αpd 1 clone j43

CD4⁺ MHC-II restricted BDC2.5 T cells, a generous gift from Dr. Kathryn Haskins (University of Colorado), were maintained in supplemented DMEM as previously described (22 (link)–25 (link)). Briefly, BDC2.5 T cells were cultured in T-25 flasks with β membrane (antigen), irradiated NOD splenocytes (antigen presenting cells: APC), and EL-4 supernatant (source of IL-2) for 2 weeks in a cell culture incubator. For mechanistic studies, a subset of flasks were treated with 5 µM PFK15 every third day over the course of the restimulation period. Day 8 and 14 T cells and culture supernatants were harvested for downstream analyses. Similarly, for reinvigoration studies, untreated and PFK15 treated T cells were put into restimulation flasks ±5µg/mL αPD-1 (clone J43; BioXCell), αLAG-3 (clone C9B7W; BioXCell), or αPD-1 + αLAG-3. Cells were treated every third day for 2 weeks.

MC38 tumor cell line was cultured in complete DMEM medium plus with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P.S). CT26 cancer cells are transfected with plasmid coding MSLN ORF, and selected by surface marker MSLN using fluorescenec-activated cell sorting (FACS). CT26-MSLN tumor cell line was cultured in complete DMEM medium plus with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P.S). For MC38 tumor model, C57BL/6 mice were injected with 1 million cells intradermally (i.d.). For CT26-MSLN tumor model, 1 million cells were injected intradermally (i.d.) into BALB/c mice. MC38 and CT26 bearing mice were randomized into four treatment cohorts: control IgG, HSA-IL21, α-PD-1 (clone J43, BioXCell) or HSA-IL21/α-PD-1. HSA-IL21 was injected by intraperitoneally (i.p.) 25 μg per mouse, α-PD-1 were injected by intraperitoneally (i.p.) 200 μg per mouse. All mice were administered on the 5th day after tumor inoculation. Tumor sizes were monitored every 2–3 days, and the tumor volume was calculated as L× W²/2.

1 x 10⁷ex vivo activated CD4⁺ T cells from BDC2.5 mice (described above) were injected i.p. into NOD.scid recipients. For initial prevention studies, recipient animals were split into two groups. One cohort of recipients received 25 mg/kg PFK15 (Selleck) dissolved in 5% DMSO + 45% PEG300 + 1% Tween80 + 49% ddH₂O prepared fresh; the other cohort received vehicle control every other day for 2 weeks. In reversibility studies, recipient animals were placed into one of the following treatment groups: 1) Vehicle Control, 2) PFK15 + IgG (Isotype controls for αPD-1 and αLAG-3 blocking antibodies; 200 μg each per treatment), or 3) PFK15 + 200 μg αPD-1 (clone J43; BioXCell), + 200 μg αLAG-3 (clone C9B7W; BioXCell) as previously described (19 (link), 20 (link)). Animals were treated every other day for two weeks, with checkpoint blockade or IgG treatment initiated during the second week. Body weights and blood glucose (BG) levels were monitored over the course of the experiments. Animals were deemed diabetic after two consecutive BG readings ≥ 350 mg/dL. Diabetic animals were sacrificed at indicated timepoints and peripheral blood, pancreata, and spleens were harvested for downstream analyses.