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

National Heart, Lung, And Blood Institute

Investigators

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Abstract

The IL-2 receptor and related cytokine/cytokine receptor systems are 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 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 induced by IL-2. 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 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 it induces IL-4R expression in a STAT5-dependent manner to prime cells for Th2 differentiation. Moreover, using genome-wide chromatin immunoprecipitation coupled to DNA sequencing (ChIP-Seq) analysis, we previously showed Th2 differentiation is regulated via STAT5A and STAT5B and that IL-2 via STAT5 induces IL-12Rb2, which is critical for Th1 differentiation and 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 previously showed 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. We also more recently 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, and we found that p73 negatively regulates IFN production and binds within or upstream of the Th1 differentiation-related genes Ifng and Il12rb2. 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 naive CD4+ T cells increased Th1 responses and disease severity. We thus had identified p73 as a negative regulator of the Th1 immune response, suggesting that p73 dysregulation contributes to autoimmune disease. In the past year, we have been working on another regulator of Th differentiation found by this approach. We previously collaborated with Dr. K. Christopher Garcia (Stanford) to study novel IL-2 variants as the first partial agonists for a type 1 cytokine. These IL-2 variants functioned as receptor signaling clamps, retaining high affinity for IL-2Rb but having weaker interaction with gc, thus attenuating IL-2Rb/gc heterodimerization. One 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 another engineered IL-2 partial agonist that we demonstrated promotes CD8 T cell stemness, clarified the basis for TSCM maintenance, and demonstrated that this partial agonist enhanced anti- tumor efficacy in adoptive transfer elimination of B16 melanoma and a second generation CAR-T model, underscoring the power of protein engineering to generate molecules with new activity and translational potential. We also collaborated with Garcia's lab, demonstrating that yet another IL-2 partial agonist promoted regulatory T cell (Treg) function and that Treg cells could be expanded using an orthogonal IL-2/IL-2 receptor system to facilitate transplantation tolerance. In the current year, we also reported that cytokines IL-21 and IL-15 could cooperatively enhance the cytolytic activity of NK cell-derived exosomes. We previously reported that IL-21 induces apoptosis of conventional dendritic cells via STAT3 and Bim. ChIP-Seq analysis had revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3, and we 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 binding STAT3 and IRF4 and that IRF4 cooperates with BATF/JUN family proteins to act via AP1-IRF composite elements (AICEs) in T cells and some B cells. We also had extended studies with H.C. Morse, showing that IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses. Moreover, we had elucidated differences in IL-2 versus IL-21, showing 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 had potent antitumor activity after adoptive transfer. Interestingly, LDH inhibition combined with IL-21 increased the formation of TSCM cells, with greater antitumor responses and host survival, indicating a key role for LDH in modulating T cell differentiation and elucidating differences between IL-2 and IL-21. Previously, we studied the role of STAT5 tetramerization in vivo by generating mice with mutant forms of STAT5A and STAT5B that can form dimers but not tetramers and showed STAT5 tetramers are critical for T cell expansion and NK survival. We are extending our studies to 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 gene in particular and super-enhancers in general. In the current year, we have significantly extended these studies of Il2ra super enhancer function. We also contributed to a study of a patient with XSCID with reversion of the mutation in T but not in NK cells, with HPV infection. Overall, these studies elucidate the mechanisms by which gc family cytokines regulate gene expression/biological processes, with relevance to normal and pathological immune function.