Jakmip1 is expressed upon T cell differentiation and has an inhibitory function in cytotoxic T lymphocytes

Jakmip1 belongs to a family of three related genes encoding proteins rich in coiled-coils. Jakmip1 is expressed predominantly in neuronal and lymphoid cells and colocalizes with microtubules. We have studied the expression of Jakmip1 mRNA and protein in distinct subsets of human primary lymphocytes. Jakmip1 is absent in naive CD8(+) and CD4(+) T lymphocytes from peripheral blood but is highly expressed in Ag-experienced T cells. In cord blood T lymphocytes, induction of Jakmip1 occurs upon TCR/CD28 stimulation and parallels induction of effector proteins, such as granzyme B and perforin. Further analysis of CD8(+) and CD4(+) T cell subsets showed a higher expression of Jakmip1 in the effector CCR7(-) and CD27(-) T cell subpopulations. In a gene expression follow-up of the development of CMV-specific CD8(+) response, Jakmip1 emerged as one of the most highly up-regulated genes from primary infection to latent stage. To investigate the relationship between Jakmip1 and effector function, we monitored cytotoxicity of primary CD8(+) T cells silenced for Jakmip1 or transduced with the full-length protein or the N-terminal region. Our findings point to Jakmip1 being a novel effector memory gene restraining T cell-mediated cytotoxicity.     

The influence of 3,3',5-triiodo-L-thyronine on human haematopoiesis

OBJECTIVES: Thyroid hormones mediate many physiological and developmental functions in humans. The role of the 3,3',5-triiodo-L-thyronine (T3) in normal human haematopoiesis at the cellular and molecular levels has not been determined. In this study, it was revealed that the human haematopoietic system might be directly depended on T3 influence. MATERIALS AND METHODS: We detected the TRalpha1 and TRbeta1 gene expression at the mRNA level in human cord blood, peripheral blood and bone marrow CD34(+)-enriched progenitor cells, using the RT-PCR method. Furthermore, we performed Western blotting to prove TRalpha1 and TRbeta1 expression occurs at the protein level in human cord blood, peripheral blood and bone marrow CD34(+) cells. In addition, the examined populations of cells were exposed in serum-free conditions to increasing doses of T3 and were subsequently investigated for clonogenic growth of granulocyte-macrophage colony-forming unit and erythrocyte burst-forming unit in methylcellulose cultures, and for the level of apoptosis, by employing annexin V staining and the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling method. We investigated expression levels of apoptosis-related Bax and antiapoptotic Bcl-2 and Bcl-x(L) genes in the examined cells. RESULTS: We found that exposure to higher and lower than normal concentration of thyroid hormone significantly influenced clonogenecity and induced apoptosis in human haematopoietic progenitor cells. CONCLUSIONS: This study expands the understanding of the role of thyroid disorders in normal human haematopoiesis and indicates a direct influence of T3 on this process.     

Polyadenylation site-specific differences in the activity of the neuronal βCstF-64 protein in PC-12 cells

Recent genome-wide analyses have implicated alternative polyadenylation — the process of regulated mRNA 3′ end formation — as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, βCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that βCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for βCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that βCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that βCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the β-adducin mRNA. Notably, we demonstrate that the activity of βCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the βCstF-64 protein. Our data address the polyadenylation functions of βCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.     

IFN-alpha and IL-10 induce the differentiation of human type 1 T regulatory cells

