Cognate Cytokine Research Articles

Refined analytical pipeline for the pharmacodynamic assessment of T-cell responses to vaccine antigens.

Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.

Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling.

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

Classic costimulatory interactions in MAIT cell responses: from gene expression to immune regulation.

Mucosa-associated invariant T (MAIT) cells are evolutionarily conserved, innate-like T lymphocytes with enormous immunomodulatory potentials. Due to their strategic localization, their invariant T cell receptor (iTCR) specificity for major histocompatibility complex-related protein 1 (MR1) ligands of commensal and pathogenic bacterial origin, and their sensitivity to infection-elicited cytokines, MAIT cells are best known for their antimicrobial characteristics. However, they are thought to also play important parts in the contexts of cancer, autoimmunity, vaccine-induced immunity, and tissue repair. While cognate MR1 ligands and cytokine cues govern MAIT cell maturation, polarization, and peripheral activation, other signal transduction pathways, including those mediated by costimulatory interactions, regulate MAIT cell responses. Activated MAIT cells exhibit cytolytic activities and secrete potent inflammatory cytokines of their own, thus transregulating the biological behaviors of several other cell types, including dendritic cells, macrophages, natural killer cells, conventional T cells, and B cells, with significant implications in health and disease. Therefore, an in-depth understanding of how costimulatory pathways control MAIT cell responses may introduce new targets for optimized MR1/MAIT cell-based interventions. Herein, we compare and contrast MAIT cells and mainstream T cells for their expression of classic costimulatory molecules belonging to the immunoglobulin superfamily and the tumor necrosis factor (TNF)/TNF receptor superfamily, based not only on the available literature but also on our transcriptomic analyses. We discuss how these molecules participate in MAIT cells' development and activities. Finally, we introduce several pressing questions vis-à-vis MAIT cell costimulation and offer new directions for future research in this area.

TLR2 dependent enhancement of bacterial clearance in macrophages via TNFR2

Abstract The public health crisis of antimicrobial resistance is especially challenging due to the rapid pace of bacterial adaptation to antibiotics. Thus, development of alternatives to antibiotic-based therapies are crucial. We found that stimulation of murine macrophage TLR2/6 heterodimer with Pam2CSK4 (PAM2), a synthetic bacterial lipoprotein, enhanced clearance of subsequent Staphylococcus aureus infection. Whole lung RNA sequencing identified tumor necrosis factor receptor superfamily member 1B (TNFRSF1B, TNFR2) and its cognate cytokine, TNFα, to be upregulated in response to PAM2. ELISA and qPCR data showed both bioactive forms of TNFα, transmembrane (tmTNFα) and soluble (sTNFα), to be upregulated. To mechanistically address the role that TNFR2 could play in PAM2 induced enhanced bacterial clearance (EBC) ability, antibody blocking of TNFR2 led to reduced macrophage S. aureus clearance. In contrast, sTNFα reduction by small molecule inhibition of ADAM17, the enzyme that cleaves tmTNFα to sTNFα, did not reduce macrophage EBC ability. These data suggest tmTNFα signaling via TNFR2 may have a protective effect which enhances S. aureus clearance. Our work aims to elucidate TLR signaling events through which enhancement of existing host bacterial resolution mechanisms can be achieved. As such, we are investigating whether/how tmTNFα:TNFR2 initiates intracellular signaling pathways that may contribute to upregulation of macrophage mechanisms to enhance pathogen clearance. An in-depth understanding of these signaling events could facilitate development of novel treatment strategies designed to modulate and enhance hostimmune responses against pathogens, which could be leveraged to circumvent antimicrobial resistance.

Accessory cells precondition naive T cells and regulatory T cells for cytokine-mediated proliferation

Abstract Naïve T cells and regulatory T cells, when purified, do not proliferate to the common cytokine receptor γ-chain family cytokines interleukin (IL)-2, IL-7 or IL-15, despite their expression of cognate cytokine receptors. Cell-to-cell contact with dendritic cells (DCs) enabled proliferation of the T cell to these cytokines, independent of antigen recognition or T cell receptor stimulation. This effect lasted after separation of T cells from DCs, enabling enhanced proliferation of the T cells in mice depleted of DCs. We propose calling this a “preconditioning effect”. Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in T cells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2-target genes. Preconditioning was necessary to activate these two pathways, and induced weak Ca 2+mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation and prolonged S6 phosphorylation occurred, and T cells underwent proliferation. Collectively, accessory cells provide T cell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of T cells.

