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/receptor systems are studied to understand the T cell immune response in normal and disease states. 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 is highly expressed by cells infected with HTLV-I, which causes adult T cell leukemia (ATL). The receptor has 3 chains: IL-2Ra, IL-2Rb, and gc. IL-2Ra and IL-2Rb are induced by IL-2. gc is shared by 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 Stat5 transgenic mice develop tumors are consistent with STAT5 being oncogenic, and STAT5 is elevated in many human tumors. Altered STAT5 expression/activation results in immunological defects. T helper differentiation is critical for normal immune responses: Th1 differentiation key for host defense to viruses/intracellular pathogens, Th2 differentiation vital in allergic disorders/helminths, and Th17 differentiation vital in inflammatory disorders. We previously showed that IL-2 promotes Th2 differentiation, inducing IL4R expression in a STAT5-dependent manner to prime cells for Th2 differentiation. Using ChIP-Seq analysis, we had shown 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/STAT5 regulates T-bet. IL-2 also inhibits IL-6Ra and gp130 expression, helping to explain its inhibition of Th17 differentiation. We previously showed a role for 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 IL-21 inducing BLIMP1 and IL-2 repressing BLIMP1. Subsequently, we 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 showed p73 negatively regulates IFN production and binds in 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 severe disease, whereas in adoptive transfer inflammatory bowel disease, transfer of p73-deficient naive CD4+ T cells increased Th1 responses and disease severity. We thus showed p73 negatively regulates the Th1 immune response, suggesting that p73 dysregulation contributes to autoimmune disease. In the past year, we have expanded our study of the role of p73 on Th cells. We previously with Dr. K. Christopher Garcia studied novel IL-2 variants as the first partial agonists for a type 1 cytokine. These IL-2 variants functioned as "receptor signaling clamps," that retained high affinity for IL-2Rb but had weakened interaction with gc, attenuating IL-2Rb/gc dimerization. A variant, H9-RETR, prolonged survival in graft-versus-host disease and blocked proliferation of smoldering ATL T cells. We also studied another engineered IL-2 partial agonist that we showed 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 2nd generation CAR-T model, underscoring the power of protein engineering to generate molecules with new activity and translational potential. In a collaboration, Garcia's lab demonstrated that 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, and we also had reported that cytokines IL-21 and IL-15 could cooperatively enhance the cytolytic activity of NK cell-derived exosomes. In the past year, we have pursued studies of other IL-2 partial agonists. We previously reported that IL-21 induces apoptosis of DCs. ChIP-Seq analysis had revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3, and we clarified roles for STAT1 vs. STAT3 in IL-21 signaling in T cells. We also showed that IL-21 regulated expression of Prdm1 encoding BLIMP1) via an element binding STAT3 and IRF4 and that IRF4 cooperates with BATF/JUN family proteins to act via AP1-IRF composite elements (AICEs) in T and some B cells. We later extended studies, 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 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, inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the TCA cycle and inhibiting terminal effector and exhaustion, with expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1. Deleting Ldha prevented antitumor effector function, but transient LDH inhibition enhanced the generation of memory cells with potent antitumor activity after adoptive transfer. 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 tetramers 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. Last year in a collaboration we reported that STAT5 tetramers drive autoimmune-mediated neuro-inflammation, and this year we reported roles for STAT5 tetramers in monocyte biology. 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. This year we significantly expanded our studies of the Il2ra super-enhancer with different biological outcomes depending on the part of the super-enhancer that was mutated/deleted. Using ChIA-PET methodology, we previously 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 substantially extended these studies using Hi-ChIP and moreover were part of a collaborative study describing a new approach termed ChIATAC. Moreover, this year we contributed to a published showing that IL-2 synergizes with PD-1 directed immunotherapy during chronic viral infection.