IL-2 Family Cytokines and Receptors-- Mechanisms of Regulation & Action

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

The IL-2 receptor and related cytokine/cytokine receptor systems are being studied to understand the T cell immune response in normal and disease. After T-cell activation, the magnitude and duration of the response is controlled by the amount of IL-2 produced, levels of IL-2 receptors, and the time course of their induction. IL-2Ra expression is highly expressed by cells infected with HTLV-I, the cause of adult T cell leukemia (ATL). There are three chains of the receptor: IL-2Ra, IL-2Rb, and gc, with IL-2Ra and IL-2Rb highly regulated at the level of transcription. gc is shared by the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors and is mutated in XSCID. We have studied the signals induced by these cytokines, particularly STAT proteins and the mechanisms by which they regulate target genes. Given our prior data that Stat5a or Stat5b transgenic mice develop tumors, consistent with STAT5 being implicated in malignant transformation and elevated in a range of human tumors, this has relevance for both normal and pathological states. Moreover, humans and mice with altered STAT protein expression or activation have a range of immunological defects. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation important for host defense to viruses and other intracellular pathogens, Th2 differentiation vital in allergic disorders/helminths, and Th17 differentiation vital in inflammatory disorders. We previously showed that IL-2 is important for Th2 differentiation and that IL-2 induces IL-4 receptor expression in a STAT5-dependent manner and controls priming for Th2 differentiation. Moreover, using genome-wide chromatin immunoprecipitation coupled to DNA sequencing (ChIP-Seq), we had shown regulation of Th2 differentiation via STAT5A and STAT5B and extended these findings to show that IL-2 via STAT5 induces IL-12Rb2, which is critical for Th1 differentiation and that IL-2 via STAT5 also regulates T-bet. We also had elucidated the mechanisms by which IL-2 inhibits Th17 differentiation. Moreover, we also reported a critical role of IL-2 in Th9 differentiation, with IL-2 inducing STAT5 binding to the Il9 promoter and IL-2 and IL-21 having opposing actions in Th9 differentiation based on induction of BCL6 by IL-21 but repression of BCL6 by IL-2. With Dr. K. Christopher Garcia (Stanford), we previously studied novel IL-2 variant partial agonists that function as "receptor signaling clamps”. One variant, H9-RETR, prolonged survival in a model of graft-versus-host disease and blocked proliferation of smoldering ATL cells. This indicated that this receptor-clamping approach was a robust and potentially general mechanism-based strategy. IL-2 REH was reported as a partial agonist that preferentially expanded Tregs, and we studied a partial agonist, H9T, that promoted a TCSM phenotype of CD8+ T cells, with enhanced efficacy in adoptive transfer treatment of B16 melanoma and in a second-generation CAR T system with efficacy for B acute lymphocytic leukemia. We discovered that STAT5 is a promoter of T cell exhaustion, and that H9T induces attenuated STAT5 activation, which results in a TSCM phenotype, and we elucidated underlying mechanisms. In the current year, we have studied other IL-2 partial agonists that we have generated in the lab as well as IL-21 mimics. IL-21 has broad actions on T- and B-cells, and we previously reported that it also induces apoptosis of conventional dendritic cells. ChIP-seq analysis had revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3, and we previously elucidated differential roles for STAT1 vs. STAT3 in IL-21 signaling in T cells. Previously, we also showed that IL-21 regulates expression of the Prdm1 gene (which encodes BLIMP1) via a response element that depends on STAT3 and IRF4, and we subsequently discovered that in contrast to its known ability to cooperate with PU.1 in B cells to act via Ets-IRF composite elements (EICEs), IRF4 cooperates with BATF/JUN family proteins to act via novel AP1-IRF composite elements (AICEs) in T cells, as well as in some B cells. In other studies, we had shown biological roles of Egr1 and Egr2 and elucidated non-immunological roles for Egr1, demonstrating that this transcription factor has a genetic-background dependent effect on eyelid development (specifically, it is required for eyelid development on the BALB/c but not C57BL/6 background). In collaborative studies, we have also contributed to studies on the role of EGR family proteins in the homing and pathogenicity of Th17 cells in the CNS (Lazarevic lab) and on key roles of EGR proteins in the CNS (Salzer lab). Importantly, this latter study revealed that Schwann cells activated clathrin-mediated endocytosis in axons, resulting in the clearance of cell adhesion molecules, which then promotes myelination. EGR2 is required for progression from the promyelinating to the myelinating stage. Previously, we studied the biological significance of STAT5 tetramerization in vivo by generating mice expressing mutant forms of STAT5A and STAT5B that can form STAT5 dimers but not tetramers. We also previously reported modeling of the 3-dimensional structure of the STAT5 tetramer and also demonstrated that STAT5 tetramers are critical for the survival of NK cells and