Accessory cells precondition naïve T cells and regulatory T cells for cytokine-mediated proliferation

Naïve Tcells and regulatory Tcells, when purified, do not proliferate to the γc-cytokines IL-2, IL-7, or IL-15, despite their expression of cognate cytokine receptors. Dendritic cells (DCs) enabled the Tcell proliferation to these cytokines, through cell-to-cell contact, but independent of Tcell receptor stimulation. This effect lasted after separation of Tcells from DCs, enabling enhanced proliferation of the Tcells in DC-depleted hosts. We propose calling this a "preconditioning effect". Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in Tcells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2 target genes. "Preconditioning" was necessary to activate these two pathways and induced weak Ca2+ mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation, and prolonged S6 phosphorylation occurred. Collectively, accessory cells provide Tcell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of Tcells.

Modular cytokine receptor-targeting chimeras for targeted degradation of cell surface and extracellular proteins

Targeted degradation of cell surface and extracellular proteins via lysosomal delivery is an important means to modulate extracellular biology. However, these approaches have limitations due to lack of modularity, ease of development, restricted tissue targeting and applicability to both cell surface and extracellular proteins. We describe a lysosomal degradation strategy, termed cytokine receptor-targeting chimeras (KineTACs), that addresses these limitations. KineTACs are fully genetically encoded bispecific antibodies consisting of a cytokine arm, which binds its cognate cytokine receptor, and a target-binding arm for the protein of interest. We show that KineTACs containing the cytokine CXCL12 can use the decoy recycling receptor, CXCR7, to target a variety of target proteins to the lysosome for degradation. Additional KineTACs were designed to harness other CXCR7-targeting cytokines, CXCL11 and vMIPII, and the interleukin-2 (IL-2) receptor-targeting cytokine IL-2. Thus, KineTACs represent a general, modular, selective and simple genetically encoded strategy for inducing lysosomal delivery of extracellular and cell surface targets with broad or tissue-specific distribution.

Cytokine Receptor Diversity in the Lamprey Predicts the Minimal Essential Cytokine Networks of Vertebrates.

The vertebrate adaptive immune systems (Agnatha and Gnathostomata) use sets of T and B lymphocyte lineages that somatically generate highly diverse repertoires of Ag-specific receptors and Abs. In Gnathostomata, cytokine networks regulate the activation of lymphoid and myeloid cells, whereas little is known about these components in Agnathans. Most gnathostome cytokines are four-helix bundle cytokines with poorly conserved primary sequences. In contrast, sequence conservation across bilaterians has been observed for cognate cytokine receptor chains, allowing their structural classification into two classes, and for downstream JAK/STAT signaling mediators. With conserved numbers among Gnathostomata, human cytokine receptor chains (comprising 34 class I and 12 class II) are able to interact with 28 class I helical cytokines (including most ILs) and 16 class II cytokines (including all IFNs), respectively. Hypothesizing that the arsenal of cytokine receptors and transducers may reflect homologous cytokine networks, we analyzed the lamprey genome and transcriptome to identify genes and transcripts for 23 class I and five class II cytokine receptors alongside one JAK signal mediator and four STAT transcription factors. On the basis of deduction of their respective orthologs, we predict that these receptors may interact with 16 class I and 3 class II helical cytokines (including IL-4, IL-6, IL-7, IL-12, IL-10, IFN-γ, and thymic stromal lymphoprotein homologs). On the basis of their respective activities in mammals, this analysis suggests the existence of lamprey cytokine networks that may regulate myeloid and lymphoid cell differentiation, including potential Th1/Th2 polarization. The predicted networks thus appear remarkably homologous to those of Gnathostomata, albeit reduced to essential functions.

Structural basis of cytokine-mediated activation of ALK family receptors.

Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGβ and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.

Anti-interferon-γ autoantibody-associated immunodeficiency.

