IL-2 Family Cytokines and their Receptors-- Biology of the IL-2 system

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The IL-2 receptor and related cytokine/cytokine receptor systems are being studied to understand the T cell immune response in normal and pathologic states. After T-cell activation, the magnitude and duration of the response is controlled in part 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 3 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 study the signals induced by these cytokines, particularly STAT proteins and the mechanisms by which they regulate target genes. Our prior data that Stat5a or Stat5b transgenic mice develop tumors are consistent with STAT5 playing a role in malignant transformation and STAT5 is elevated in a range of human tumors. Moreover, humans and mice with altered STAT protein expression or activation have immunological defects. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation important for host defense to viruses/intracelllular pathogens, Th2 differentiation vital in allergic disorders/helminths, and Th17 differentiation vital in inflammatory disorders, including psoriasis and inflammatory bowel disease. We previously showed that IL-2 is important for Th2 differentiation and that IL-2 induces IL-4R expression in a STAT5-dependent manner and controls priming of cells for Th2 differentiation. Moreover, using genome-wide chromatin immunoprecipitation coupled to DNA sequencing (ChIP-Seq) analysis, we previously found broad regulation of Th2 differentiation via STAT5A and STAT5B and extended these findings by showing that IL-2 via STAT5 induces IL-12Rb2, which is critical for Th1 differentiation. We also showed that IL-2 via STAT5 regulates T-bet. Interestingly, IL-2 also inhibits expression of IL-6Ra and gp130, helping to explain the inhibition of Th17 differentiation. We also previously reported ed a key role of IL-2 in Th9 differentiation, with IL-2 inducing STAT5 binding to the Il9 promoter, and that IL-2 and IL-21 had opposing actions in Th9 differentiation, with BCL6 induction by IL-21 but repression by IL-2. In the current year, we have studied the role of new molecules, identified by a computational genomics approach, in Th differentiation, analyzing in vitro differentiated Th1 cells from 16 inbred mouse strains. Haplotype-based computational genetic analysis implicated the p53 family protein, p73, in Th1 differentiation. In vitro, p73 negatively regulates IFN production. p73 binds within, or upstream of, and modulates the expression of Th1 differentiation-related genes Ifng and Il12rb2. Furthermore, in mouse experimental autoimmune encephalitis, p73-deficient mice had increased IFN production and less disease severity, whereas in adoptive transfer inflammatory bowel disease, transfer of p73-deficient nave CD4+ T cells increases Th1 responses and augments disease severity. We thus identified p73 as a negative regulator of the Th1 immune response, suggesting that p73 dysregulation may contribute to susceptibility to autoimmune disease. We previously collaborated with Dr. K. Christopher Garcia (Stanford), generating novel IL-2 variants, which represent the first partial agonists for a type 1 cytokine. These next-generation IL-2 variants function as "receptor signaling clamps," retaining high affinity for IL-2Rb but having weaker interaction with gc, thus attenuating IL-2Rb/gc heterodimerization. We previously showed that a variant, H9-RETR, prolonged survival in graft-versus-host disease and blocked proliferation of smoldering adult T cell leukemia (ATL) T cells. During the past year, we continued our study of these molecules and additionally have studied a new IL-2 partial agonist. IL-21 has broad actions on T- and B-cells, and we previously reported that it induces apoptosis of conventional dendritic cells via STAT3 and Bim, and that this is inhibited by GM-CSF. ChIP-Seq analysis had revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3, and we had elucidated roles for STAT1 vs. STAT3 in IL-21 signaling in T cells. We had also demonstrated that IL-21 regulates expression of the Prdm1 gene (encoding BLIMP1) via a response element that depends on STAT3 and IRF4 and found that in contrast to its known ability to cooperate with PU.1 in B cells to act via Ets-IRF composite elements, IRF4 cooperates with BATF/JUN family proteins to act via novel AP1-IRF composite elements (AICEs) in T cells, as well as in B cells. In the previous year, we extended our studies with H.C. Morse, reporting that the transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses. We also had reported a role for IL-21 in neutrophil biology and shown its cooperative interplay with type I interferon in regulating the innate immune response to S. aureus. In the current year, we elucidated mechanisms underlying actions of IL-2 versus IL-21, reporting that they dichotomously shape CD8+ T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (TSCM) and antitumor responses. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1. Deletion of Ldha prevented development of cells with antitumor effector function, but transient LDH inhibition enhanced the generation of memory cells that could trigger robust antitumor responses after adoptive transfer. LDH inhibition caused major transcriptomic changes, and LDH inhibition combined with IL-21 increased the formation of TSCM cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy. Previously, we studied the biological significance of STAT5 tetramerization in vivo by generating mice expressing mutant forms of STAT5A and STAT5B that could form dimers but not tetramers, and we also previously reported a critical role for STAT5 tetramers for the survival of NK cells. In the current year, we now have extended our studies of tetramers within other cell types as well. 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 ChIA-PET methodology, we had defined long-distance chromatin interactions and used CRISPR-Cas9 technology to functionally dissect elements of this super-enhancer, providing new insights into the molecular regulation of the Il2ra in particular and super-enhancers in general. In the current year, we have significantly extended these studies. Overall, these studies enhance our understanding of the mechanism by which gc family cytokines regulate gene expression/biological processes and are relevant to normal and pathological immune function.