Inflammatory cytokines determine the susceptibility of human CD8 T cells to Fas-mediated activation-induced cell death through modulation of FasL and c-FLIP(S) expression

The nature of inflammatory signals determines the outcome of T cell responses. However, little is known about how inflammatory cytokines provided to human CD8 T cells during activation affects their susceptibility to post-activation cell death. We have examined and compared the effects of the inflammatory cytokine IL-12, as well as the combination of IL-1, IL-6, and IL-23 (IL-1/6/23) on the susceptibility of primary human CD8 T cells to post-activation cell death. Human CD8 T cells activated in the presence of IL-1/6/23 underwent significantly less cell death after activation as compared with those activated in IL-12. This was due to reduced susceptibility to Fas-mediated activation-induced cell death (AICD). Mechanistically, the reduced level of cell death in CD8 T cells activated in IL-1/6/23 was a result of a low level of FasL expression and high level of c-FLIP(S) expression. When the effect of IL-1, IL-6, and IL-23 individually was examined, IL-1 or IL-6 alone was sufficient to inhibit CD8 T cell death that occurs after activation in IL-12. IL-1, but not IL-6, inhibited expression of FasL, whereas IL-6, but not IL-1, increased c-FLIP(S) expression. Our findings show that the presence of IL-1 and/or IL-6 during activation of human CD8 T cells attenuates Fas-mediated AICD, whereas IL-12 increases the susceptibility of activated CD8 T cells to this form of cell death.     

利用复感儿T淋巴细胞作为外源性ADA基因表达效应细胞的实验探讨

对４１例健康儿童和１７例反复呼吸道感染患儿（复咸儿）的外周血淋巴细胞ＡＤＡ活性进行了检测．两例ＡＤＡ活性明显低下的复感儿的外周血Ｔ淋巴细胞被选作实验样品,经体外培养后,采用ｌｉｐｏｆｅｃｔｉｎ介导的的方法导入外源性ＡＤＡ基因．结果表明：体外培养的两例复感儿淋巴细胞中ＡＤＡ活性较基因转移前升高;细胞免疫学特性也在一定程度上获得改善     

Functional involvement of dual specificity phosphatase 16 (DUSP16), a c-Jun N-terminal kinase-specific phosphatase, in the regulation of T helper cell differentiation

Naïve CD4⁺ T helper (Th) cells differentiate into distinct subsets of effector cells (Th1, Th2, Th17, and induced regulatory T cells (iTreg)) expressing different sets of cytokines upon encounter with presented foreign antigens. It has been well established that Th1/Th2 balance is critical for the nature of the following immune responses. Previous reports have demonstrated important roles of c-Jun N-terminal kinase (JNK) in Th1/Th2 balance, whereas the regulatory mechanisms of JNK activity in Th cells have not been elucidated. Here, we show that dual specificity phosphatase 16 (DUSP16, also referred to as MKP-M or MKP-7), which preferentially inactivates JNK, is selectively expressed in Th2 cells. In the in vitro differentiation assay of naïve CD4⁺ cells, DUSP16 expression is up-regulated during Th2 differentiation and down-regulated during Th1 differentiation. Chromatin immunoprecipitation revealed the increased acetylation of histone H3/H4 at the dusp16 gene promoter in CD4⁺ T cells under the Th2 condition. Adenoviral transduction of naïve CD4⁺ T cells with DUSP16 resulted in increased mRNA expression of IL-4 and GATA-3 in Th2 and decreased expression of IFNγ and T-bet in Th1 differentiation. In contrast, transduction of a dominant negative form of DUSP16 had the reverse effects. Furthermore, upon immunization, T cell-specific dusp16 transgenic mice produced antigen-specific IgG2a at lower amounts, whereas DN dusp16 transgenic mice produced higher amounts of antigen-specific IgG2a accompanied by decreased amounts of antigen-specific IgG1 and IgE than those of control mice. Together, these data suggest the functional role of DUSP16 in Th1/Th2 balance.     

Signaling events involved in interleukin 27 (IL-27)-induced proliferation of human naive CD4+ T cells and B cells

IL-27 induces stronger proliferation of naive than memory human B cells and CD4(+) T cells. In B cells, this differential response is associated with similar levels of IL-27 receptor chains, IL-27Rα and gp130, in both subsets and stronger STAT1 and STAT3 activation by IL-27 in naive B cells. Here, we show that the stronger proliferative response of CD3-stimulated naive CD4(+) T cells to IL-27 is associated with lower levels of IL-27Rα but higher levels of gp130 compared with memory CD4(+) T cells. IL-27 signaling differs between naive and memory CD4(+) T cells, as shown by more sustained STAT1, -3, and -5 activation and weaker activation of SHP-2 in naive CD4(+) T cells. In the latter, IL-27 increases G0/G1 to S phase transition, cell division and, in some cases, cell survival. IL-27 proliferative effect on naive CD4(+) T cells is independent of MAPK, but is dependent on c-Myc and Pim-1 induction by IL-27 and is associated with induction of cyclin D2, cyclin D3, and CDK4 by IL-27 in a c-Myc and Pim-1-dependent manner. In BCR-stimulated naive B cells, IL-27 only increases entry in the S phase and induces the expression of Pim-1 and of cyclins A, D2, and D3. In these cells, inhibition of Pim-1 inhibits IL-27 effect on proliferation and cyclin induction. Altogether, these data indicate that IL-27 mediates proliferation of naive CD4(+) T cells and B cells through induction of both common and distinct sets of cell cycle regulators.     

