Heparin tubes

Peripheral blood was collected by venipuncture in heparin tubes (Greiner-BioOne, Monroe, NC, USA). Whole blood from each individual was split and cultured in the presence or absence of PHA. All lymphocyte cultures were prepared in RPMI 1640 (Lonza, Walkersville, MD, USA) reconstituted with: 10 % heat inactivated fetal bovine serum (FBS – Sigma-Aldrich, St Louis, MO, USA), 2 % L-glutamine (Thermo-Fisher, Waltham, MA, USA) and 1 % penicillin-streptomycin solution (Thermo-Fisher, Waltham, MA, USA). All cultures had a total volume of 5 ml and for those that PHA was added, they were reconstituted with 100 μl of PHA, (45 mg/vial) (Remel Inc, Lenexa, KS, USA) 0.8–1.0 ml of blood was incubated for 71 h at 37 °C (5 % CO₂). Following lymphocyte culture incubation (71 h), lymphocytes were prepared following standard karyotyping protocols. Proliferating cells in metaphase were arrested using 0.2 μg colcemid (Thermo-Fisher, Waltham, MA, USA) for 30 min at 37 °C, followed by standard hypotonic conditions to allow separation of white blood cells from anucleate erythrocytes (0.075 M of KCL - Thermo-Fisher, Waltham, MA, USA) for 45 min at 37 °C. White blood cells were subsequently fixed in 3:1 (v/v) of methanol:acetic acid solution to clean and fix the preparation. All cultures were stored at -20 °C immediately following the harvesting procedure.

A fasting intravenous glucose tolerance test (IV-GTT) was performed at day 133 ± 0.25 of age. The necks of lambs were shaved and cleaned with chlorhexidine (Durvet, Beaver Dam, WI) followed by 70% ethanol (Fisher Bioreagents, Pittsburgh, PA). A cannula (18 g × 2.5 in; Exel International, Quebec, Canada) was inserted into a jugular vein of each lamb 1 h before GTT to allow lambs time to recover. A single bolus injection of glucose (0.25 g/kg BW of a 50% dextrose solution; VetOne, Boise, ID) was infused via the jugular cannula. Blood samples (3 mL) were collected via the cannula at −30, −15, 0, 2, 5, 10, 15, 30, 60, and 120 min relative to glucose infusion, placed into heparin tubes (Greiner Bio-one), and stored on ice. Blood was centrifuged (3,000 × g for 30 min at 4 °C), and plasma was stored at −20 °C for insulin and glucose analysis.

Blood from healthy donors was collected after informed consent in heparin tubes (Greiner Bio-One, Alphen a/d Rijn, The Netherlands) for peripheral blood mononuclear cell (PBMC) and neutrophil isolation by density gradient centrifugation on Lymphoprep (d = 1.077 ± 0.001 g/ml; Axis-Shield, Oslo, Norway). Subsequently, PBMCs were separated into monocyte and peripheral blood lymphocyte (PBL) fractions by density gradient centrifugation on Percoll (GE Healthcare, Hoevelaken, The Netherlands). Monocyte-derived DCs were generated from the monocyte fraction, as described previously (9 (link)), and the purity of DCs was 95% or higher. CD4⁺CD45RA⁺ naive cells were subsequently purified by negative selection and CD4⁺CD45RO⁺ memory cells by positive selection from the PBL fraction by magnetic cell separation, as described previously (9 (link)). The purity of obtained naive T cells always exceeded 98%, and T cells were stored in liquid nitrogen until use in co-culture. Neutrophils were isolated fresh on the day of co-culture initiation from the erythrocyte pellet of Lymphoprep density centrifugation, as described previously (9 (link)). The purity of neutrophils was always more than 98%. The purity of all cell types was analyzed by flow cytometry and is shown in Supplementary Figure 1.