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. There are three forms of IL-2 receptors, binding IL-2 with high affinity (Kd ~ 10-11M, comprising IL-2Ra, IL-2Rb, and IL-2Rg), intermediate affinity (Kd ~ 10 -9M, comprising IL-2Rb and IL-2Rg), and low affinity (Kd ~10-8M, comprising IL-2Ra alone). High and intermediate affinity receptors are functional. High affinity receptors are expressed on activated T cells and other lymphocytes, on regulator T cells (Tregs) and ILC2s. Intermediate affinity receptors are on NK cells and CD8+ T cells. IL-2Ra is also highly expressed by cells infected with HTLV-I, the virus that causes adult T cell leukemia (ATL). We demonstrated that IL-2Rg is mutated in humans with X-linked SCID (XSCID) and is a common cytokine receptor gamma chain (gc) that we and others collectively showed is shared by the receptors for IL-4, IL-7, IL-9, IL-15, and IL-21. We also discovered JAK3-deficient human SCID and IL7R-deficient human SCID. We study the signals induced by gc family cytokines, including STAT proteins and mechanisms by which they regulate target genes. Our prior data that Stat5 transgenic mice develop tumors are consistent with the observations that STAT5 is elevated in many human tumors and is oncogenic. Our studies also indicated that inhibiting JAK3 would be immunosuppressive, providing the basis for the development of JAK3 inhibitors, with more than 10 JAK inhibitors approved for treatment of a range of human diseases. T helper differentiation is critical for normal immune responses, with Th1 cells being key for host defense to viruses/intracellular pathogens, Th2 cells being vital in allergic disorders/helminths, and Th17 cells being vital in inflammatory disorders. We previously showed that IL-2 primes for Th2 differentiation, in part by inducing IL4R expression in a STAT5-dependent manner. Using ChIP-Seq analysis, we had shown Th2 differentiation is regulated via STAT5A/STAT5B, 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 why IL-2 can inhibit Th17 differentiation. We previously showed that IL-2 promotes Th9 differentiation, with IL-2 inducing STAT5 binding to the Il9 promoter, and that IL-2 and IL-21 have opposing actions in Th9 differentiation, with IL-21 inducing and IL-2 repressing BLIMP1. We also investigated the role of new molecules in Th differentiation that we identified by a computational genomics approach, 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 IFNg production and binds at or upstream of the Th1 differentiation-related genes, Ifng and Il12rb2. In mouse experimental autoimmune encephalitis (EAE, a model of human multiple sclerosis), 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. Thus, p73 negatively regulates the Th1 immune response, suggesting that p73 dysregulation contributes to autoimmune disease. With Dr. K. Christopher Garcia (Stanford), we previously studied novel IL-2 variants on the “super-IL-2” background as the first partial agonists for a type 1 cytokine. These functioned as receptor signaling clamps that had super-high affinity for IL-2Rb but diminished 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 had studied another engineered IL-2 partial agonist, H9T, that we showed promotes CD8+ T cell stemness, clarified the basis for the maintenance of T-stem like memory cells (TSCM), and showed that H9T enhanced antitumor efficacy in adoptive transfer-mediated treatment of B16 melanoma and in a 2nd generation CAR-T model against B lymphocytic leukemia. Thus, we demonstrated that protein engineering could be used to generate molecules with new actions and translational potential, and we also contributed to a study of another IL-2 partial agonist that promoted regulatory T cell (Treg) function; moreover, Treg cells could be expanded using an orthogonal IL-2/IL-2R system to facilitate transplantation tolerance. We also previously reported that IL-21 and IL-15 cooperatively enhanced the cytolytic activity of NK cell-derived exosomes, and with Jamie Spangler, that an engineered IL-2-antibody fusion immunocytokine can preferentially expands T effector cells, with enhanced antitumor activity and synergy with checkpoint inhibitors, indicating the power of such immunocytokines. In the current year, we have continued our studies of H9T, with new evolving findings, and we have developed new IL-2 partial agonists with exciting actions for these molecules in ongoing work. We also previously elucidated key differences in the properties of 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 of BLIMP1 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. We also more recently demonstrated that IL-21 + LDHi treatment