Pc 61

Selected mice with long-surviving allografts (n = 6) were i.p. injected with 500 μg of purified CD25 mAb (PC61.5.3, Bio X Cell, West Lebanon, NH, USA) to deplete CD25⁺ T cells on day 60 post transplantation, as previously described^{16 (link)}. The depletion was confirmed using the 7D4 antibody (BD PharMingen, San Diego, CA, USA), which recognizes a different epitope of CD25 than PC61.5.3 Ab, to stain splenic lymphocytes on day 7. Groups of mice were also i.p. injected with 500 μg of an isotype IgG (n = 5) or PBS (n = 6) on day 60 post transplantation as a control. The blood glucose levels of the recipients were monitored to evaluate the survival of the islet allografts.

CD4⁺ T cells were isolated from the spleen of naïve mice by MACS (Miltenyi Biotec, Gaithersburg, MD, U.S.). Equivalent numbers (5 × 10⁴–5 × 10⁵) of naïve OTII cells were transferred (i.v.) into naïve congenic recipients. For some experiments, transferred donor OTII cells were sorted from recipient mice after staining with fluorochrome-conjugated anti-B220, anti-CD4, anti-CD44, and anti-CD45.1. All sorting experiments were performed using a FACSAria (BD Biosciences) sorter at the University of Alabama at Birmingham Flow Cytometry core. Sorted cells were more than 98% pure, as determined by flow cytometry. For in vitro cultures, CD4⁺ T cells were labeled for 10 min at 37 °C with CellTrace Violet CTV (Molecular Probes, Thermo Fisher Scientific) and then activated with plate-bound anti-CD3 (clone 145-2C11; 1.5 μg/ml) and soluble anti-CD28 (clone 37.51; 1.5 μg/ml) Abs for 48 hours at 37 °C in flat-bottom 96-well plates. The indicated concentrations of an anti-IL-2 neutralizing Ab (JES6-1A12 and S4B6-1; BioXCell), an anti-CD25 neutralizing Ab (PC-61.5.3; BioXCell) and rIL-6 (PeproTech) were added for an additional 72 hours. Complete medium comprised RPMI 1640 medium supplemented with sodium pyruvate, HEPES (pH range, 7.2 to 7.6), nonessential amino acids, penicillin, streptomycin, 2-mercaptoethanol and 10% heat-inactivated FBS (all from Gibco, Thermo Fisher Scientific).

HDM (Dermatophagoides pteronyssinus and D. farina) extract (<30 EU/mg endotoxin) was obtained from Greer Laboratories (Lenoir, NC, U.S.). Endotoxin quantification was performed using the Pierce Chromogenic Endotoxin Quant Kit (A39552S; Thermo Fisher Scientific). Mice were intranasally administered (i.n.) 100 µg of HDM extract, +/- 5 µg of LPS-free EndoFit OVA ( < 0.1 EU/mg endotoxin; InvivoGen, San Diego, CA, U.S.) +/- LPS from Escherichia coli 0111:B4 (Sigma‒Aldrich, Saint Louis, MO, U.S.) +/- 100 ng of rIL-6 (PeproTech, Cranberry, NJ, U.S.) daily for 3 days and challenged (i.n.) with 100 µg of HDM + /− 5 µg of LPS-free EndoFit OVA for 3 days. The i.n. administrations were given in 100 µl of PBS. In some experiments, mice were intraperitoneally administered (i.p.) 250 μg of a mix of anti–IL-6/R (15A7; BioXCell, Lebanon, NH, U.S.) and anti-IL-6 (MP5-20F3; BioXCell) neutralizing Abs, 250 µg of anti-CD25 neutralizing Ab (PC-61.5.3; BioXCell), 250 μg of a mix of anti-IL-2 neutralizing Abs (JES6-1A12 and S4B6-1; BioXCell), or 60,000 U rIL-2 (National Cancer Institute, Bethesda, MD, U.S.) at the indicated time points. In some experiments, mice were orally treated with the JAK1 inhibitor upadacitinib (ABT-494; Selleck Chemicals, Radnor, PA, U.S.) at a dose of 20 mg/kg per day in palm oil at the indicated time points.

Antibodies directed against the following mouse markers were used: CD3 (145–2C11, Tonbo Biosciences 60–0031), CD4 (RM4–5, BioLegend 100559; GK1.5, BioXCell BE0003–1), CD8 (53–6.7, Tonbo Biosciences 75–0081; 53.6.72, BioXCell BE0004), CD11b (M1/70, BioLegend 101204; M1/70, BioXCell BE0007), CD11c (N418, BioLegend 117322), CD19 (6D5, BioLegend 115534), CD25 (PC61, BioLegend 102024; PC61.5.3, BioXCell BE0012), CD45 (30-F11, BioLegend 103138), CD45.1 (A20, BioLegend 110714), CD69 (H1.2F3, BioLegend 104504), CD80 (16–10A1, BioLegend 104724), B220 (RA3–6B2, BioLegend 103232; RA3.3A1/6.1, BioXCell BE0067), AIRE (5H12, eBioscience 53–5934–82), EpCAM (G8.8, BioLegend 118206), F4/80 (BM8, BioLegend 123116), Gr-1 (RB6–8C5, BioLegend 108410; RB6–8C5, BioXCell BE0075), I-A/I-E (M5/114.15.2, BioLegend 107628), NK1.1 (PK136, BioLegend 108716), PDCA1 (eBio927, eBioScience 12–3172–82), Sirpα (P84, BioLegend 144008), TER-119 (TER-119, BioLegend 116210; TER-119, BioXCell BE0183), Vα2 (B20.1, BioLegend 127818), Vβ5 (MR9–4, BioLegend 139504), and XCR1 (ZET, BioLegend 148212). For immunostaining ~10⁷ cells in 100 μL of PBS + 2% bovine calf serum (BCS), fluorochrome-conjugated antibodies were diluted from stock concentrations of 0.5 mg mL⁻¹ and incubated with cells for 20 min on ice, unless specified.