CD4(+) T regulatory type 1 (Tr1) cells suppress Ag-specific immune responses in vitro and in vivo. Although IL-10 is critical for the differentiation of Tr1 cells, the effects of other cytokines on differentiation of naive T cells into Tr1 cells have not been investigated. Here we demonstrate that endogenous or exogenous IL-10 in combination with IFN-alpha, but not TGF-beta, induces naive CD4(+) T cells derived from cord blood to differentiate into Tr1 cells: IL-10(+)IFN-gamma(+)IL-2(-/low)IL-4(-). Naive CD4(+) T cells derived from peripheral blood require both exogenous IL-10 and IFN-alpha for Tr1 cell differentiation. The proliferative responses of the Tr1-containing lymphocyte populations, following activation with anti-CD3 and anti-CD28 mAbs, were reduced. Similarly, cultures containing Tr1 cells displayed reduced responses to alloantigens via a mechanism that was partially mediated by IL-10 and TGF-beta. More importantly, Tr1-containing populations strongly suppressed responses of naive T cells to alloantigens. Collectively, these results show that IFN-alpha strongly enhances IL-10-induced differentiation of functional Tr1 cells, which represents a first major step in establishing specific culture conditions to generate T regulatory cells for biological and biochemical analysis, and for cellular therapy to induce peripheral tolerance in humans.     

Maturation of adult peripheral blood CD38(+)CD4(+) T cells demonstrated by cytokine production in response to a superantigen,TSST-1

This study looks at immunoincompetent CD4(+) T cells in adult peripheral blood (APB) using cytokine production in response to a superantigen as a measure of function. We compared the function of APB CD38(+)CD4(+) and CD38(-/low)CD4(+) T cells to that of cord blood (CB) CD4(+) T cells. APB CD4(+) T cell blasts produce substantial amounts of IL-2 in response to TSST-1 restimulation, while CB CD4(+) T cell blasts produce less. APB CD38(+)CD4(+) T cells produce low levels of IL-4 and IFN-gamma in response to TSST-1, even after activation, while APB CD38(-/low)CD4(+) T cells retain their ability to produce high levels of these cytokines despite high CD38 expression. These results suggest that the developmental stage of APB CD38(+)CD4(+) T cells lies between that of CB CD4(+) T cells and APB CD38(-/low)CD4(+) T cells and that APB CD38(+)CD45RO(-)CD4(+) T cells gradually cease to express CD38 as they acquire full function. We reconsider CD4(+) cell maturation and response to TSST-1 and discuss the implications of T cell maturity on infectious diseases.     

Differential regulation of human blood dendritic cell subsets by IFNs

Based on the relative expression of CD11c and CD1a, we previously identified subsets of dendritic cells (DCs) or DC precursors in human peripheral blood. A CD1a(+)/CD11c(+) population (CD11c(+) DCs), also called myeloid DCs, is an immediate precursor of Langerhans cells, whereas a CD1a(-)/CD11c(-) population (CD11c(-) DCs), sometimes called lymphoid DCs but better known as plasmacytoid DCs, is composed of type I IFN (IFN-alpha beta)-producing cells. Here, we investigate the effects of IFN-alpha beta and IFN-gamma as well as other cytokines on CD11c(+) and CD11c(-) DC subsets, directly isolated from the peripheral blood, instead of in vitro-generated DCs. IFN-gamma and IFN-alpha, rather than GM-CSF, were the most potent cytokines for enhancing the maturation of CD11c(+) DCs. Incubation of CD11c(+) DCs with IFN-gamma also resulted in increased IL-12 production, and this IL-12 allowed DCs to increase Th1 responses by alloreactive T cells. In contrast, IFN-alpha did not induce IL-12 but, rather, augmented IL-10 production. IFN-alpha-primed matured CD11c(+) DCs induced IL-10-producing regulatory T cells; however, this process was independent of the DC-derived IL-10. On the other hand, IFN-alpha by itself neither matured CD11c(-) DCs nor altered the polarization of responding T cells, although this cytokine was a potent survival factor for CD11c(-) DCs. Unlike IFN-alpha, IL-3 was a potent survival factor and induced the maturation of CD11c(-) DCs. The IL-3-primed CD11c(-) DCs activated T cells to produce IL-10, IFN-gamma, and IL-4. Thus, CD11c(+) and CD11c(-) DC subsets play distinct roles in the cytokine network, especially their responses to IFNs.     

CD69 is expressed on Daudi cells in response to interferon-alpha.