Anticytokine autoantibodies are an emerging disease etiology, through the disturbance of physiological functions of cognate cytokines. Anti-interferon (IFN)-γ autoantibodies (AIGAs) were first identified in patients with severe mycobacterial infections, and were considered to be an autoimmune phenocopy of inborn genetic errors of the IL-12/IFN-γ axis. More than 600 reported cases, most originating from Southeast Asia, have been diagnosed over the last decade. Specific HLA class II molecules are associated with these autoantibodies, which provide a genetic basis for the high prevalence of this immunodeficiency syndrome in certain ethnic groups. Salmonellosis and herpes zoster reactivation are observed in more than half the patients with AIGAs. Moreover, AIGAs have been shown to underlie severe Taralomyce marneffei infection in HIV-negative patients. AIGAs may, thus, be considered a new form of late-onset immunodeficiency conferring a predisposition not only to severe mycobacterial, but also to some bacterial and fungal infections.

Administration of fusion cytokines induces tumor regression and systemic antitumor immunity.

It is difficult to improve the curative effects of cancer immunotherapy on solid tumors. Cytokines, as powerful immune regulators, show potential in activating host antitumor immunity. We have previously found that the administration of certain cytokine combinations induces complete tumor clearance. Here, we constructed cognate fusion cytokines and evaluated their antitumor effects in various mouse tumor models. The in situ induction of the expression of the fusion cytokine IL12IL2GMCSF caused tumor eradication, including that of the tumors at advanced stages. An immune memory against unrelated syngeneic tumors was also elicited. Furthermore, flow cytometry analysis revealed that tumor‐infiltrating CD3+ cells were greatly increased in the treated tumors and were accompanied by an elevation of CD8+/CD4+ ratios. This fusion protein exhibited superior immune activating capability compared to that of cytokine mixtures, in the experiments done in vitro. We also induced tumor regression in various immunocompetent tumor models via intratumoral injection. To improve its translational potential for clinical application, a systemically‐administered immunocytokine, IL12IL2DiaNFGMCSF, was constructed by inserting a tumor‐targeting diabody in the fusion protein. This protein also displayed good immune stimulating activities in vitro. Intravenous infusion of IL12IL2DiaNFGMCSF induced tumor‐infiltrating immune cell alterations like IL12IL2GMCSF, with moderate serum IFNγ increment. Therapeutic effects were observed in the various tumor models after systemic administration of IL12IL2DiaNFGMCSF, but with slight toxicity. These results show the feasibility of developing a versatile cancer immunotherapy.

Targeted DNA methylation profiling reveals epigenetic signatures in peanut allergy.

DNA methylation (DNAm) has been shown to play a role in mediating food allergy; however, the mechanism by which it does so is poorly understood. In this study, we used targeted next-generation bisulfite sequencing to evaluate DNAm levels in 125 targeted highly informative genomic regions containing 602 CpG sites on 70 immune-related genes to understand whether DNAm can differentiate peanut allergy (PA) versus nonallergy (NA). We found PA-associated DNAm signatures associated with 12 genes (7 potentially novel to food allergy, 3 associated with Th1/Th2, and 2 associated with innate immunity), as well as DNAm signature combinations with superior diagnostic potential compared with serum peanut–specific IgE for PA versus NA. Furthermore, we found that, following peanut protein stimulation, peripheral blood mononuclear cell (PBMCs) from PA participants showed increased production of cognate cytokines compared with NA participants. The varying responses between PA and NA participants may be associated with the interaction between the modification of DNAm and the interference of environment. Using Euclidean distance analysis, we found that the distances of methylation profile comprising 12 DNAm signatures between PA and NA pairs in monozygotic (MZ) twins were smaller than those in randomly paired genetically unrelated individuals, suggesting that PA-related DNAm signatures may be associated with genetic factors.

Utilizing Immunocytokines for Cancer Therapy.

Cytokine therapy for cancer has indicated efficacy in certain diseases but is generally accompanied by severe toxicity. The field of antibody–cytokine fusion proteins (immunocytokines) arose to target these effector molecules to the tumor environment in order to expand the therapeutic window of cytokine therapy. Pre-clinical evidence has shown the increased efficacy and decreased toxicity of various immunocytokines when compared to their cognate unconjugated cytokine. These anti-tumor properties are markedly enhanced when combined with other treatments such as chemotherapy, radiotherapy, and checkpoint inhibitor antibodies. Clinical trials that have continued to explore the potential of these biologics for cancer therapy have been conducted. This review covers the in vitro, in vivo, and clinical evidence for the application of immunocytokines in immuno-oncology.

