (Invitrogen). The expression of FoxP3 was measured by real-time RT-PCR with primers 5'-GGCCCTTCTCCAGGACAGA-3' and 5'-ACAACCCAGCCATGATCAGC-3' at a final concentration of 300 nM, and the internal TaqMan probe 5'-FAM-ACTTCATGCATCAGCTCTCCAC-TAM-1-3' at a final concentration of 125 nM. 2 microglobulin (2M) was used as an internal reference and was measured by using primers 5'-GCTATCCAGAAAACCCCTCAAA-3' and 5'-CTGTGTTACGTAGCAGTTCAGTATGTTC-3' at a final concentration of 300 nM, and the TaqMan probe 5'-FAM-AGTATACTCACGCCACCCACCGGAGAAT-TAM-1-3' at a final concentration of 200 nM (all primers and probes were synthesized by Sigma, except the 2M probe, which was synthesized by Applied Biosystems). mRNA levels were quantified by using the ABI 7000 Sequence Detection System (Applied Biosystems). Samples were run in duplicate, and their relative expression of FoxP3 was determined by normalizing expression to 2M.

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