An approach to obtain monoclonal antibodies directed against cell surface proteins induced by interferon has been developed in order to characterize such proteins and determine their role. Hybridomas obtained by fusion of murine myeloma cells and spleen cells of mice immunized with interferon-alpha-treated Daudi cells were screened for the production of antibodies reacting differentially with interferon-alpha-treated and untreated Daudi cells. One such hybridoma, 2D5, produced an antibody reacting with a 28/32 kDa homodimeric protein (p28/32) expressed at the surface of Daudi cells in response to IFN-alpha treatment. IFN-alpha treatment also increased the basal level of p28/32 detected on peripheral blood leukocytes (PBL). 2D5 Antibody was used to probe the expression of p28/32 on different cells and in response to various inducers. It appears that 2D5 reacted in fact with CD69, a marker of leukocyte activation and that, following IFN-alpha treatment, CD69 was not induced on all cultured cell lines tested. Interestingly, IFN-gamma was also able to induce CD69 expression on a restricted number of cell lines but the induction pattern only partially overlapped that of IFN-alpha. As expected, activation of cells with phorbol myristate acetate (PMA) resulted in a notable increase in the level of CD69 on all cell lines considered except for the epithelial and fibroblastic types.     

Evidence for a selective migration of fetus-specific CD4+CD25bright regulatory T cells from the peripheral blood to the decidua in human pregnancy

During pregnancy, the maternal immune system has to tolerate the persistence of fetal alloantigens. Many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. Murine studies suggest that CD4(+)CD25(+) T cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. Previous studies by our group demonstrate that a significantly higher percentage of activated T cells and CD4(+)CD25(bright) T cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy. In this study, we examined the phenotypic and functional properties of CD4(+)CD25(bright) T cells derived from maternal peripheral blood and decidual tissue. Depletion of CD4(+)CD25(bright) T cells from maternal peripheral blood demonstrates regulation to third party umbilical cord blood cells comparable to nonpregnant controls, whereas the suppressive capacity to umbilical cord blood cells of her own child is absent. Furthermore, maternal peripheral blood shows a reduced percentage of CD4(+)CD25(bright)FOXP3(+) and CD4(+)CD25(bright)HLA-DR(+) cells compared with peripheral blood of nonpregnant controls. In contrast, decidual lymphocyte isolates contain high percentages of CD4(+)CD25(bright) T cells with a regulatory phenotype that is able to down-regulate fetus-specific and fetus-nonspecific immune responses. These data suggest a preferential recruitment of fetus-specific regulatory T cells from maternal peripheral blood to the fetal-maternal interface, where they may contribute to the local regulation of fetus-specific responses.     

Enterovirus 71 3C Protease Cleaves a Novel Target CstF-64 and Inhibits Cellular Polyadenylation

Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell–virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3C^pro) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3′ pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C^pro. CstF-64 was cleaved in vitro by 3C^pro but neither by mutant 3C^pro (in which the catalytic site was inactivated) nor by another EV71 protease 2A^pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C^pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3′-end pre-mRNA processing and polyadenylation in 3C^pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C^pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.     

CD4+CD25bright regulatory T cells actively regulate inflammation in the joints of patients with the remitting form of juvenile idiopathic arthritis

