Tolerability and clinical signs of toxicity Clinical signs of toxicity related to treatment such as weight gain/loss, abnormal behavior, discoloration of urine, stool and fur monitored at least 3 times per week and continued throughout the study. Body weight was recorded before initiation of the study and measured 1–2 times weekly thereafter. Induction of diabetes by i.p injection of streptozotocin (STZ) STZ (Sigma-Aldrich, St. Louis, MO) was freshly prepared in citrate buffer (pH = 4.5) just before injection and final volume was adjusted by adding sterile PBS. BALB/c mice (n = 10) were received 5 dose of STZ (50 mg/kg) by i.p injection in 5-consecitive days. Diabetic mice were treated one week post diabetes onset with either AG490 or DMSO (control). Purification of CD4+, CD4+CD25+/2 T-cells and dendritic cells Purification of CD4+CD25+ T-cells was performed from pooled splenocytes using a CD4+CD25+ isolation kit (Miltenyi Biotec, Auburn, AL) and autoMACS magnetic separator, accord- ing to instructions provided by the manufacturer. Purity of purified CD4+CD25+ and CD4+CD252 T-cells were greater than 85– 90% and 95%, respectively, as judged by flow cytometry analysis. Splenic CD4+ T-cells and dendritic cells (from spleen and bone marrow derived DCs) were enriched with CD4+ and CD11c MicroBeads, respectively (Miltenyi Biotech) using autoMACS instrument according to manufacturer’s instructions. Generation of bone marrow-derived dendritic cells Dendritic cells (DC) were generated from bone marrow in 5days. In brief, mice were euthanized and femur and tibiae removed and cleaned from other tissues. Both ends of each bone were cut and marrow flushed with RPMI 1640 containing 10% serum. Red blood cells were lysed with an ammonium chloride lysis solution (Sigma-Aldrich), and cells were plated at 16106 cells/ ml, 2 ml/well in 12-well plate (Falcon; BD Biosciences) in RPMI 1640 medium, supplemented with 1% penicillin and streptomycin, 10% heat-inactivated and filtered FCS containing rmGM-CSF (500 U/ml R&D systems) and IL-4 (1000 U/ml, BD Biosciences). On day 2, non-adherent and loosely adherent cells were harvested by replacing one-half of the cell-containing supernatant from each well and replaced with 1 ml of fresh medium containing cytokines. On day 5, DC maturation proceeded by adding of LPS and those cells that did not receive LPS were considered as immature DC. Daily microscopic inspection of cell culture by microscopic examination was performed until the end of the DC culture. T- cell culture, Proliferation/Suppression assays The AG490/DMSO treated bone marrow derived DC (BMDC) were cultured at different ratios, with constant number of CD4+CD252 T-cells (16105), in 96-well round bottom plates containing soluble anti-CD3 (2.5 mg/ml) for 72 hourrs. Prolifer- ation of responding populations were measured in the last 16 hours of cell culture by measuring 3H-thymidine incorporation into the DNA using Scintillation Counter (Beckman). TaqMan mouse Immune Response Array and Real-Time TaqMan Gene expression analyses Total RNA was extracted from CD4 and dendritic cells using RNAeasy kit (QIAGEN). CD4+ T-cells was purified from 5 individual mice using mouse CD4+ T-cell isolation kit (Miltenyi Biotech) followed by autoMACS separation according to the AG490 Prevents and Reverses Diabetes manufacturer’s instructions. To screen the Taqman Array Mouse Immune Response, 200 ng of total RNA was converted to cDNA using High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The cDNA from 5 mice were pooled together (one microgram of total RNA) and then used to screen 96- immune response related genes including 4 housekeeping genes. In the case of Real-Time TaqMan Gene expression (Assay-On-Demand), 100 ng of total RNA was used for reverse transcription followed by PCR amplification using 7300 Applied Biosystems PCR machine. All primers and probes were purchased from Applied Biosystems (Assays-on-Demand). Adoptive transfer Female NOD mice (4 weeks old) were treated with AG490 or DMSO (n = 5 per group) for 5 consecutive week, with three injections per week. Mice were sacrificed one week after the last injection (i.e., week 10). Cell suspensions of splenocytes from pool mice were prepared, with dead cells and red blood cells removed by Histopaque (Sigma) gradient separation. Cell viability was .99% as judged by trypan blue exclusion. 56106 splenocytes were transferred by i.p injection into female NOD.Scid recipient mice. Flow cytometry analysis Cell surface staining was performed using anti-mouse CD4 (L3T4; BD Pharmingen, San Diego, CA), CD25 (3C7; BD Pharmingen), CD11c (HL3; BD Pharmingen), CD80 (16-10A1; eBioscience, San Diego, CA), biotin-conjugated MHC II (M5/ 114.15.2; eBioscience) and anti-hTGFb- Biotin (BAF240; R & D Systems) and biotin labeled anti chicken IgY (BAF010; R & D System) for isotype control. Purified rat anti-mouse CD16/CD32 (2.4G2; BD Pharmingen) was used to block Fc receptor in myeloid cell lineages. Intra-cellular staining of Foxp3 (FJK, 16S), IL-10 (JES5-16E3), IFNc (XMG1.2) was performed using Foxp3 intracellular staining kit (eBioscience), according to instructions provided by the manufacturer. Viable cells were gated and a minimum of 30,000 events were acquired using a FACScaliber (BD Biosciences). Data was analyzed using FCS Express (v3) software (De Novo Software, Los Angeles CA). Histology and immunofluorescence histochemistry (IFHC) Pancreata were fixed in 4% PFA (paraformaldehyde) and microscopic sections prepared with 100 mm interval from paraffin embedded tissue, and stained with H & E, at the Pathology Core Laboratories at University of Florida. Insulitis scoring was performed in a blinded fashion. A numeric code was given to each slide and then scored by two different investigators. A three point scoring method was used where 0 was given to pre-insulitis or intact islets, 1 was given to islets with ,50% infiltration and 2 where infiltration was .50%. IFHC was performed on 4 mm paraffin sections of pancreas from the NOD mice. The concen- tration of primary antibodies was based on recommendation of the manufacturers. Stat1p91 (SC-591), Stat3 (SC-7179), Stat5B (SC- 835), IFNc (SC-8308), insulin (SC-7838), Jak1 (SC-277), JAk2 (SC- 1656), Jak3 (SC-6932), Tyk2 (SC-136265), were from Santa Cruz (Santa Cruz, CA) and were diluted 1: 50 and TNFa-PE conjugated Ab (Pharmingen) was diluted 1:100. Secondary fluorescence conjugated antibodies with Alexa Fluor 488 (green) (1: 200) or Alexa Fluor 594 (red) (1: 500) (Invitrogen, Carlsbad, CA) against the host primary antibody were used for signal detection. Anti-fade solution (Invitrogen) containing DAPI was used for counter staining and mounting. PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e36079