of CD8+ T cells results in potent expression of the LIM-domain-only protein (LMO4) and that it enhances T cell stemness and tumor rejection by enhancing IL-21-STAT3 signaling (see IL-21 report). A patent applicant was filed for using LMO4 as a new way of boosting antitumor activity. Previously, we also studied the role of STAT5 tetramers in vivo by generating mice with mutated STAT5A and STAT5B that form dimers but not tetramers and demonstrated that STAT5 tetramers are critical for T-cell proliferation, including during LCMV infection of mice, as well as for NK-cell survival, and that STAT5 tetramers also are important in promoting autoimmune-mediated neuro-inflammation as well as playing roles in monocyte biology. We also previously characterized super-enhancers regulated by IL-2 (via STAT5) and IL-21 (via STAT3) and studied their relationship to highly inducible genes. Interestingly, we identified that the Il2ra gene contains the most highly ranked STAT5-dependent super enhancer in both mice and humans and within both CD4+ and CD8+ T cells. We then expanded our studies of the Il2ra super-enhancer, used CRISPR-Cas9 technology to further functionally dissect elements of this super-enhancer, and showed that there are different biological outcomes depending on the part of the super-enhancer that is deleted, providing new insights into Il2ra gene regulation in particular and super-enhancers in general. We found provocative cell-type specific effects of deleting intronic vs. promoter regions, particularly related to differential effects on effector T cells and Treg cells. We also used Hi-ChIP to elucidate chromosomal looping and contributed to a new approach termed ChIATAC that extends DNA-looping of ChIA-PET. In the current period, we have continued our functional analysis of the IL2Ra super-enhancer (detailed in gene regulation report). In the past year, we also reported critical roles for tyrosine phosphorylation of STAT5A and STAT5B in vivo by using CRISPR-Cas9 to generate knockin mice that had tyrosine-to-phenylalanine mutations in these STAT5 proteins. These studies also revealed that STAT5A and STAT5B form heterodimers and elucidated new features of STAT5 biology. We found a more robust role for STAT5B then STAT5A, but in both knockins, CD8+ T cell numbers were greatly diminished, with markedly defective proliferation. This was due to reduced induction of MYC, pRB, cyclins, and CDKs, with a partial defect in the G1 to S phase transition. Moreover, the cells had defective IL-2-mediated phosphorylation of ERK and AKT. By coupling RNA-seq to mass spectrometry data, we also elucidated the molecular basis of IL-2-mediated proliferation of these cells. We also reported in the current period a genomic-wide CRISPR-knockout screen of IL-2-dependent cells that were derived from a patient with HTLV-I-induced adult T-cell leukemia (ATL). In the screen, among other “hits”, we identified a known negative regulator of IL-2 signaling (PTEN) as well as BLIMP1 (B lymphocyte-induced maturation protein1), which is encoded by the PRDM1 gene, as a novel negative regulator of IL-2 signaling. BLIMP1 was known to inhibit the production of IL-2 but the effect we identified on IL-2 signaling is new. We showed that deleting PRDM1 expression augmented IL-2 in T cells and regulatory T cells as well as during adoptive T cell transfer-induced colitis and in T cells from influenza-infected mice. Moreover, cells from patients with ATL had diminished expression of BLIMP1 and correspondingly had enhanced IL-2 signaling, where this signaling was diminished when BLIMP1 was overexpressed. We therefore have now identified BLIMP1 as a negative regulator of IL-2 signaling in normal cells and in disease-related situations, indicating possible therapeutic potential in manipulating this critical factor. Finally, related to our continued interest in immunodeficiency states, we contributed to a study that found that cutaneous squamous-cell carcinoma associated with β-HPV infection in an individual with mutations in the ZAP70 adapter (which is required for T-cell receptor (TCR) signaling) was resolved following restoration of TCR signaling by allogeneic hematopoietic-cell transplantation; this revealed a role of β-HPV in skin carcinogenesis in hosts with defective adaptive T-cell responses. Overall, our studies have significantly expanded our knowledge related to IL-2, with new insights into translational potential. Our findings range from the study of new cytokine partial agonists to the identification of new regulatory proteins and ways to control gene expression, with translational implications in cancer and autoimmune disease.