This study investigates the role of CD4(+)CD25(+) regulatory T cells during the clinical course of juvenile idiopathic arthritis (JIA). Persistent oligoarticular JIA (pers-OA JIA) is a subtype of JIA with a relatively benign, self-remitting course while extended oligoarticular JIA (ext-OA JIA) is a subtype with a much less favorable prognosis. Our data show that patients with pers-OA JIA display a significantly higher frequency of CD4(+)CD25(bright) T cells with concomitant higher levels of mRNA FoxP3 in the peripheral blood than ext-OA JIA patients. Furthermore, while numbers of synovial fluid (SF) CD4(+)CD25(bright) T cells were equal in both patient groups, pers-OA JIA patients displayed a higher frequency of CD4(+)CD25(int) T cells and therefore of CD4(+)CD25(total) in the SF than ext-OA JIA patients. Analysis of FoxP3 mRNA levels revealed a high expression in SF CD4(+)CD25(bright) T cells of both patient groups and also significant expression of FoxP3 mRNA in the CD4(+)CD25(int) T cell population. The CD4(+)CD25(bright) cells of both patient groups and the CD4(+)CD25(int) cells of pers-OA JIA patients were able to suppress responses of CD25(neg) cells in vitro. A markedly higher expression of CTLA-4, glucocorticoid-induced TNFR, and HLA-DR on SF CD4(+)CD25(bright) T regulatory (Treg) cells compared with their peripheral counterparts suggests that the CD4(+)CD25(+) Treg cells may undergo maturation in the joint. In correlation with this mature phenotype, the SF CD4(+)CD25(bright) T cells showed an increased regulatory capacity in vitro compared with peripheral blood CD4(+)CD25(bright) T cells. These data suggest that CD4(+)CD25(bright) Treg cells play a role in determining the patient's fate toward either a favorable or unfavorable clinical course of disease.     

Edmonston measles virus prevents increased cell surface expression of peptide-loaded major histocompatibility complex class II proteins in human peripheral monocytes

Gamma interferon (IFN-gamma) induces expression of the gene products of the major histocompatibility complex (MHC), whereas IFN-alpha/beta can interfere with or suppress class II protein expression. In separate studies, measles virus (MV) was reported to induce IFN-alpha/beta and to up-regulate MHC class II proteins. In an attempt to resolve this paradox, we examined the surface expression of MHC class I and class II proteins in MV-infected peripheral monocytes in the presence and absence of IFN-alpha/beta. Infection of purified monocytes with Edmonston B MV resulted in an apparent increase in cell surface expression of HLA-A, -B, and -C class I proteins, but it had no effect on the expression of HLA-DR class II proteins. MV-infected purified monocytes expressed IFN-alpha/beta, but no measurable IFN-gamma expression was detected in supernatant fluids. Class II protein expression could be enhanced by coculture of purified monocytes with uninfected peripheral blood mononuclear cell (PBMC) supernatant. MV infection of PBMCs also did not affect expression of class II proteins, but the expression of HLA-A, -B, and -C class I proteins was increased two- to threefold in most donor cells. A direct role for IFN-alpha/beta suppression of MHC class II protein expression was not evident in monocytes since MV suppressed class II protein expression in the absence of IFN-alpha/beta. Taken together, these data suggest that MV interferes with the expression of peptide-loaded class II complexes, an effect that may potentially alter CD4(+)-T-cell proliferation and the cell-mediated immune responses that they help to regulate.     

Excessive production of IFN-gamma in patients with systemic lupus erythematosus and its contribution to induction of B lymphocyte stimulator/B cell-activating factor/TNF ligand superfamily-13B

Expression and immunological significance of IFN-gamma, a pivotal cytokine in murine lupus, have not been clearly demonstrated in human systemic lupus erythematosus (SLE). In the present study we investigated the expression of IFN-gamma in peripheral blood T cells from patients with SLE and its role in the production of the soluble B lymphocyte stimulator (sBLyS). Peripheral blood T cells from patients with SLE expressed significantly larger amounts of IFN-gamma in response to stimulation with anti-CD3 mAb plus anti-CD28 mAb than those from normal controls as shown by three analytical methods, including ELISA, flow cytometry, and quantitative RT-PCR. The ratio of IFN-gamma-producing T cells to effector memory T cells in CD3(+)CD4(+) and CD3(+)CD8(+) populations in patients with SLE was significantly higher than that of normal controls. The T-box expressed in T cells (T-bet) mRNA/GATA-binding protein-3 (GATA-3) mRNA ratio was significantly higher in patients with SLE than in normal controls. T cell culture supernatants from patients with SLE contained significantly higher sBLyS-inducing activity than normal controls; this was almost completely inhibited by the addition of anti-human IFN-gamma mAb. Percentages of BLyS-expressing peripheral blood monocytes in patients with SLE were significantly higher than those of normal controls. Monocytes from patients with SLE produced significantly larger amounts of sBLyS in response to IFN-gamma than those from normal controls. Taken together, these data strongly indicate that the overexpression of IFN-gamma in peripheral blood T cells contributes to the immunopathogenesis of SLE via the induction of sBLyS by monocytes/macrophages, which would promote B cell activation and maturation.     

