Th1 and Th17 cells are distinct lineages of effector/memory cells, imprinted for reexpression of , by upregulated expression of T-bet and retinoic acid-related orphan receptor ct (RORct) , respectively. Apparently, Th1 and Th17 cells share tasks in the control of inflammatory immune responses. Th cells coexpressing IFN-c and IL-17 have been observed in vivo, but it remained elusive, how these cells had been generated and whether they represent a distinct lineage of Th differentiation. It has been shown that ex vivo isolated Th1 and Th17 cells are not interconvertable by TGF-b/IL-6 and IL-12, respectively. Here, we show that ex vivo isolated Th17 cells can be converted into Th1/Th17 cells by combined IFN-c and IL-12 signaling. IFN-c is required to upregulate expression of the IL-12Rb2 chain, and IL-12 for Th1 polarization. These Th1/Th17 cells stably coexpress RORct and T-bet at the single-cell level. Our results suggest a molecular pathway for the generation of Th1/Th17 cells in vivo, which combine the pro-inflammatory potential of Th1 and Th17 cells. IntroductionTh1 cells, with a memory for IFN-g expression and determined by the master transcription factor T-bet, are considered to be essential for protection against intracellular pathogens, and had been viewed as the major pathogenic drivers of chronic autoimmune inflammation, e.g. EAE [1][2][3], uveitis [4] or colitis [5]. Recently, Th17 cells, with a memory for expression of IL-17 and determined by the transcription factor retinoic acid related orphan receptor gt (RORgt), have been identified as another pathogenic Th-cell lineage driving pathogenesis in these autoimmune models [6,7]. Th17 cells contribute to inflammation through the recruitment of neutrophils and the induction of secretion of pro-inflammatory mediators such as IL-6, IL-8, TNF-a, IL-1b, CXCL1, CXCL10 and matrix metalloproteinases from tissue cells (reviewed in [8]). Th1 cells contribute to inflammation by activation of macrophages [9]. The concerted action of IFN-g and IL-17 has been shown to be essential in the effective induction and maintenance of autoimmunity [10,11], e.g. Th1 cells being required for the recruitment of Th17 cells into the central nervous system in EAE.In inflamed tissue of autoimmune patients, Th cells coexpressing IFN-g and IL-17 have been identified [12][13][14].Ã These authors have contributed equally to this study. ResultsIn vivo, Th17 cells do not express IL-12Rb2 and do not respond to IL-12Although IL-17-expressing cells isolated from cultures stimulated in vitro respond to subsequent stimulation with IL-12 with gain of IFN-g expression and loss of IL-17 expression (Fig. 1A, upper panel), IL-17 expressing cells directly isolated ex vivo maintained IL-17 expression and could not be induced to express IFN-g in the presence of IL-12 (Fig. 1A, lower panel) [17,19]. To identify the molecular mechanism of refraction of in vivo generated Th17 cells to conversion by IL-12, we here compared the expression of genes relevant for IL-12 signaling by Th17 cells generated in vit...

Th1 cells are prominent in inflamed tissue, survive conventional immunosuppression, and are believed to play a pivotal role in driving chronic inflammation. Here, we identify homeobox only protein (Hopx) as a critical and selective regulator of the survival of Th1 effector/memory cells, both in vitro and in vivo. Expression of Hopx is induced by T-bet and increases upon repeated antigenic restimulation of Th1 cells. Accordingly, the expression of Hopx is low in peripheral, naïve Th cells, but highly up-regulated in terminally differentiated effector/memory Th1 cells of healthy human donors. In murine Th1 cells, Hopx regulates the expression of genes involved in regulation of apoptosis and survival and makes them refractory to Fas-induced apoptosis. In vivo, adoptively transferred Hopxdeficient murine Th1 cells do not persist. Consequently, they cannot induce chronic inflammation in murine models of transfer-induced colitis and arthritis, demonstrating a key role of Hopx for Th1-mediated immunopathology.Key words: Cell survival . CD4 T cells . Inflammation . Memory cells Supporting Information available onlineIntroduction T-helper type 1 (Th1) cells mediate immune responses to intracellular pathogens, such as viruses, and produce IFN-g as their signature cytokine [1]. IFN-g, together with IL-12 and the transcription factors STAT1, STAT4 and T-bet, promotes the development of Th1 cells [2][3]. T-bet (T-box 21) is considered to act as the master transcription factor critically regulating Th1 lineage commitment [3][4][5]. Apart from their protective role in clearing infections, Th1 cells can initiate and maintain chronic inflammatory diseases, e.g. inflammatory bowel disease [6][7][8][9], uveitis [10], EAE [11,12] and arthritis [13]. In vitro, Th1 cells are much more sensitive to Fas-mediated apoptosis than Th2 or Th17 cells [14][15][16][17][18][19][20]. In vivo, however, effector/memory Th1 cells are abundant in chronically inflamed tissue [21][22][23] and persist over long time periods [24][25][26], suggesting that their sensitivity to Eur. J. Immunol. 2010. 40: 2993-3006 DOI 10.1002 HIGHLIGHTS 2993 FrontlineFas-mediated apoptosis is strictly regulated. Here, we demonstrate that among CD4 1 T cells, the transcriptional cofactor homeobox only protein (Hopx) is expressed by repeatedly restimulated Th1 cells, but not by Th2, Th17 or regulatory T cells. Hopx regulates Fas-mediated apoptosis of effector/memory Th1 cells and is critically required for their persistence in vivo.In vertebrates, Hopx expression originally was detected in the myocardium [27,28]. There, expression of Hopx is induced by the cardiac transcription factor Nkx2-5. Hopx-deficient mice show a complex, incompletely penetrant phenotype. Some Hopxdeficient embryos have a poorly developed myocardium with reduced cell numbers, others show normal, and still others show increased numbers of cardiomyocytes after birth [27,28]. Hopx does not bind to homeobox consensus binding sequences, and it has been postulated that Hopx acts indirectly, partnering with ...

