Anti il 9

LAG3^IC and PD-1^IC expression were analyzed on 5,000 bulk and 5,000 naïve T cells after incubation with various cytokines and patient plasma (100 μL). Bulk and naïve CD8⁺ T cells and CD4⁺ T cells were sorted as specified and incubated in complete RPMI media (with components as above) with low-dose IL2 (1,000 IU/mL), low-dose IL7 (1 ng/mL; IL2 and IL7 from R&D Systems; 202-IL-050/CF, BT-007-AFL), with no TCR stimulation. These assays included incubation with 45 individual cytokines as per the R&D kit in addition to the IL2 and IL7 indication above at their corresponding EC₅₀ concentrations (R&D Systems; LKTM014), as well as plasma samples from the fresh patient PBL cohorts, with both low and high LAG3 CD8⁺ T-cell subsets. Samples (5,000 T cells) were also incubated in complete RPMI media as above with 100 μL patient plasma and cytokine blockade was performed using anti-IL6 (EC₅₀ = 0.1 ng/mL), anti-IL8 (EC₅₀ = 0.1 ng/mL), anti-IL22 (EC₅₀ = 0.1 ng/mL), anti-IL1β (EC₅₀ < 12 pg/mL), anti-IL9 (EC₅₀ = 1 ng/mL), anti-IL10 (EC₅₀ = 1.95 ng/mL), anti-IL15 (EC₅₀ = 0.13 ng/mL), and anti-IL21 (EC₅₀ = 0.5 ng/mL; Abcam). Cells were incubated for 60 hours and analyzed by flow cytometry for IR expression as described above.