IL-2 Family Cytokines and their Receptors-- Biology of the IL-21 system

National Heart, Lung, And Blood Institute

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Abstract

IL-2 and related cytokine systems are studied to clarify the immune response in normal, neoplastic, immunodeficient, and other disease states. Following activation by antigen, the magnitude and duration of the T-cell immune response is determined by the IL-2 produced, levels of receptors expressed, and time course of each event. The IL-2 receptor contains three chains, IL-2Ra, IL-2Rb, and gc. Dr. Leonard cloned IL-2Ra in 1984, the lab co-discovered IL-2Rb in 1986 and then reported in 1993 that mutation of the gc chain results in X-linked severe combined immunodeficiency (XSCID, which has a T-B+NK- phenotype) in humans. We reported in 1995 that mutations of the gc-associated kinase, JAK3, result in autosomal recessive SCID that phenocopies XSCID and in 1998 that T-B+NK+ SCID results from mutations in the IL7R gene. Based on our work and that of others, gc was previously shown to be shared by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. We previously cloned IL-21R, generated IL-21 transgenic and Il21r knockout mice, elucidated mechanisms of IL-21 signaling, showed with Dan Littman that IL-21 promotes Th17 differentiation as part of a cytokine cascade, and showed that IL-21 regulates immunoglobulin production. We showed that IL-21 serves key roles in autoimmune disease, including in mouse models of lupus, type 1 diabetes, and experimental autoimmune uveitis, and indicated potential for IL-21 as an anti-tumor agent. We showed a role for IL-21 in the development of T follicular helper cells and that IL-21 signaling is required for CD8+ T cell survival and memory cell formation. We previously found that IL-21 promotes the pathogenic response to pneumonia virus of mice and that IL-21 induces IL-10 and with Tom Tedder that IL-21 could expand regulatory B cells (Breg cells) that produce IL-10 (B10 cells). Moreover, we demonstrated that IL-2 drives the terminal differentiation of B cells and is pro-apoptotic for B cells. This action may be analogous to that of IL-2 in the process of activation-induced cell death, with IL-21 potentially serving to prevent the accumulation of autoreactive B cells. We previously also 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 had shown 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; this is distinct from the ability of IRF4 to cooperate with PU.1 in B cells via ETS-IRF composite elements (EICEs). IL-21 broadly acts on T- and B-cells and has actions in innate immunity. Previously, we showed IL-21 induces apoptosis of conventional dendritic cells (cDCs) via STAT3 and BIM, and this was inhibited by GM-CSF. We also previously showed that IL-21 induces IL-1 production in cDCs via a STAT3-dependent but NF-kB-independent pathway. STAT3-dependent IL-1 expression in cDCs partially explains the IL-21-mediated pathologic response occurring during infection with PVM. We also previously showed that IL-1 signaling is critical for the development of autoimmune uveitis, a process we showed was dependent on IL-21. Mechanistically, IL-21 activates several STATs, particularly STAT3 and STAT1. STAT3 is the major STAT activated, but STAT1 is also important, and we had found that Tbx21 (encoding Tbet) and Ifng are differentially regulated by these STATs, with opposing actions of STAT1 and STAT3 on IFNg expression in CD4(+) T cells during chronic LCMV infection. Moreover, concordant actions of IL-21 related to IFNG and TBX21 expression was found using CD4+ T cells from patients with hyper-IgE syndrome, a disease caused STAT3 mutations, as well as in cells from STAT1 gain-of-function patients. Thus, STAT1 vs. STAT3 activation can fine-tune the actions of IL-21. We also previously found opposing roles for IL-21 and IL-2 in Th9 differentiation and contributed to studies showing that IL-21 with IFNg and IL-4 can govern TBET and CD11c expression in TLR-activated B cells, and that IL-21 signaling in B cells but not T cells is essential for the development of collagen-induced arthritis in mice. We previously also reported an interplay between IL-21 and type I IFN that regulates neutrophil-dependent responses to Staphylococcus aureus. Interestingly, in investigating the effect of cytokines on the cytolytic activity of exosomes derived from NK cells, we showed extracellular vesicles (EVs) that were derived from NK cells (NK-EVs), including exosomes, possessed cytotoxic capacity against tumor cells and found that EVs from human NK-92 cells stimulated with IL-15 + IL-21 had enhanced cytotoxic capacity and that granzymes B and H were induced by IL-15 + IL-21 stimulation in NK-EVs but were not required for the cytotoxic capacity. Overall, we elucidated novel properties of NK-EVs. We also previously contributed to a study showing that proliferation, together with increased apoptosis, causes T-cell lymphopenia in endogenous Cushings syndrome patients. Specifically, IL-21 was decreased in high-dose glucocorticoid environments, and adding IL-21 reversed glucocorticoid therapy-induced apoptosis. Thus, administering IL-21 in patients receiving long-term, high-dose glucocorticoid therapy might be beneficial. We also previously collaborated with Brian Annex’s lab to show that the loss of IL-21 receptor activation in hypoxic endothelial cells impaired perfusion recovery after hindlimb ischemia and showed there is increased endothelial IL-21 receptor expression in peripheral artery disease and furthermore with him reported that microRNA-30b is necessary and sufficient for IL-21-mediated angiogenesis in peripheral arterial disease. We previously showed that IL-2 and IL-21 differentially influence CD8+ T cell differentiation and that IL-2 drives terminal differentiation to cells poorly effective against tumors, but 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 more quiescent state. 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 NR4A family nuclear receptors, as well as PRDM1 and XBP1. Deleting Ldha prevented development of cells with antitumor effector function, but transient LDH inhibition (LDHi) increased memory cells that exhibited antitumor efficacy after adoptive transfer. Moreover, combining LDH inhibition with IL-21 increased TSCM cells, with greater antitumor responses and host survival, showing LDH modulates cytokine-mediated T cell differentiation, with translational potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy. We also discovered that treatment of CD8+ T cells with IL-21 and LDHi resulted in robust expression of the LIM-domain only protein, LMO4. Expression of LMO4 resulted in a TSCM like phenotype to CD8+ T cells and augmented antitumor immunity. A patent application was filed; LMO4 may be a way of effectively enhancing antitumor immunity. In the past year, we have also generated exciting work on IL-21 neokines/mimetics with David Baker, worked on the biological role of IL-21 in other cell types, and investigated mechanisms of IL-21-induced signaling and aspects of IL-21 biology. Related to IL-21/mimetics, in collaboration with David Baker as well as the laboratories of Michael and Stephanie Dougan, we have extensively studied these new IL-21 mimics. Previously, the Baker lab reported IL-2/IL-15 mimics that had an up-down-up-down configuration in contrast to the up-up-down-down four-alpha helical bundle configuration of native IL-2 and IL-15. The IL-2 mimic was more compact and heat stable, with greater antitumor efficacy. We have now studied an IL-21 mimic with a similar topological fold that exhibits greater stability and is a potent mimic of the activities of native IL-21 on both mouse and human cells. Moreover, it exhibits robust antitumor activity, for example in a melanoma model. A manuscript related to this molecule has been submitted. Overall, our studies have elucidated the biology/mechanisms of IL-21, expanding our knowledge of normal and pathological immune cell function, with relevance to autoimmunity and cancer, as well as to the basic control of T-cell and B-cell actions, with a range of potential therapeutic implications.