SUMMARY Long-term survival and anti-tumor immunity of adoptively-transferred CD8+ T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically-robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8+ T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8+, CD4+ T-cell subsets and long-term hematopoietic stem cells. This metabolism-based approach to cell-selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

Interleukin-15 (IL-15) and IL-2 possess distinct immunological functions despite both signaling through IL-2Rβ and the common cytokine receptor γ-chain, γc, We find that in the IL-15—IL-15Rα—IL-2Rβ—γc quaternary complex structure, IL-15 heterodimerizes IL-2Rβ and γc identically to the IL-2—IL-2Rα—IL-2Rβ—γc complex, despite differing receptor-binding chemistries. IL-15Rα dramatically increases the affinity of IL-15 for IL-2Rβ, and this allostery is required for IL-15 trans-signaling versus IL-2 cis-signaling. Consistent with the identical IL-2Rβ—γc dimer geometry, IL-2 and IL-15 exhibited similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induce similar signals, and the cytokine-specificity of IL-2Rα versus IL-15Rα determines cellular responsiveness. These results provide important new insights for specific development of IL-15-versus IL-2-based immunotherapeutics.

SUMMARY Interleukin-2 (IL-2) regulates lymphocyte function by signaling through heterodimerization of the IL-2Rβ and γc receptor subunits. IL-2 is of considerable therapeutic interest, but harnessing its actions in a controllable manner remains a challenge. Previously, we have engineered an IL-2 “superkine” with enhanced affinity for IL-2Rβ. Here, we describe next-generation IL-2 variants that function as “receptor signaling clamps.” They retained high-affinity for IL-2Rβ, inhibiting binding of endogenous IL-2, but their interaction with γc was weakened, attenuating IL-2Rβ-γc heterodimerization. These IL-2 analogues acted as partial agonists and differentially affected lymphocytes poised at distinct activation thresholds. Moreover, one variant, H9-RETR, antagonized IL-2 and IL-15 better than blocking antibodies against IL-2Rα or IL-2Rβ. 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 may be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation.

Interleukin 9 (IL-9) is a γ c -family cytokine that is highly produced by T-helper 9 (Th9) cells and regulates a range of immune responses, including allergic inflammation. Here we show that IL-2-JAK3-STAT5 signaling is required for Th9 differentiation, with critical STAT5 binding sites in the Il9 (the gene encoding IL-9) promoter. IL-2 also inhibited B cell lymphoma 6 (BCL6) expression, and overexpression of BCL6 impaired Th9 differentiation. In contrast, IL-21 induced BCL6 and diminished IL-9 expression in wild-type but not Bcl6 −/− cells, and Th9 differentiation was increased in Il21and Il21r −/− T cells. Interestingly, BCL6 bound in proximity to many STAT5 and STAT6 binding sites, including at the Il9 promoter. Moreover, there was increased BCL6 and decreased STAT binding at this site in cells treated with blocking antibodies to IL-2 and the IL-2 receptor, suggesting a possible BCL6-STAT5 binding competition that influences IL-9 production. BCL6 binding was also increased when cells were Th9-differentiated in the presence of IL-21. Thus, our data reveal not only direct IL-2 effects via STAT5 at the Il9 gene, but also opposing actions of IL-2 and IL-21 on BCL6 expression, with increased BCL6 expression inhibiting IL-9 production. These data suggest a model in which increasing BCL6 expression decreases efficient Th9 differentiation, indicating possible distinctive approaches for controlling this process.T cells can differentiate into an array of specialized T-helper populations, including Th1 cells, which mediate antiviral responses; Th2 cells, which mediate host defense to parasites and allergic inflammation; and Th17 cells, which are involved in inflammatory processes and diseases such as psoriasis and inflammatory bowel disease (1-5). Th9 cells are a population of cells differentiated in the presence of IL-4 and TGF-β to secrete IL-9 and mediate allergic inflammation and immunity to intestinal parasites (6-9). The IL-9 receptor consists of IL-9R and the common cytokine receptor γ chain, γ c , which is shared by the receptors for IL-2, IL-4, IL-7, IL-15, and IL-21 (10) and mutated in humans with X-linked severe combined immunodeficiency (11). IL-9R is broadly expressed, including on hematopoietic progenitors, mast cells, macrophages, dendritic cells, B cells, airway epithelial cells, immature neurons, eosinophils, natural killer T (NKT) cells, natural killer (NK) cells, Th9 cells, Th17 cells, and Treg cells (6-9, 12, 13). This distribution helps to explain diverse actions of IL-9. IL-9 increases CD4 + T-cell growth, IgE production by B cells, Treg function, Th17 differentiation, mast cell growth and survival, expression of FceR1α, production of IL-6 by mast cells, and the maturation of hematopoietic progenitor cells (8,9,13). IL-9 also induces the production of IL-8, IL-13, and eotaxin by airway smooth muscle cells and goblet cell metaplasia in airway epithelial cells (14). Recently, IL-9-producing cells have also been shown to exhibit robust antitumor immunity for melanoma (15,16).Like IL-9, IL-2 is a type ...