Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells.

Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rß1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rß1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rß2/IL-12Rß1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rß1 directly engages the common p40 subunit. We targeted the shared IL-12Rß1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.

Transformation of hematopoietic cells and activation of JAK2-V617F by IL-27R, a component of a heterodimeric type I cytokine receptor.

From a patient with acute myeloid leukemia (AML), we have identified IL-27Ra (also known as TCCR and WSX1) as a gene whose expression can induce the transformation of hematopoietic cells. IL-27Ra (IL-27R) is a type I cytokine receptor that functions as the ligand binding component of the receptor for IL-27 and functions with the glycoprotein 130 (gp130) coreceptor to induce signal transduction in response to IL-27. We show that IL-27R is expressed on the cell surface of the leukemic cells of AML patients. 32D myeloid cells transformed by IL-27R contain elevated levels of activated forms of various signaling proteins, including JAK1, JAK2, STAT1, STAT3, STAT5, and ERK1/2. Inhibition of JAK family proteins induces cell cycle arrest and apoptosis in these cells, suggesting the transforming properties of IL-27R depend on the activity of JAK family members. IL-27R also transforms BaF3 cells to cytokine independence. Because BaF3 cells lack expression of gp130, this finding suggests that IL-27R-mediated transformation of hematopoietic cells is gp130-independent. Finally, we show that IL-27R can functionally replace a homodimeric type I cytokine receptor in the activation of JAK2-V617F, a critical JAK2 mutation in various myeloproliferative disorders (MPDs). Our data demonstrate that IL-27R possesses hematopoietic cell-transforming properties and suggest that, analogous to homodimeric type I cytokine receptors, single-chain components of heterodimeric receptors can also enhance the activation of JAK2-V617F. Therefore, such receptors may play unappreciated roles in MPDs.

Expression and functional activity of the IL-8 receptor type CXCR1 and CXCR2 on human mast cells.

To further elucidate mechanisms involved in mast cell accumulation at sites of cutaneous inflammation, we have studied the ability of human leukemic mast cells (HMC-1 cells) to express functionally active IL-8 receptors. Expression of mRNA for both types of IL-8 receptors (CXCR1 and CXCR2) was demonstrated by PCR and of both proteins by flow cytometry. Binding and competition studies with 125I-labeled IL-8 and its homologue melanoma growth stimulating activity (125I-labeled MGSA) revealed two specific binding sites for IL-8, K1 = 1.1 x 10(11) M(-1) and K2 = 5 x 10(7) M(-1); and for MGSA, K1 = 2.8 x 10(10) M(-1) and K2 = 5 x 10(7) M(-1). This finding was supported by a dose-dependent rise of cytosolic free calcium concentration ([Ca2+]i) induced by both chemokines and to a lesser extent by the homologue neutrophil-activating peptide-2 (NAP-2). A significant migratory response of human leukemic mast cells (HMC-1) was observed with all three chemokines at a range from 10(-8) M to 10(-9) M. Moreover, the formation of cellular F-actin was induced in a rapid, dose-dependent fashion, with a maximally 1.7-fold increase at 10(-7) M. Using postembedding immunoelectron microscopy, we could show the expression of CXCRI on the cytoplasmatic membrane of isolated human skin mast cells whereas CXCR2 was located in mast cell-specific granules. These findings demonstrate for the first time the functional expression of both types of IL-8 receptors on human mast cells, suggesting a role for their ligands during mast cell activation and recruitment.

Crystal structure of Epstein-Barr virus protein BCRF1, a homolog of cellular interleukin-10.

The crystal structure of Epstein-Barr virus protein BCRF1, an analog of cellular interleukin-10 (IL-10), has been determined at the resolution of 1.9 A and refined to an R-factor 0.191. The structure of this cytokine is similar to that of human IL-10 (hIL-10), forming an intercalated dimer of two 17 kDa polypeptides related by a crystallographic 2-fold symmetry axis. BCRF1 exhibits novel conformations of the N-terminal coil and of the loop between helices A and B compared to hIL-10. These regions are likely to be involved in binding of one or more components of the IL-10 receptor system, and thus the structural differences may account for the lower binding affinity and limited spectrum of biological activities of viral IL-10, compared to hIL-10.

Solution structure of recombinant human interleukin-6.

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.

Cytokine receptor architecture, structure and genetic assembly.

In this review major structural and molecular characteristics of interleukin-6-type cytokine receptors consisting of ligand-specific (e.g., IL-6 receptor) and public (gp130) elements are outlined. The peculiar shedding feature of the ligand-binding receptor subunit provides a possibility to form a receptor-ligand complex in the soluble phase, followed by an autocrine or paracrine re-attaching to the membrane bound gp130. This situation provides a dynamic 4-chain model for IL-6-type receptors, depending on a critical balance between membrane bound and soluble cytokine receptors. The generation and transduction of intracellular signal for IL-6-type cytokine receptors based primarily on generation of phospho-tyrosine proteins. In this set of events kinases of the JAK family are basically involved. Although not all primary substrates are uncovered, gp130 and stat proteins are phosphorylated. The variability of the JAK/Stat system and its still not clear relation to the specificity of cytokine actions are discussed.