Homologous elements hs3a and hs3b in the 3' regulatory region of the murine immunoglobulin heavy chain (Igh) locus are both dispensable for class-switch recombination

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.     

Ubiquitination Regulates Expression of the Serine/Arginine-rich Splicing Factor 1 (SRSF1) in Normal and Systemic Lupus Erythematosus (SLE) T Cells

T cells from patients with systemic lupus erythematosus (SLE) exhibit reduced expression of the critical T cell receptor (TCR)-associated CD3ζ signaling chain and are poor producers of the vital cytokine IL-2. By oligonucleotide pulldown and mass spectrometry discovery approaches, we identified the splicing regulator serine/arginine-rich splicing factor (SRSF) 1 or splicing factor 2/alternative splicing factor (SF2/ASF) to be important in the expression of CD3ζ chain. Importantly, increases in the expression of SRSF1 rescued IL-2 production in T cells from patients with SLE. In this study, we investigated the regulation of SRSF1 expression in resting and activated human T cells. We found that T cell stimulation induced a rapid and significant increase in mRNA expression of SRSF1; however, protein expression levels did not correlate with this increase. Co-engagement of CD28 induced a similar mRNA induction and reduction in protein levels. Proteasomal but not lysosomal degradation was involved in this down-regulation as evidenced by blocking with specific inhibitors MG132 and bafilomycin, respectively. Immunoprecipitation studies showed increased ubiquitination of SRSF1 in activated T cells. Interestingly, T cells from patients with SLE showed increased ubiquitination of SRSF1 when compared with those from healthy individuals. Our results demonstrate a novel mechanism of regulation of the splicing factor SRSF1 in human T cells and a potential molecular mechanism that controls its expression in SLE.     

Ikaros Sets the Potential for Th17 Lineage Gene Expression through Effects on Chromatin State in Early T Cell Development

Th17 cells are important effectors of immunity to extracellular pathogens, particularly at mucosal surfaces, but they can also contribute to pathologic tissue inflammation and autoimmunity. Defining the multitude of factors that influence their development is therefore of paramount importance. Our previous studies using Ikaros^−/− CD4+ T cells implicated Ikaros in Th1 versus Th2 lineage decisions. Here we demonstrate that Ikaros also regulates Th17 differentiation through its ability to promote expression of multiple Th17 lineage-determining genes, including Ahr, Runx1, Rorc, Il17a, and Il22. Ikaros exerts its influence on the chromatin remodeling of these loci at two distinct stages in CD4+ T helper cell development. In naive cells, Ikaros is required to limit repressive chromatin modifications at these gene loci, thus maintaining the potential for expression of the Th17 gene program. Subsequently, Ikaros is essential for the acquisition of permissive histone marks in response to Th17 polarizing signals. Additionally, Ikaros represses the expression of genes that limit Th17 development, including Foxp3 and Tbx21. These data define new targets of the action of Ikaros and indicate that Ikaros plays a critical role in CD4+ T cell differentiation by integrating specific cytokine cues and directing epigenetic modifications that facilitate activation or repression of relevant genes that drive T cell lineage choice.     

Interleukin-27 and interferon-gamma are involved in regulation of autoimmune arthritis

Inflammation underlying immune pathology and tissue damage involves an intricate interplay between multiple immunological and biochemical mediators. Cytokines represent the key immune mediators that trigger a cascade of reactions that drive processes such as angiogenesis and proteolytic damage to tissues. IL-17 has now been shown to be a pivotal cytokine in many autoimmune diseases, supplanting the traditional Th1-Th2 paradigm. Also, the dual role of proinflammatory IFN-γ has unraveled new complexities in the cytokine biology of such disorders. A major hurdle in fully understanding the effector pathways in these disorders is the lack of information regarding the temporal kinetics of the cytokines during the course of the disease, as well as the interplay among the key cytokines. Using an experimental model of arthritic inflammation, we demonstrate that the temporal expression of cytokines during the incubation phase is a critical determinant of disease susceptibility. The susceptible rats raised a vigorous IL-17 response early, followed by IFN-γ and IL-27 response in that sequence, whereas the resistant rats displayed an early and concurrent response to these three cytokines. Accordingly, treatment with exogenous IFN-γ/IL-27 successfully controlled arthritic inflammation and inhibited the defined mediators of inflammation, angiogenesis, cell survival, apoptosis, and tissue damage. Furthermore, IFN-γ enhanced IL-27 secretion, revealing a cooperative interplay between the two cytokines. Our results offer a novel immunobiochemical perspective on the pathogenesis of autoimmune arthritis and its therapeutic control.     

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane

β₂-Adrenergic receptors (β₂-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β₂-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β₂-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β₂-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β₂-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β₂-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.