Increasing evidence has linked dysregulated interleukin (IL)-10 production by IL-10+ve B cells to autoimmunity, highlighting the importance of improving the understanding of the regulation of IL-10 production in these cells. In both B cells and myeloid cells, IL-10 can be produced in response to Toll-like receptor (TLR) agonists. In macrophages, previous studies have established that mitogen- and stress-activated protein kinases (MSKs) regulate IL-10 production via the phosphorylation of cAMP response element–binding (CREB) protein on the IL-10 promoter. We found here that although MSKs are activated in peritoneal B cells in response to TLR4 agonists, neither MSKs nor CREB are required for IL-10 production in these cells. Using a combination of chemical inhibitors and knockout mice, we found that IL-10 induction in B cells was regulated by an ERK1/2- and p90 ribosomal S6 kinase-dependent mechanism, unlike in macrophages in which p90 ribosomal S6 kinase was not required. This observation highlights fundamental differences in the signaling controlling IL-10 production in B cells and macrophages, even though these two cell types respond to a common TLR stimulus.

Cytokines are soluble factors vital for mammalian physiology. Cytokines elicit highly pleiotropic activities, characterized by their ability to induce a wide spectrum of functional responses in a diverse range of cell subsets, which makes their study very challenging. Cytokines activate signalling via receptor dimerization/oligomerization, triggering activation of the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling pathway. Given the strong crosstalk and shared usage of key components of cytokine signalling pathways, a long-standing question in the field pertains to how functional diversity is achieved by cytokines. Here, we discuss how biophysicalfor example, ligand-receptor binding affinity and topologyand cellularfor example, receptor, JAK and STAT protein levels, endosomal compartmentparameters contribute to the modulation and diversification of cytokine responses. We review how these parameters ultimately converge into a common mechanism to fine-tune cytokine signalling that involves the control of the number of Tyr residues phosphorylated in the receptor intracellular domain upon cytokine stimulation. This results in different kinetics of STAT activation, and induction of specific gene expression programs, ensuring the generation of functional diversity by cytokines using a limited set of signalling intermediaries. We describe how these first principles of cytokine signalling have been exploited using protein engineering to design cytokine variants with more specific and less toxic responses for immunotherapy.

Cytokines dimerize/oligomerize surface receptors to activate signaling. While cytokine receptors preferentially bind only one member of the JAK family, ancestral cytokine receptors, such as Gp130, promiscuously recruit different JAKs to elicit their activities. Here, we have explored how the identity of JAKs in Gp130 signaling complexes can regulate functional outcomes. Using a synthetic biology approach, we show that Gp130 bound to different JAKs propagates distinct STAT activation profiles. While Gp130-JAK1 complexes activated both, STAT1 and STAT3 very potently, Gp130-JAK2 complexes exhibited a clear preference for STAT3 activation. Gp130-TYK2 complexes triggered overall weaker signaling but with diminished STAT specificity. The three JAKs competed for binding to Gp130 and led to differential activation of phospho-Tyr in the Gp130 intracellular domain. JAK1, JAK2 and to a lower extent TYK2 bound with comparable affinities to Gp130, and in response to IL-6 stimulation efficiently drove Gp130 dimerization. However, the three JAKs differentially affected Gp130 surface expression, identifying JAK-dependent receptor trafficking as a critical determinant of signaling plasticity. Our results provide new mechanistic insights into how differential functional coupling in Gp130-JAK complexes translates into unique signaling signatures that likely contribute to its large functional diversity.

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