have a role in the regulation of monocyte differentiation. Moreover, we reported that tyrosine phosphorylation of both STAT5A and STAT5B is critical for IL-2-mediated gene expression and proliferation, but that there is a greater role for STAT5B. We also previously contributed to studies of an IL-4 mimetic that specifically acted via type 1 IL-4 receptors (Spangler and Baker labs). We also previously contributed to a collaborative study an efficient new approach, ChIATAC, for the multiomics mapping of 3D epigenomes and reported a new user-friendly R Shiny application for RNA-sequencing analysis and biomarker discovery. We also previously globally characterized super-enhancers regulated by IL-2-activated STAT5 and IL-21-activated STAT3 and their relationship to highly inducible genes and had found that the Il2ra gene contains the most highly ranked STAT5-dependent super enhancer. Using CRISPR-Cas9 technology, we previously functionally dissected the elements of the Il2ra super-enhancer, providing key new insights into the molecular regulation of the Il2ra in particular and super-enhancers in general, and we subsequently significantly extended these studies, clarifying the differential importance of different super-enhancer elements in different cell types. We also showed that different parts of the Il2ra super-enhancer are functionally distinct, with the intronic region required for normal CD25 expression in effector T cells, and the upstream region critical for expression of CD25 on DN2/DN3 thymocytes as well as thymic and peripheral Treg cells. Strikingly, deletion of the upstream region resulted in autoimmune alopecia. Overall, these studies underscore the compartmentalization of different functions within different regions of the super-enhancer and indicate ways of affecting T effector versus Treg differentiation. Moreover, in the current period, we have actively further extended studies of the IL2Ra super-enhancer. We also have previously contributed to a study reporting that immunomodulatory imide drugs can degrade IKZF1 and IKZF3 binding to super-enhancers, resulting in the downregulation of MYC and IRF4 and the enhanced control and killing of multiple myeloma. We also demonstrated that clusters of TCR-regulated genes colocalized with TAD rearrangements, linking changes in chromatin structure to function. Moreover, in the current period, we reported a new pipeline, denoted SPICE (for Spacing Preference Identification of Composite Elements) that can systematically predict binding partners for transcription factors. Using this pipeline, we not only de novo “re-discovered” known composite elements/binding partners, but we discovered a novel JUN-IKZF1 composite element at the CNS9 element in the human IL10 gene. This approach thus not only extended specific information but can be used in new discovery-based investigation. In the current period, we also performed a genomic-wide CRISPR-knockout screen in IL-2-dependent cells derived from a patient with HTLV-I-induced adult T-cell leukemia (ATL). We discovered and reported that BLIMP1 is a novel negative regulator of IL-2 signaling. BLIMP1 was previously known to inhibit the production of IL-2 but the effect on signaling was unknown. Besides acting in primary mouse and human cells, BLIMP1 also negatively regulates IL-2 signaling in cells from patients with adult T cell leukemia. Interestingly, by ChIP-seq, BLIMP1 binds not only to the IL2 locus but also to the loci for IL2RA and IL10, providing additional insights into its mechanisms of regulating gene expression, with BLIMP1 potentially either directly negatively regulating (e.g., IL2RA) or positively regulating (e.g., IL10), depending on the gene. We therefore have now identified BLIMP1 as a negative regulator of IL-2 signaling in normal cells and in disease-related situations and indicated possible therapeutic potential in manipulating this critical factor. We also participated in collaborative studies with Weiping Zou in the field of immune checkpoint blockade (ICB), which has transformed cancer therapy. In general, dendritic cells (DCs) present tumor antigens, allowing T cell priming and activation. It was found that there is an interplay between STAT3- and STAT5-dependent transcriptional pathways in DCs, where STAT3 limits the STAT5 pathway, thereby rewiring DC function. Activated STAT3 is typically found in the tumor microenvironment; by degrading STAT3 with PROTACS (proteolysis-target chimeras), the DCs better supported immunogenicity, and degrading STAT3 also promoted antitumor activity, including in ICB-resistant tumors. Accordingly, the interplay between STAT5 and STAT3 transcriptional pathways influences the DC phenotype, including in the tumor microenvironment, with promise for cancer immunotherapy. It is noteworthy that these observations echo some of the cross-talk we previously demonstrated related to the actions of GM-CSF and IL-21 on DCs in the context of apoptosis, where ChIP-seq analysis had revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3 binding in these cells. Overall, our broad-ranging studies have markedly enhanced our understanding of mechanisms by which gc family cytokines regulate gene expression and biological processes. These are relevant to normal and pathological immune cell function, and our findings have clear therapeutic implications as discussed above.