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T. Shahin et al.
protein structure that is conserved across species (Figure 1E and F).19 We found GP130 protein expressed in PP498L fibroblasts, albeit at lower levels (Figure 1G), and in a CRISPR-engineered HEK293-IL6ST knockout (KO) cell line with transient overexpression of the GP130N404Y mutant.5
Functional assessment of GP130P498L mutation in primary and patient-derived cells
Phosphorylation of STAT3 (p-STAT3) is a direct down- stream effect of GP130 activation. To assess the impact of the p.P498L substitution, we studied STAT3 phosphoryla- tion in primary T cells from PP498L and observed markedly decreased p-STAT3 levels upon stimulation with IL-6 and IL-27 compared to stimulation with IL-10 or IL-21 and to healthy donors (Figure 2A). Our previous study5 on PN404Y showed that IL-6 signaling had been abolished but a smaller reduction in p-STAT3 after stimulating primary T cells with IL-27 (CD3+, CD4+, and CD8+ T cells) (Figure 2B), indicating a mutation-dependent effect on the severity of downstream signaling through selected cytokines. Furthermore, IL-6 sig- naling was shown to be defective in both EBV-LCLs and T lymphoblasts derived from PBMCs of PP498L, and IL-27 (that
stimulates T lymphoblasts) was aberrant in PP498L (Figure 2C and D). In addition, we tested the effect of the new p.P498L substitution on the activation of other STAT family tran- scription factors in T lymphoblasts. Stimulation with IL-6 mainly activated STAT3 in healthy donors with no com- pensatory increase in activation of STAT1 in the patient cells (Online Supplementary Figure S1A), whereas phosphory- lation of STAT1, STAT3 and STAT4 was abolished in patient T lymphoblasts upon stimulation with IL-27 com- pared to healthy donor-derived cells (Online Supplementary Figure S1B). Activation of STATs by GP130-independent cytokines including IL-4, IL-21 and IFNb was unaffected in patient cells, except for STAT4 which showed increased phosphorylation upon IL-12 stimulation of PP498L T lym- phoblasts (Online Supplementary Figure S1C-F).
To further evaluate the spectrum of mutation-dependent signaling defects, we analyzed p-STAT3 responses in fibroblasts from PP498L and a healthy donor, after overnight starvation. IL-6 or IL-11 stimulation demonstrated signifi- cantly reduced p-STAT3 levels (Figure 3A and B), with the aberrant IL-11 signaling likely to underlie the majority of bone manifestations in PP498L.14 OSM stimulation resulted in a partial and statistically significant reduction in p-STAT3
AB
CD
E
Figure 4. Functional assessment of GP130P498L variant in GP130-KO HEK293 cell line. (A-E) Relative mean fluorescence intensity (rMFI) of p-STAT3 in GP130 CRISPR- knockout HEK293 cells that were transfected with a plasmid coding for the GP130P498L (GP130 KO + p.P498L), wild-type GP130 (GP130 KO + WT GP130) or trans- fected with the empty plasmid (GP130 KO + eV), after stimulation with (A) IL-6, (B) IL-11, (C) IL-27, (D) LIF, (E) OSM. From left to right: dose-escalation curves, stacked histograms displaying shifts in p-STAT3 signals, and bar graphs showing rMFI of fibroblasts upon stimulation with the highest concentration of the corresponding cytokine. (6 replicates of 3 independent experiments are shown; Wilcoxon matched-pairs signed rank test; *P<0.05.)
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haematologica | 2019; 104(3)