技术资料

CD46 is a glycoprotein with important functions in innate and adaptive immune responses. Functionally different isoforms are generated by alternative splicing at exons 7-9 (BC and C isoforms) and exon 13 (CYT-1 and CYT-2 isoforms) giving rise to BC1,BC2,C1 and C2. We developed a novel real-time PCR assay that allows quantitative comparisons between these isoforms. Their relative frequency in CD4(+) T cells from 100 donors revealed a distribution with high interpersonally variability. Importantly,the distribution between the isoforms was not random and although splicing favoured inclusion of exon 8 (BC isoforms),exclusion of exon 8 (C isoforms) was significantly linked to exclusion of exon 13 (CYT-2 isoforms). Despite inter-individual differences,CD4(+) and CD8(+) T cells,B cells,NK cells and monocytes expressed similar isoform profiles intra-individually. However,memory/effector CD4(+) T cells had a significantly higher frequency of CYT-2 when compared with naïve CD4(+) T cells. Likewise,in vitro activation of naïve and total CD4(+) T cells increased the expression of CYT-2. This indicates that although splicing factors determine a certain expression profile in an individual,the profile can be modulated by external stimuli. This suggests a mechanism by which alterations in CD46 isoforms may temporarily regulate the immune response. View Publication

T lymphocytes require signals from self-peptides and cytokines,most notably interleukins 7 and 15 (IL-7,IL-15),for survival. While mouse T cells die rapidly if IL-7 or IL-15 is withdrawn,human T cells can survive prolonged withdrawal of IL-7 and IL-15. Here we show that IL-7 and IL-15 are required to maintain human T cell proliferative capacity through the STAT5 signaling pathway. T cells from humanized mice proliferate better if stimulated in the presence of human IL-7 or IL-15 or if T cells are exposed to human IL-7 or IL-15 in mice. Freshly isolated T cells from human peripheral blood lose proliferative capacity if cultured for 24 hours in the absence of IL-7 or IL-15. We further show that phosphorylation of STAT5 correlates with proliferation and inhibition of STAT5 reduces proliferation. These results reveal a novel role of IL-7 and IL-15 in maintaining human T cell function,provide an explanation for T cell dysfunction in humanized mice,and have significant implications for in vitro studies with human T cells. View Publication

Retinoic acid (RA) imprints gut-homing specificity on T cells upon activation by inducing the expression of chemokine receptor CCR9 and integrin α4β7. CCR9 expression seemed to be more highly dependent on RA than was the α4β7 expression,but its molecular mechanism remained unclear. In this article,we show that NFAT isoforms NFATc1 and NFATc2 directly interact with RA receptor (RAR) and retinoid X receptor (RXR) but play differential roles in RA-induced CCR9 expression on murine naive CD4(+) T cells. TCR stimulation for 6-24 h was required for the acquisition of responsiveness to RA and induced activation of NFATc1 and NFATc2. However,RA failed to induce CCR9 expression as long as TCR stimulation continued. After terminating TCR stimulation or adding cyclosporin A to the culture,Ccr9 gene transcription was induced,accompanied by inactivation of NFATc1 and sustained activation of NFATc2. Reporter and DNA-affinity precipitation assays demonstrated that the binding of NFATc2 to two NFAT-binding sites and that of the RAR/RXR complex to an RA response element half-site in the 5'-flanking region of the mouse Ccr9 gene were critical for RA-induced promoter activity. NFATc2 directly bound to RARα and RXRα,and it enhanced the binding of RARα to the RA response element half-site. NFATc1 also bound to the NFAT-binding sites and directly to RARα and RXRα,but it inhibited the NFATc2-dependent promoter activity. These results suggest that the cooperativity between NFATc2 and the RAR/RXR complex is essential for CCR9 expression on T cells and that NFATc1 interferes with the action of NFATc2. View Publication

T-cell-mediated processes play an essential role in the pathogenesis of several inflammatory skin diseases such as atopic dermatitis,allergic contact dermatitis and psoriasis. The aim of this study was to investigate the role of the IL-2-inducible tyrosine kinase (Itk),an enzyme acting downstream of the T-cell receptor (TCR),in T-cell-dependent skin inflammation using three approaches. Itk knockout mice display significantly reduced inflammatory symptoms in mouse models of acute and subacute contact hypersensitivity (CHS) reactions. Systemic administration of a novel small molecule Itk inhibitor,Compound 44,created by chemical optimization of an initial high-throughput screening hit,inhibited Itk's activity with an IC50 in the nanomolar range. Compound 44 substantially reduced proinflammatory immune responses in vitro and in vivo after systemic administration in two acute CHS models. In addition,our data reveal that human Itk,comparable to its murine homologue,is expressed mainly in T cells and is increased in lesional skin from patients with atopic dermatitis and allergic contact dermatitis. Finally,silencing of Itk by RNA interference in primary human T cells efficiently blocks TCR-induced lymphokine secretion. In conclusion,Itk represents an interesting new target for the therapy of T-cell-mediated inflammatory skin diseases. View Publication

Regulatory T cells (Tregs) contribute significantly to the tolerogenic nature of the liver. The mechanisms,however,underlying liver-associated Treg induction are still elusive. We recently identified the vitamin A metabolite,retinoic acid (RA),as a key controller that promotes TGF-β-dependent Foxp3(+) Treg induction but inhibits TGF-β-driven Th17 differentiation. To investigate whether the RA producing hepatic stellate cells (HSC) are part of the liver tolerance mechanism,we investigated the ability of HSC to function as regulatory APC. Different from previous reports,we found that highly purified HSC did not express costimulatory molecules and only upregulated MHC class II after in vitro culture in the presence of exogenous IFN-γ. Consistent with an insufficient APC function,HSC failed to stimulate naive OT-II TCR transgenic CD4(+) T cells and only moderately stimulated α-galactosylceramide-primed invariant NKT cells. In contrast,HSC functioned as regulatory bystanders and promoted enhanced Foxp3 induction by OT-II TCR transgenic T cells primed by spleen dendritic cells,whereas they greatly inhibited the Th17 differentiation. Furthermore,the regulatory bystander capacity of the HSC was completely dependent on their ability to produce RA. Our data thus suggest that HSC can function as regulatory bystanders,and therefore,by promoting Tregs and suppressing Th17 differentiation,they might represent key players in the mechanism that drives liver-induced tolerance. View Publication

How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice,N-glycan branching by the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity,cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis,spontaneous inflammatory demyelination and neurodegeneration,the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3),including genetic variants in interleukin-7 receptor-α (IL7RA*C),interleukin-2 receptor-α (IL2RA*T),MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3),optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway,namely N-glycosylation. View Publication