Regulation of CD28 expression on umbilical cord blood and adult peripheral blood CD8+ T cells by interleukin(IL)-15/IL-21

Interleukin (IL)-15 and IL-21, both belonging to common γ-chain-signaling cytokine family, have an important role to maintain homeostatic proliferation of CD8(+) T cells. CD28, an essential co-stimulatory molecule on T cells, may be a marker of replicative senescence. We investigated the effect of IL-15 and IL-21, alone or in combination, on activation, apoptosis, cytokine production and cytotoxic function of magnetic bead purified umbilical cord blood (UCB) and adult peripheral blood (APB) CD8(+) T cells with regards to their CD28 expression. We established that (1) IL-15-induced CD8(+) T cell proliferation was associated with a preferential expansion of CD28(-) population in UCB, which could be partially counteracted by IL-21; (2) UCB CD8(+) T cells were more readily responsive to IL-15 compared to their adult counterparts in terms of CD69 expression, with the majority of CD69-bearing CD8(+) T cells were CD28(-); (3) IL-21 further promoted interferon-gamma, but not tumor necrosis factor-alpha production from IL-15 treated CD8(+) T cells; (4) IL-21 also synergized with IL-15 to enhance perforin and granzyme B expression of CD8(+) T cells, especially in APB CD8(+)CD28(-) subsets; (5) IL-21 resulted in CD8(+) T cells apoptosis both in APB and UCB cells, mainly in CD8(+)CD28(-) subsets. Taken together, we demonstrate differential IL-15/IL-21 response in UCB CD8(+) T cells with regards to CD28 expression. Our results suggest that combining IL-21 and IL-15 immunotherapy may be better than IL-15 alone to ameliorate graft-versus-host disease while preserving antitumor effect in the post-UCB transplantation period.     

Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus

CD4(+)CD25(+) T regulatory cells (Tregs) play an essential role in maintaining immunologic homeostasis and preventing autoimmunity. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear components. We hypothesized that altered function of CD4(+)CD25(high) Tregs might play a role in the breakdown of immunologic self-tolerance in patients with SLE. In this study, we report a significant decrease in the suppressive function of CD4(+)CD25(high) Tregs from peripheral blood of patients with active SLE as compared with normal donors and patients with inactive SLE. Notably, CD4(+)CD25(high) Tregs isolated from patients with active SLE expressed reduced levels of FoxP3 mRNA and protein and poorly suppressed the proliferation and cytokine secretion of CD4(+) effector T cells in vitro. In contrast, the expression of FoxP3 mRNA and protein and in vitro suppression of the proliferation of CD4(+) effector T cells by Tregs isolated from inactive SLE patients, was comparable to that of normal individuals. In vitro activation of CD4(+)CD25(high) Tregs from patients with active SLE increased FoxP3 mRNA and protein expression and restored their suppressive function. These data are the first to demonstrate a reversible defect in CD4(+)CD25(high) Treg function in patients with active SLE, and suggest that strategies to enhance the function of these cells might benefit patients with this autoimmune disease.     

P-selectin enhances generation of CD14+CD16+ dendritic-like cells and inhibits macrophage maturation from human peripheral blood monocytes

Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcgammaRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14(+)CD16(+)CD64(-) monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4(+) T cells to produce significantly less IFN-gamma than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14(+)CD16(+) DLC generation were completely abrogated by pretreatment of cells with the protein kinase C delta inhibitor rottlerin, but not by classical protein kinase C inhibitor G?6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.