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 pathologic 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, the cause of 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 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 being oncogenic, and STAT5 is elevated in a range of human tumors. Moreover, humans and mice with altered STAT expression/activation have immunological defects. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation important for host defense to viruses/intracellular 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 promotes Th2 differentiation and that it induces IL-4R expression in a STAT5-dependent manner to prime cells for Th2 differentiation. Using 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/STAT5 regulates T-bet. IL-2 also inhibits expression of IL-6Ra and gp130, helping to explain the 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. We 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 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 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 worked on another regulator Th differentiation found by this approach. We previously collaborated with Dr. K. Christopher Garcia 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," that retained high affinity for IL-2Rb but had weaker interaction with gc, thus attenuating IL-2Rb/gc dimerization. Variant H9-RETR prolonged survival in graft-versus-host disease and blocked proliferation of smoldering adult T cell leukemia (ATL) T cells. We then 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. In a collaboration, Garcia's lab demonstrated 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, and we also had 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 DCs 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 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, 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. In a collaboration this year, we reported that STAT5 tetramers drive autoimmune-mediated neuro-inflammation and 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 extended these studies. We also contributed to a study showing that synthetic IL-9 receptor signaling endows T cells with a combination of stem cell memory and effector antitumor activity and to a study showing how IL-2 synergizes with PD-1 directed immunotherapy during chronic viral infection. In the current year, we contributed to studies showing that IFNg regulates NAD+ metabolism to promote the respiratory burst in human monocytes. In the previous year, we helped in a study of a patient with XSCID with reversion of the mutation in T but not in NK cells, with HPV infection, and this year, we co-reported a SCID patient with a T-