The IL-2 receptor and related cytokine receptor systems are being studied to clarify the T cell immune response in normal, neoplastic, and immunodeficient states. Following T-cell activation by antigen, the magnitude and duration of the T-cell immune response is determined by the amount of 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, and our lab discovered IL-2Rb in 1986, and 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 an autosomal recessive form of SCID indistinguishable from XSCID and in 1998 that T-B+NK+ SCID results from mutations in the IL7R gene. Prior work in our lab and others together showed that gc is shared by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. We also previously characterized genes that were induced or repressed by IL-2, IL-4, IL-7, and IL-15. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation being important for host defense to viruses and other intracellular pathogens, Th2 differentiation being vital in allergic disorders and related to helminths, and Th17 differentiation being vital in a range of inflammatory disorders, including psoriasis and inflammatory bowel disease. We previously showed that IL-2 is important for Th2 differentiation and that IL-2 regulates expression of the IL-4 receptor in a STAT5-dependent manner and critically controls priming of cells for Th2 differentiation. Moreover, using genome-wide ChIP-Seq (chromatin immunoprecipitation coupled to DNA sequencing) analysis, we had discovered broad regulation of Th2 differentiation via STAT5A and STAT5B and that IL-2-mediated IL-4Ra induction was critical in priming cells for Th2 differentiation. We then had extended these findings by showing that IL-2 via STAT5 induces expression of IL-12Rb1 and IL-12Rb2 and that the induction of IL-12Rb2 is critical for Th1 differentiation. We also previously showed that IL-2 via STAT5 also regulates the T box protein, T-bet, and that in contrast to the induction of IL-12R proteins, IL-2 inhibits expression of IL-6Ra and gp130, helping to explain the inhibition of Th17 differentiation. Consistent with the ability of Tbx21 to inhibit Th17 differentiation, expression of Tbx21 in Th17 cells resulted in increased IFNg but decreased expression of IL-17A. These results indicated a very broad effect of IL-2 via STAT5 on T helper cell differentiation. We also previously demonstrated a direct effect of IL-2 on Th9 differentiation via and showed opposing actions of IL-2 and IL-21 in Th9 differentiation based on their differential regulation of BCL6, which is induced by IL-21 but repressed by IL-2. We previously collaborated with Dr. K. Christopher Garcia (Stanford) on a project in which the three dimensional structure of IL-2 complexed to its receptor was compared to that of IL-15 bound to its receptor. These studies provided key mechanistic and structural insights into the functional differences between IL-2 and IL-15. We more recently markedly extended this earlier collaboration, studying the actions of wild type IL-2 versus novel IL-2 variants that represent the first partial agonists for a type 1 cytokine. IL-2 is of considerable therapeutic interest, but harnessing its actions in a controllable manner remains a challenge. We had modified an IL-2 superkine with enhanced affinity for IL-2R in order to generate next-generation IL-2 variants that function as receptor signaling clamps. that retain high affinity for IL-2R, inhibiting binding of endogenous IL-2, but their interaction with c was weakened, attenuating IL-2R-c heterodimerization. These IL-2 analogs acted as partial agonists and differentially affected lymphocytes poised at distinct activation thresholds. Moreover, we previously showed that one variant, H9-RETR, antagonized IL-2 and IL-15 better than blocking antibodies against IL-2R or IL-2R. Furthermore, this mutein prolonged 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 for engineering cytokine partial agonists for therapeutic immunomodulation, with applications to other type 1 cytokines as well. In the current year, we have extended our initial studies with H9-RETR in collaboration with Tom Waldmann and studied other novel IL-2 variants with distinctive properties. Previously, we studied the biological significance of STAT5 tetramerization in vivo by generating mice expressing mutant forms of STAT5A and STAT5B that could form dimers but not tetramers. This past year we reported intricate modeling of the 3-dimensional structure of the tetramer, providing new insights into its modeling. We also substantially extended our analysis of the basis of defective NK cell development in the double knockin mice and have studied effects related to both IL-2 and IL-15. Moreover, we generated mice with other mutations in the Stat5a and Stat5b genes and also have furthered study STAT biology using next generation sequencing approaches. With Axel Kallies, we identified a program of CD8 T effector cell differentiation that is regulated by IL-2 and IL-12 signaling and the combined activities of the transcriptional regulators Blimp-1 and T-bet. Moreover, we have generated a range on new data on the signaling by IL2 and IL-21. Finally, we have the long-term goal of identifying new causes of inherited human immunodeficiency that are based on defects in pathways that relate to the gc family of cytokines. Overall, these studies help to improve our understanding of signaling by gc family cytokines. These findings clarify basic molecular mechanisms that are relevant to normal and pathological immune cell function, including in allergy, autoimmunity, and cancer.