Human il 2 duoset elisa

Functional activity of ipilimumab was evaluated by IL-2 production. Briefly, frozen human PBMCs were thawed and stimulated by 2.5 μg/mL Phytohemagglutinin-L (Thermo Fisher Scientific) for 6 days. CD3+cells were isolated by CD3 MicroBeads (Myltenyi Biotech) and co-cultured with engineered Raji cells, expressing an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner and endogenously expressing CTLA-4 ligands CD80 and CD86, and ipilimumab mIgG2a or ipilimumab mIgG2a LALAPG for 24 h. Culture supernatants were collected, and IL-2 production was evaluated by human IL-2 DuoSet ELISA (R&D Systems) according to the manufacturer’s protocol.

Briefly, 0.5 × 10⁴ SUDHL-10 or Raji cells were used as target cells with tumor specific cytotoxic T cells as effector cells at a 1:5 ratio. T cells were pre-incubated with indicated concentrations of antibodies, cell culture supernatant was collected after 24 hours of incubation and assayed for IL-2 release using Human IL-2 DuoSet ELISA (R&D Biosystems, Minneapolis, MN, USA) following manufacturer provided instructions. Anti-PD-1, anti-TIM-3, anti-LAG-3 compounds for in vitro assays were generously provided by Tesaro.

Cytokine production by CAR-T cells was quantified by ELISA according to the manufacturer’s instructions. Human IL-2 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) and Human IFN-gamma DuoSet ELISA (R&D Systems) were used to measure the concentration of IL-2 and IFN-γ, respectively. Mesothelin-targeting CAR-T cells were restimulated by K562-mesothelin, and the culture supernatants were collected 48 h after stimulation. The concentration of samples under the detection limit was regarded as zero for statistical analysis.

For activation by SEE: Raji B cells were resuspended at 10⁶ cells/ml, and 50 μl were seeded in 96-well flat-bottom plates. Jurkat wt or IRAP ko cells were resuspended at 10⁶ cells/ml, and 100 μl were added to the Raji B cells. In total, 50 μl SEE (Toxin Technology) was added at the indicated final concentrations, and supernatants were harvested after 6 h incubation at 37 °C, 5% CO₂.
For activation by MART1 peptide: 15 × 10³ Daju-A2 cells per well were cultured in a 96-well flat-bottom plate overnight. The day after, MART1 peptide (ProImmune) was added at the indicated final concentrations, as well as 200 × 10³ Jurkat wt or IRAP ko cells expressing TCR-MelanA2. Supernatants were harvested after overnight incubation at 37 °C, 5% CO₂.
IL-2 was measured by ELISA with the kit Human IL-2 DuoSet ELISA (R&D Systems) on a Tecan Infinite 200 microplate reader.

The Ph.D.-12 phage display peptide library kit was purchased from New England Biolabs. Histopaque-1077 (10771) was from Sigma-Aldrich. CD4⁺ T-cell isolation kit (130-096-533) and Mo-DC Generation Toolbox I (130-093-568) were obtained from Miltenyi Biotech. The Human IFN-gamma DuoSet ELISA (DY285B) and Human IL-2 DuoSet ELISA (DY202) were from R&D Systems. The TMB substrate single solution (D0022) was purchased from Yacoo. Opdivo (NDC 0003-3772-11) was obtained from Bristol-Myers Squibb. Gal-9 fusion protein (CHI-HF-210GAL9) and TIM3 fusion protein (CHI-MF-210T3-C050) were obtained from Chimerigen. The anti-TIM3 monoclonal antibody C23 was generated by our laboratory. Anti-GAPDH (10494-1-AP) was purchased from Proteintech. HRP-anti-mouse (ab6789) was obtained from Abcam.