NOD mice and NOD/severe combined immunodeficiency (SCID) mice were purchased from Clea Japan (Tokyo, Japan). IRF4-deficient mice had been previously generated . These mice were maintained by breeding at the Laboratory Animal Center for Animal Research at Nagasaki University under specific pathogen-free conditions. Only female mice were used in this study.
Establishment of IRF4-deficient NOD mice
IRF4-deficient mice were backcrossed with NOD mice for 15 successive generations. These mice were selected by a PCR analysis of tail DNA, as previously described [37, 38]. An analysis of the microsatellite markers of the diabetes susceptibility (Idd1-14) loci by PCR of the tail DNA, as previously described , showed that the mice were homozygous for all of the NOD alleles. Genotyping of chromosome 13 confirmed that the NOD/B6/129 polymorphic markers D13Mit80 (8.7 cM from IRF4 loci), D13Mit218 (21.8 cM), D13Mit163 (25.4 cM), D13Mit84 (25.7 cM) and D13Mit61 (Idd14) (41.0 cM) were all derived from the NOD background.
Homozygous IRF4-deficient (Irf4
−/−), heterozygous IRF4-deficient (Irf4
+/−) and wild-type (WT) NOD mice were established by intercrossing heterozygotes of the 15th generation. All animal experiments described in this study were approved by the institutional animal experimentation committee and were conducted in accordance with the Guidelines for Animal Experimentation.
Monitoring for spontaneous diabetes
Mouse blood glucose levels were monitored for spontaneous diabetes using the OneTouch Ultra blood glucose monitoring system (Johnson & Johnson, Tokyo, Japan). Monitoring was conducted weekly when the mice were 12–20 weeks old and then every other week from 20 to 50 weeks of age. Monitoring was terminated when the mice became moribund. Mice with blood glucose levels of more than 13.9 mmol/l for two consecutive measurements were considered diabetic.
Measurement of serum IgG and insulin autoantibodies
We performed an ELISA to measure IgG in the serum of mice at 4 weeks of age using the Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX, USA). We evaluated the levels of insulin autoantibody (IAA) in serum using a 96-well filtration plate micro IAA assay, as previously described .
Pancreatic sections from 18-week-old mice were histologically analysed by staining paraffin-embedded samples with haematoxylin and eosin. A minimum of 30 islets from each mouse were evaluated under a microscope by two different observers. The severity of insulitis was scored as follows: 0, no lymphocytic infiltration; 1, lymphocytic infiltration occupying <25% of the total islet cell area; 2, lymphocytic infiltration occupying 25–50% of the total islet cell area; 3, lymphocytic infiltration occupying 50–75% of the total islet cell area; 4, lymphocytic infiltration occupying >75% of the total islet cell area, or small retracted islets.
Adoptive transfer experiments
CD4+CD25− T cells or CD8+ T cells were purified from the spleens of 10- to 12-week-old prediabetic Irf4
+/− and WT NOD mice using magnetic bead cell sorting (Miltenyi Biotech, Auburn, CA, USA). The purity of the CD4+CD25− and CD8+ T cells was at least 92% and 98%, respectively, as confirmed by flow cytometry analysis. Purified CD4+CD25− (1.0 × 107) and CD8+ (0.5 × 107) T cells were combined and i.p. injected into 10-week-old NOD/SCID mice, and the mice were then monitored for blood glucose twice weekly after the adoptive transfer.
Flow cytometric analysis
Single-cell suspensions were prepared from mouse spleens, pancreatic lymph nodes and inguinal lymph nodes. Red blood cells were lysed in ammonium chloride buffer. For surface staining, cells were stained for 20 min with the corresponding fluorescent-labelled or biotin-conjugated antibodies against surface molecules: CD3e (145-2C11), CD4 (GK1.5), CD8 (53-6.7), B220 (RA3-6B2), CD44 (IM7), CD62L (MEL-14), I-Ag7 (I-Ak, 10-3.6) and CD11c (N418).
For intracellular IRF4 staining of T cells, splenocytes (SPCs) were stimulated with plate-bound anti-CD3 (BD Biosciences/BD Pharmingen, San Diego, CA, USA) and soluble anti-CD28 (37.51) (2 μg/ml) (eBioscience, San Diego, CA, USA) for 36 h and then stained with phycoerythrin (PE)-conjugated anti-IRF4 (3E4) antibodies. For the intracellular cytokine staining, SPCs were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 500 ng/ml ionomycin (both from Sigma, St Louis, MO, USA) in the presence of 2 μM monensin for 5 h. Thereafter, the cells were stained with allophycocyanin-conjugated anti-CD4, followed by intracellular staining with PE-Cy7-conjugated anti-IL-17 (eBio17B7) and PerCP-Cy5.5-conjugated anti-IFN-γ (XMG1.2) antibodies (all from eBioscience). For intracellular IRF4 staining of DCs, cells were directly stained without culture.
For intracellular staining of granzyme B and perforin, purified CD8+ T cells were activated with plate-bound anti-CD3 and anti-CD28 for 48 h, followed by incubation with recombinant mouse IL-2 (100 U/ml; eBioscience) for 72 h. Before intracellular staining, the activated CD8+ T cells were re-stimulated with PMA and ionomycin in the presence of monensin during the final 5 h. Thereafter, the cells were stained with PE-conjugated anti-granzyme B (16G6) and FITC-conjugated anti-perforin (eBioOMAK-D) antibodies. For intracellular forkhead box protein P3 (FOXP3) staining, cells were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD25 (PC61), followed by intracellular FOXP3 staining with PE-Cy5-conjugated anti-FOXP3 (FJK-16s). All cells were analysed on a FACSCanto II flow cytometry system using FACSDiva software (BD Biosciences).
Data are expressed as means (SD). Group differences were analysed using Student’s t test. Differences between Kaplan–Meier survival curves were evaluated by the log-rank test, using SPSS II software for Windows (SPSS, Chicago, IL, USA). p values of less than 0.05 were considered significant.