The IL-2 receptor and related cytokine/cytokine receptor systems are being studied to understand the T cell immune response in normal and neoplastic cells. After T-cell activation, the magnitude and duration of the response is controlled by IL-2, levels of IL-2 receptors, and the time course of their induction. IL-2Ra expression is very high in 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 being regulated at the level of transcription. gc is shared by the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, and is mutated in XSCID. We have focused on the signals induced by these cytokines, particularly activation of STAT proteins and the mechanisms by which they regulate target genes. Given our prior data that STAT5A or STAT5B transgenic mice develop tumors, consistent with STAT5 being implicated in malignant transformation and elevated in a range of human tumors, this is an important area for both normal and pathological states. Moreover, humans and mice with defective STAT protein expression have immunological defects. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation important for host defense to viruses and other 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 IL-2 induces IL-4 receptor expression in a STAT5-dependent manner and controls priming of cells for Th2 differentiation. Moreover, using genome-wide chromatin immunoprecipitation coupled to DNA sequencing (ChIP-Seq) analysis, we previously found broad regulation of Th2 differentiation via STAT5A and STAT5B. We previously extended these findings by showing that IL-2 via STAT5 induces IL-12Rb2, which is critical for Th1 differentiation. We also showed that IL-2 via STAT5 also regulates T-bet. Interestingly, IL-2 also inhibits expression of IL-6Ra and gp130, helping to explain the inhibition of Th17 differentiation. We also had reported a critical role of IL-2 in Th9 differentiation, with a direct effect of IL-2 on Th9 differentiation via its induction of STAT5 binding to the Il9 promoter and that IL-2 and IL-21 had opposing actions in Th9 differentiation based on induction of BCL6 by IL-21 but repression by IL-2. We also have continued studies of STAT5 tetramerization in vivo. We previously collaborated with Dr. K. Christopher Garcia at Stanford, comparing IL-2 and IL-15 receptor structures, providing mechanistic and structural insights into the functional differences between IL-2 and IL-15. During the current year, we extended this earlier collaboration, studying the actions of wild type IL-2 versus novel IL-2 variants, which represent the first partial agonists for a type 1 cytokine. Importantly, IL-2 regulates lymphocyte function by signaling through heterodimerization of the IL-2Rb and gc receptor subunits. IL-2 is of considerable therapeutic interest, but harnessing its actions in a controllable manner is challenging. We modified an IL-2 superkine with enhanced affinity for IL-2Rb; in order to generate next-generation IL-2 variants that function as receptor signaling clamps. They retained high affinity for IL-2Rb, inhibiting binding of endogenous IL-2, but their interaction with gc was weakened, attenuating IL-2Rb/gc heterodimerization. These IL-2 analogs acted as partial agonists and differentially affected lymphocytes at distinct activation thresholds with different potency for STAT activation. One variant, H9-RETR could prolong survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy with applications to other type 1 cytokines as well. We also unexpectedly found that EGR2 promotes peripheral nave T-cell proliferation and differentiation, with less T-cell receptor-induced IL-2 production in Egr2-deficient nave T cells and diminished cytokine production in T-helper differentiated cells. Previously, we demonstrated that IL-21 regulated expression of the Prdm1 gene that encodes BLIMP1 via a response element that depends on STAT3 and IRF4 and subsequently discovered that in contrast to its known ability to cooperate with PU.1 in B cells to act via Ets-IRF composite elements (EICEs), IRF4 cooperates with BATF/JUN family proteins to act via novel AP1-IRF composite elements (AICEs) in T cells, as well as in B cells. We demonstrated cooperative regulation of important genes via these AICEs and cooperative binding of IRF4, BATF, and JUN family proteins, with markedly diminished IRF4 binding in Batf-deficient cells and markedly diminished BATF binding in Irf4-deficient cells. We have continued studies of AICEs and IRF4/BATF/JUN/STAT3 complexes. IL-21 has broad actions on T- and B-cells, but its actions in innate immunity are poorly understood. We previously