Figure 4. Cell-based cytokine receptor-blocking assays.

(A) FACS plots of IFN-α2, IL-6, and GM-CSF signaling assays. Cells were treated with media only; commercial blocking antibody or 10% positive control serum from a patient with atypical mycobacterial infection (AMI); 10% healthy control serum; or 10% test serum. Cells were treated with patient serum or a control in the unstimulated condition and with both cytokine and patient serum or a control in the stimulated condition. (B) Blocking activity of patient serum on cells in cytokine signaling assays, reported as percentage of pSTAT⁺ cells in the unstimulated and stimulated condition. Patient sera were from patients with influenza (n_Marburg = 4, n_Athens = 5), Stanford ICU (n_infected = 19, n_noninfected = 2), or ARDS (n = 8) . For IFN-α2 and IFN-α8, results shown represent 2 independent experiments (Supplemental Figure 7). HC and positive controls (PC; commercially available antibody or prototype patient serum with known blocking activity) are also included. (C) Neutralization activity to IFN-α2, IFN-γ, and IFN-λ3 in the serum samples of 2 patients. IFN-α2, IFN-γ, and IFN-λ3 were incubated with heat-inactivated serum from donor AMI (PC) and donor SU047 (infected Stanford ICU cohort) and added to HAP1 reporter cells. The serum samples were prepared and tested with a 5-fold serial dilution on HAP1 reporter cells. Final concentrations of IFN-α2, IFN-γ, and IFN-λ3 in the culture were 40 U/mL, 8 U/mL, and 1 ng/mL, respectively (Supplemental Figure 8). The percentages of GFP⁺ HAP1 reporter cells were evaluated 22–24 hours after the incubation with flow cytometry.