This article is available online at http://www.jlr.org usually determines isotope enrichment by measuring the derivatized forms of D0 and trideuterated leucine (D3-Leu) ( 2, 3 ), a method with high cost and low sensitivity and specifi city. Recently, proteomics-based triple quadrupole multiple reaction monitoring (MRM) permitted a more practical and highly specifi c multipeptide approach to in vivo kinetic studies ( 4, 5 ). However, MRM relies on low-resolution readouts (unit mass resolution) that do not readily permit precise quantifi cation of tracer enrichment that is lower than 1%, which is common in apolipoprotein kinetics ( 5, 6 ). Factors contributing to low precision include interference by not only the sister isotope 13C15N M3 ion but also background ions. In this study, we aim to extend further the scope of in vivo kinetics by exploiting the recently developed highresolution/accurate mass parallel reaction monitoring (HR/AM-PRM) method performed on the quadrupole Orbitrap mass spectrometer ( 7,8 ). The HR/AM fragment ion scan feature has the potential to measure D3-Leu enrichment between 0.03% and 1.0%, a low incorporation range that is a consequence of a bolus-administered tracer, useful in revealing tracer-tracee relationships. (Nagoya, Japan; M.A.) and the National Institutes of Health [ R01HL107550 (M.A.); UL1 RR 025758-01 ; and R01HL095964 (F.M.S.)]. Abstract Endogenous labeling with stable isotopes is used

Interleukin-21 (IL-21) has broad actions on T- and B-cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) 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. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive-transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon (IFN)-γ producing CD4+ T cells in Il21r−/−Rag2−/− mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2−/− mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.

IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4 + T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4 + T cells. RNA-Seq analysis of CD4 + T cells from Stat1-and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4 + T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4 + T cells from patients with autosomal dominant hyperIgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.) is a type I cytokine that signals via a receptor composed of IL-21R and the common cytokine receptor γ-chain, γ c (1). γ c is also shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 and is mutated in humans with X-linked severe combined immunodeficiency (XSCID), a disease characterized by the absence of T and natural killer (NK) cells and the presence of nonfunctional B cells (2). IL-21 is primarily produced by CD4 + T cells and natural killer T (NKT) cells, but it has pleiotropic actions on both adaptive and innate immune cells, including T, B, NK, NKT, and dendritic cells (1). In T cells, IL-21 can act as a comitogen and cooperates with IL-7 and IL-15 to expand CD8 + T cells (3), promotes Th17 differentiation (4-6), and induces BCL6 expression (7) to promote T follicular helper cell development (8, 9). In B cells, IL-21 promotes plasma cell differentiation (10, 11), and in combination with IL-4, drives IgG1 and IgG3 class switch (11,12). Defective signaling by IL-21 appears to substantially explain the B-cell defect observed in patients with XSCID (11, 13). Furthermore, IL-21 can enhance the cytotoxic activity of NK and NKT cells (1) and induce the apoptosis of conventional dendritic cells (14).IL-21 activates multiple signaling pathways, including the JAK-STAT, PI 3-kinase (PI3K), and MAPK pathways (15). Of these, the JAK-STAT pathway has been most extensively studied. IL-21 induces phosphorylation of JAK1 and JAK3, which in turn leads to phosphorylation and nuclear translocation of STAT3, which then binds to IFN-γ-activated sequence (GAS) motifs and modulates expression of IL-21-responsive genes. IL-21 also activates STAT1, but the function of IL-21-activated STAT1 is largely unknown, although IL-21 was suggested to use STAT1 to promote CD8 + T-cell cytotoxicity and ap...