The inhibitor of jB kinase e (IKKe) is pivotal for an efficient innate immune response to viral infections and has been recognized as breast cancer oncogene. The antiviral function of IKKe involves activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-jB, thus inducing the expression of type I IFN. Here, we have identified two novel splice variants of human IKKe, designated IKKe-sv1 and IKKe-sv2, respectively. Interestingly, RT-PCR revealed quantitatively different isoform expression in PBMC from different individuals. Moreover, we found cell type-and stimulus-specific protein expression of the various splice variants. Overexpression of full-length wt IKKe (IKKe-wt) leads to the activation of NF-jB-as well as IRF3-driven luciferase reporter genes. Although none of the splice variants activates IRF3, IKKe-sv1 still activates NF-jB, whereas IKKe-sv2 is also defective in NF-jB activation. Both splice variants form dimers with IKKe-wt and inhibit IKKe-wt-induced IRF3 signaling including the antiviral activity in a dominant-negative manner. The lack of IRF3 activation is likely caused by the failure of the splice variants to interact with the adapter proteins TANK, NAP1, and/or SINTBAD. Taken together, our data suggest alternative splicing as a novel regulatory mechanism suitable to shift the balance between different functions of IKKe.Key words: IFN . Innate immunity . NF-kB pathway . Protein/protein interactions . Signal transductionSupporting Information available online IntroductionViral infections are recognized by the innate immune system, which is essential for the subsequent initiation of adaptive immunity. Invading viruses are sensed by pattern-recognition receptors (PRR) recognizing pathogen-associated molecular patterns such as singleor double-stranded RNA. These PRR comprise TLR with endosomal/lysosomal localization like TLR3 and cytoplasmic receptors such as the retinoic acid-inducible protein I and melanoma differentiation-associated gene 5. Activation of these PRR engages intracellular signaling cascades leading to the secretion of type I IFN, which are important anti-viral cytokines ultimately facilitating viral clearance [1,2]. The signal transduction pathways leading to type I IFN expression involve activation of the serine/threonine kinases TANK-binding kinase 1 (TBK-1), also known as NF-kB activating kinase NAK [3], and inhibitor of kB kinase e (IKKe), also known as IKKi [4]. After virus infection, both kinases are activated and phosphorylate the transcription factor IFN regulatory factor 3 (IRF3), leading to homo-dimerization, translocation into the nucleus, and activation of promoters containing the IRF3 binding site termed IFN-stimulated response element [5,6]. For IRF3 activation after triggering of different PRR, the three related scaffold proteins NAP1, TANK, and SINTBAD are essential [7][8][9], whereas the use of a distinct scaffold protein depends on the respective stimulus activating the TBK1/IKKe pathway [10]. Ultimately, the formation of a multisubunit complex c...

Caspases are essential mediators of cytokine release and apoptosis. Additionally, caspase activity is required for the proliferation of naive T lymphocytes. It remained unclear how proliferating cells are able to cope with the pro-apoptotic activity especially of effector caspases-3 and -7. Possible reasons might include limited subcellular localization of active caspases or inhibition by endogenous caspase inhibitors. Here, we compared the activation of various caspases in proliferating human T cells with that in apoptotic cells. We show that cleaved caspases-3/-7 appear to be widely distributed in apoptotic cells while they are largely confined to the cytoplasm in proliferating cells. Additionally, in proliferating T cells caspase-3 remains incompletely cleaved, while in apoptotic cells fully mature caspase-3 is generated. We provide evidence that during T cell proliferation the intracellular caspase inhibitor X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspases-3/-7, thereby blocking their full activation, substrate cleavage, and cell death. The lack of substrate cleavage might also lead to the observed limited subcellular distribution of caspases-3/-7. After induction of apoptosis, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/ DIABLO) is released from mitochondria, resulting in the abrogation of the inhibitory effect of XIAP, full activation of caspases-3/-7, and apoptosis.