Perspectives on long pentraxin 3 and rheumatoid arthritis: several potential breakthrough points relying on study foundation of the past

Rheumatoid arthritis (RA) is a systemic chronic autoimmune inflammatory disease which is mainly characterized by synovitis and results in a severe burden for both the individual and society. To date, the underlying mechanisms of RA are still poorly understood. Pentraxin 3 (PTX3) is a typical long pentraxin protein which has been highly conserved during evolution. Meanwhile, functions as well as properties of PTX3 have been extensively studied. Several studies identified that PTX3 plays a predominate role in infection, inflammation, immunity and tumor. Interestingly, PTX3 has also been verified to be closely associated with development of RA. We therefore accomplished an elaboration of the relationships between PTX3 and RA. Herein, we mainly focus on the associated cell types and cognate cytokines involved in RA, in combination with PTX3. This review infers the insight into the interaction of PTX3 in RA and aims to provide novel clues for potential therapeutic target of RA in clinic.

An engineered 4-1BBL fusion protein with “activity on demand”

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display "activity on demand" properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

Analysis of associations of CXCR3 ligands with immune microenvironment and aggressiveness in murine and human pancreatic ductal adenocarcinoma.

762 Background: The complex milieu of cytokines within pancreatic ductal adenocarcinoma (PDAC) promotes tumor progression and immune suppression thereby contributing to the dismal prognosis of patients with PDAC. However, the roles of many cytokines, including CXCR3 ligands, in PDAC have not been thoroughly investigated. Methods: Bioinformatics analyses of PDAC microarray and TCGA datasets were used to identify cytokines overexpressed in PDAC, their association with patient survival as well as the expression of cognate cytokine receptors. Comparative analysis of cytokine expression in KrasLSL-G12D-P53LSL-R172H-Pdx1-Cre (KPC) and KrasLSL-G12D-Pdx1-Cre (KC) murine PDAC models were used to validate these findings. Pathway and CIBERSORT analyses were employed to determine mechanistic basis of altered survival associated with cytokines of interest. Results: Of the 149 cytokines analyzed, CXCR3 ligands CXCL9 and CXCL10 were highly and consistently overexpressed in PDAC datasets. Concurrently, CXCL9, CXCL10 and PF4 were overexpressed in the aggressive KPC murine model compared to the indolent KC model. CXCR3 showed robust expression in PDAC in microarray, TCGA and IHC analyses. Interestingly, high expression of CXCR3 ligands was associated with shorter overall survival (p = 0.04 for CXCL9, 10 and 11 and p = 0.02 for PF4) while high expression of CXCR3 was associated with increased overall survival (p = 0.03). Pathway analysis of genes correlated with CXCR3 and/or its ligands showed that CXCR3 ligands may promote T-cell exhaustion (p < 0.001). Finally, CIBERSORT analysis of TCGA data demonstrated that high CXCR3 expression was associated with increased CD8 T-cell and naïve B-cell signatures and loss of plasma cells signatures. High CXCR3 ligand expression was associated with increased CD8 T-cell, and M1 macrophage, and loss of NK-cell signatures(p < 0.05). Conclusions: CXCR3 ligands are overexpressed in PDAC and are associated with poor survival, likely related to alterations in tumor immune infiltrate/activity and may represent targets to augment anti-tumor immunity.

Deciphering Binding Interactions of IL-23R with HDX-MS: Mapping Protein and Macrocyclic Dodecapeptide Ligands.

Molecular characterization of the binding epitope of IL-23R and its cognate cytokine IL-23 is paramount to understand the role in autoimmune diseases and to support the discovery of new inhibitors of this protein-protein interaction. Our results revealed that HDX-MS was able to identify the binding epitope of IL-23R:IL-23, which opened the way to evaluate a peptide macrocycle described in the literature as disrupter of this autoimmune target. Thus, the characterization of the interactions of this chemotype by HDX-MS in combination with computational approaches was achieved. To our knowledge, this is the first reported structural evidence regarding the site where a small compound binds to IL-23R.