reported that IL-21 induced apoptosis of conventional dendritic cells (cDCs) occurs via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. This past year, we also elucidated roles for STAT1 vs. STAT3 in IL-21 signaling in T cells. Overall, the above findings enhance our understanding of mechanisms by which gc family cytokines regulate gene expression and biological processes. In addition, they may have implications related to the treatment of cancer, autoimmune, and other diseases. Key parts of the genome including microRNAs and noncoding RNAs went unnoticed for years. Despite extensive annotation/assembly of the human genome, RNA-sequencing continues to yield millions of unmappable reads. We examined >300 billion reads from 536 normal donors and 1,873 patients spanning 21 cancer types, identified 300 million uncharacterized reads, and used a new approach to assemble 2,550 novel human transcripts, which mainly are long noncoding RNAs; 230 had relatively specific expression or non-expression in certain cancer types, whereas 183 exhibited tissue specificity. Moreover, lentiviral-mediated expression of 3 select transcripts had higher expression in normal than in cancer patients and inhibited the growth of HepG2 cells. Our analysis provides a comprehensive resource of unmapped human transcripts and reveals associations with specific cancers, providing new genes for potential therapeutic targeting. Viruses are causally associated with a number of human malignancies. We sought to identify new virus-cancer associations by searching RNA-seq data from >2,000 patients, spanning 21 cancers from The Cancer Genome Atlas (TCGA). As expected, we found human papillomavirus 16 (HPV16) and HPV18 in oropharyngeal cancer and hepatitis B and C viruses in liver cancer. Unexpectedly, we found HPV38, a cutaneous form of HPV in 32 of 168 samples from endometrial cancer. In 12 of the HPV38+ samples, there was at least one paired read mapping to both human and HPV38 genomes, indicating viral integration. Expression of HPV38 transcripts were relatively low, and all 32 HPV38+ samples were in the same batch of 40 samples, whereas none of the other 128 endometrial carcinoma samples were HPV38+, raising doubts about the validity of the HPV38 association. The HPV38+ samples contained the same 10 novel single nucleotide variations (SNVs), leading us to hypothesize that one patient was infected with this new isolate of HPV38, which was integrated into his/her genome and may have cross-contaminated other TCGA samples, indicating caution is needed in analyzing TCGA samples.

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24
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2015
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U.S. National Heart Lung and Blood Inst
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Lin, Jian-Xin; Leonard, Warren J (2018) The Common Cytokine Receptor ? Chain Family of Cytokines. Cold Spring Harb Perspect Biol 10:
Spolski, Rosanne; Li, Peng; Leonard, Warren J (2018) Biology and regulation of IL-2: from molecular mechanisms to human therapy. Nat Rev Immunol :
Mitra, Suman; Leonard, Warren J (2018) Biology of IL-2 and its therapeutic modulation: Mechanisms and strategies. J Leukoc Biol 103:643-655
Shin, Hyun Mu; Kapoor, Varun N; Kim, Gwanghun et al. (2017) Transient expression of ZBTB32 in anti-viral CD8+ T cells limits the magnitude of the effector response and the generation of memory. PLoS Pathog 13:e1006544
Oh, Jangsuk; Wang, Yujuan; Chen, Shida et al. (2017) Genetic background-dependent role of Egr1 for eyelid development. Proc Natl Acad Sci U S A 114:E7131-E7139
Spolski, Rosanne; Gromer, Daniel; Leonard, Warren J (2017) The ? c family of cytokines: fine-tuning signals from IL-2 and IL-21 in the regulation of the immune response. F1000Res 6:1872
Li, Peng; Mitra, Suman; Spolski, Rosanne et al. (2017) STAT5-mediated chromatin interactions in superenhancers activate IL-2 highly inducible genes: Functional dissection of the Il2ra gene locus. Proc Natl Acad Sci U S A 114:12111-12119
Lin, Jian-Xin; Du, Ning; Li, Peng et al. (2017) Critical functions for STAT5 tetramers in the maturation and survival of natural killer cells. Nat Commun 8:1320
Xin, Annie; Masson, Frederick; Liao, Yang et al. (2016) A molecular threshold for effector CD8(+) T cell differentiation controlled by transcription factors Blimp-1 and T-bet. Nat Immunol 17:422-32
Sukumar, Madhusudhanan; Liu, Jie; Mehta, Gautam U et al. (2016) Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy. Cell Metab 23:63-76

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