Kinetics of cytokine receptor trafficking determine signaling and functional selectivity

Abstract

Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.

Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

“…Indeed, several studies have reported the ability of STAT3 to inhibit transcription induced by other STATs (Costa-Pereira et al, 2002;Ray et al, 2014;Yang et al, 2011), suggesting that STAT3 activating cytokines may elicit their functions by disrupting transcriptional programs induced by other cytokines. In agreement with this model, we recently reported that IL-6, another STAT3 activating cytokine, promoted strong STAT3 binding to chromatin, but poor gene expression (Martinez-Fabregas et al, 2019).…”

“…WTM showed a poor activation of STAT1 and STAT3 with amplitudes of activation reaching less than fifty percent of those elicited by WTD ( Figure 3b). While WTD, R5A11D and R5A11M showed a similar pSTAT1 to pSTAT3 ratio, WTM's clear bias towards pSTAT3 (Figure 3d), agrees with previous observations from our laboratory describing biased signaling by short-lived cytokine-receptor complexes (Martinez-Fabregas et al, 2019). R5A11M induced activation of both STAT1 and STAT3 to levels comparable to those induced by the dimeric cytokines at saturating doses, suggesting that the defective signaling elicited by WTM results from its weak IL-10Rβ binding affinity (Figure 3b and 3c).…”

“…We recently showed that the number of phospho-tyrosines available in cytokine receptor intracellular domains critically contribute to defining signaling identity by cytokines (Martinez-Fabregas et al, 2019). IL-6 variants that triggered partial phosphorylation of Tyr available in the gp130 intracellular domain exhibited a biased STAT3 versus STAT1 activation (Martinez-Fabregas et al, 2019). A similar model could be invoked to explain functional differences between monomeric and dimeric IL-10 ligands.…”

“…Overall these observations suggest that in addition to receptor binding affinity, the stoichiometry of the IL-10-receptor complex contributes to fine-tune IL-10 bioactivity potencies. We recently showed that the number of phospho-tyrosines available in cytokine receptor intracellular domains critically contribute to defining signaling identity by cytokines (Martinez-Fabregas et al, 2019). IL-6 variants that triggered partial phosphorylation of Tyr available in the gp130 intracellular domain exhibited a biased STAT3 versus STAT1 activation (Martinez-Fabregas et al, 2019).…”

“…The tags were designed to specifically recognise either one of two different anti-GFP nanobodies (Kirchhofer et al, 2010). These nanobodies (NBs) were conjugated to photostable organic fluorophores ATTO Rho11 and ATTO 643 suitable for simultaneous dualcolour single molecule tracking of IL-10Rα and IL-10Rβ in the plasma membrane of live cells as shown previously in other cytokine receptor systems (Martinez-Fabregas et al, 2019;Moraga et al, 2015a;Wilmes et al, 2020) (Figure 2a and Sup. Figure 3a).…”