Abstract 5563: Kinetically-controlled release of blocking molecules for the targeted enhancement of therapeutic activity in antibody-cytokine fusion proteins

Abstract Cytokines are able to mediate a potent anticancer activity but have significant side effects that prevent dose escalation to therapeutically-active regimens. Targeting the cytokines to the site of disease helps to improve their therapeutic index while decreasing associated toxicities. However, peak concentrations in blood are still associated with side-effects. We have developed a novel technology (termed “CORK Technology”), which consists in masking the undesired activity of a cytokine in circulation. The “cork” can be either an antibody fragment or a small molecule, which specifically binds to the cytokine thus preventing the interaction with its receptor. Once the complex accumulates in the tumor, the cork dissociate from the cytokine allowing it to exert its function. Capitalizing on the fact that the kinetic dissociation constant koff of the complex is identical in different species, this technology can be easily translated from mouse to man with comparable results. In this poster we describe the experimental implementation of CORK Technology to improve the therapeutic index of the clinical stage L19-IL2 (L19 is an antibody fragment specific to the alternatively-spliced EDB domain of fibronectin, a marker of tumor angiogenesis). Two classes of IL2 inhibitors were discovered and characterized: (i) a fully human scFv fragment, and (ii) a small organic ligand, featuring a modified methylindole moiety. Both types of agents reduced IL2 activity in vitro, were able to dissociate from the cognate cytokine in a kinetically-tuned process and allowed the administration of the L19-IL2 fusion protein at higher doses, both in normal and in tumor-bearing mice. The technology is applicable to other cytokine payloads and facilitates the development of targeted cytokine products with “activity on demand” (i.e., with increased activity at the tumor site, promoting an activation and proliferation of tumor-resident lymphocytes). Albeit still in its infancy, this elegant approach provides promising results and a strong rationale to further investigate this therapeutic power of the CORK Technology. Citation Format: Tiziano Ongaro, Martina Bigatti, Filippo Sladojevich, Etienne Donckele, Alessandra Micaela Villa, Dario Neri. Kinetically-controlled release of blocking molecules for the targeted enhancement of therapeutic activity in antibody-cytokine fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5563.

The Thrombopoietin Receptor: Structural Basis of Traffic and Activation by Ligand, Mutations, Agonists, and Mutated Calreticulin.

A well-functioning hematopoietic system requires a certain robustness and flexibility to maintain appropriate quantities of functional mature blood cells, such as red blood cells and platelets. This review focuses on the cytokine receptor that plays a significant role in thrombopoiesis: the receptor for thrombopoietin (TPO-R; also known as MPL). Here, we survey the work to date to understand how this receptor functions at a molecular level throughout its lifecycle, from traffic to the cell surface, dimerization and binding cognate cytokine via its extracellular domain, through to its subsequent activation of associated Janus kinases and initiation of downstream signaling pathways, as well as the regulation of these processes. Atomic level resolution structures of TPO-R have remained elusive. The identification of disease-causing mutations in the receptor has, however, offered some insight into structure and function relationships, as has artificial means of receptor activation, through TPO mimetics, transmembrane-targeting receptor agonists, and engineering in dimerization domains. More recently, a novel activation mechanism was identified whereby mutated forms of calreticulin form complexes with TPO-R via its extracellular N-glycosylated domain. Such complexes traffic pathologically in the cell and persistently activate JAK2, downstream signal transducers and activators of transcription (STATs), and other pathways. This pathologic TPO-R activation is associated with a large fraction of human myeloproliferative neoplasms.

Acetylation Modulates IL-2 Receptor Signaling in T Cells.

Ligand binding to the cognate cytokine receptors activates intracellular signaling by recruiting protein tyrosine kinases and other protein modification enzymes. However, the roles of protein modifications other than phosphorylation remain unclear. In this study, we examine a novel regulatory mechanism of Stat5, based on its acetylation. As for phosphorylation, IL-2 induces the acetylation of signaling molecules, including Stat5, in the murine T cell line CTLL-2. Stat5 is acetylated in the cytoplasm by CREB-binding protein (CBP). Acetylated Lys696 and Lys700 on Stat5 are critical indicators for limited proteolysis, which leads to the generation of a truncated form of Stat5. In turn, the truncated form of Stat5 prevents transcription of the full-length form of Stat5. We also demonstrate that CBP physically associates with the IL-2 receptor β-chain. CBP, found in the nucleus in resting CTLL-2 cells, relocates to the cytoplasm after IL-2 stimulation in an MEK/ERK pathway-dependent manner. Thus, IL-2-mediated acetylation plays an important role in the modulation of cytokine signaling and T cell fate.