International Immunology - current issue

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2009-06-21

Synthetic methylated CpG ODNs are potent in vivo adjuvants when delivered in liposomal nanoparticles

2009-06-21

Although it is well documented that the immunological activity of cytosine–guanine (CpG) motifs is abrogated by 5' methylation of the cytosine residue, encapsulation within stabilized lipid nanoparticles endows these methylated cytosine–guanine- (mCpG-) containing oligonucleotides (ODNs) with potent immunostimulatory activity in murine animal models. Surprisingly, not only do liposomal nanoparticulate (LN) mCpG ODN possess immunostimulatory activity, their potency is found to be equivalent and often greater than the equivalent unmethylated form, as judged by a number of ex vivo innate and adaptive immune parameters and anti-tumor efficacy in murine models. Preliminary data indicate that both methylated and unmethylated CpG ODN act through a common receptor signaling pathway, specifically via toll-like receptor (TLR) 9, based on observations of up-regulated TLR9 expression, induction of nitric oxide and dependence on endosomal maturation. This is confirmed in TLR9 knockout animals which show no immunostimulatory activity following treatment with LN-mCpG ODN. These data, therefore, indicate that the mCpG DNA is fully competent to interact with TLR9 to initiate potent immune responses. Furthermore, this work implicates an as yet unidentified mechanism upstream of TLR9 which regulates the relative activities of free methylated versus unmethylated CpG ODN that is effectively bypassed by particulate delivery of CpG ODN.

Constitutively active aryl hydrocarbon receptor expressed in T cells increases immunization-induced IFN-{gamma} production in mice but does not suppress Th2-cytokine production or antibody production

2009-06-21

The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) has been implicated in various immune functions. Our previous studies have shown that AhR activation by exposure of ovalbumin (OVA)-immunized mice to the potent ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases immunization-induced IFN- production in the spleen and suppresses the production of T_h2 cytokines and OVA-specific antibodies. In the present study, we used transgenic (Tg) mice that express a constitutively active mutant of aryl hydrocarbon receptor (CA-AhR) specifically in T-lineage cells to clarify the role of AhR activation in T cells in these reactions. The results of this study clearly demonstrated that AhR activation only in the T cells augments IFN- production upon OVA immunization. By contrast, production of T_h2 cytokines and antibodies were not significantly suppressed by CA-AhR in the T cells. These results suggest that suppression of T_h2 cytokines and antibodies production require AhR activation not only in T cells but also in other cell types as caused by TCDD exposure. Alternatively, these results may indicate that IFN- augmentation and T_h2 cytokines and antibodies suppression depend on different ways of functions of AhR in the T cells and that CA-AhR does not replicate the suppressive effect of TCDD-activated AhR on T_h2 cytokines and antibodies. Expression of CA-AhR in the T cells was also shown to increase the percentage of CD25⁺ cells among CD4⁺ cells in the thymus and spleen. Thus, studies using T-cell-specific CA-AhR Tg mice provide a way to dissect the role of AhR in individual cell types and how the AhR functions.

Functionally relevant decreases in activatory receptor expression on NK cells are associated with pulmonary tuberculosis in vivo and persist after successful treatment

2009-06-21

Correlates for the initiation of Mycobacterium tuberculosis hominis (Mth) replication from latency are needed in order to improve Mth control. In order to analyze if perturbations of peripheral NK cells may be associated with exit from Mth latency, sequential patients with newly diagnosed lung tuberculosis (TB) were studied. Peripheral NK cells were analyzed by cytofluorometry, in vitro culture and functional assays. At the onset of lung TB, imbalances in NK cell subsets were evident. Decreased CD56^brightCD16^+/– subsets with significantly compromised NKp30 and NKp46 expression and with specifically decreased -IFN production upon triggering were evident. These features were not completely restored when purified NK cells were cultured in vitro. Culture supplementation with -IFN increased only NKp30 expression in TB and healthy donors. Extensive peripheral NK cell triggering was evident in these patients, as shown by the expression of NK cell activation markers and of the lymph node-homing chemokine receptor CCR7 on CD16⁺ CD56^dull cells. Significant persistence of decreased NKp30 and NKp46 after successful treatment with a standard four-drug regimen was detected after full recovery. NK cell function is deeply affected in patients at the onset of pulmonary TB. The involvement of multiple activatory receptors may provide a relevant contribution to the spread of mycobacteria exiting from latency.

Natural killer cells kill human melanoma cells with characteristics of cancer stem cells

2009-06-21

Experimental and clinical data suggest that tumours harbour a cell population retaining stem cell characteristics that can drive tumorigenesis. CD133 is considered an important cancer stem cells (CSC)-associated marker. In a large variety of human malignancies, including melanoma, CD133⁺ cells have been reported to comprise CSC. In this study, we show that melanoma cell lines are highly heterogeneous for the expression of several stem cell-associated markers including CD133, c-kit/CD117 and p75 neurotrophin receptor/CD271. Since no information is available on the ability of NK cells to recognize and lyse melanoma stem cells, we assessed whether melanoma cell lines, characterized by stem cell-like features, were susceptible to lysis by IL-2-activated NK cells. We show that activated NK cells efficiently kill malignant melanoma cell lines that were enriched in putative CSC by the use of different selection methods (i.e. CD133 expression, radioresistance or the ability to form melanospheres in stem cell-supportive medium). NK cell-mediated recognition and lysis of melanoma cells involved different combinations of activating NK receptors. Since CSC have been reported to be both drug resistant and radioresistant, our present data suggest that NK-based adoptive immunotherapy could represent a novel therapeutic approach to possibly eradicate metastatic melanoma.

Roles of PU.1 in monocyte- and mast cell-specific gene regulation: PU.1 transactivates CIITA pIV in cooperation with IFN-{gamma}

2009-06-21

Over-expression of PU.1, a myeloid- and lymphoid-specific transcription factor belonging to the Ets family, induces monocyte-specific gene expression in mast cells. However, the effects of PU.1 on each target gene and the involvement of cytokine signaling in PU.1-mediated gene expression are largely unknown. In the present study, PU.1 was over-expressed in two different types of bone marrow-derived cultured mast cells (BMMCs): BMMCs cultured with IL-3 plus stem cell factor (SCF) and BMMCs cultured with pokeweed mitogen-stimulated spleen-conditioned medium (PWM-SCM). PU.1 over-expression induced expression of MHC class II, CD11b, CD11c and F4/80 on PWM-SCM-cultured BMMCs, whereas IL-3/SCF-cultured BMMCs expressed CD11b and F4/80, but not MHC class II or CD11c. When IFN- was added to the IL-3/SCF-based medium, PU.1 transfectant acquired MHC class II expression, which was abolished by antibody neutralization or in Ifngr^–/– BMMCs, through the induction of expression of the MHC class II transactivator, CIITA. Real-time PCR detected CIITA mRNA driven by the fourth promoter, pIV, and chromatin immunoprecipitation indicated direct binding of PU.1 to pIV in PU.1-over-expressing BMMCs. PU.1-over-expressing cells showed a marked increase in IL-6 production in response to LPS stimulation in both IL-3/SCF and PWM-SCM cultures. These results suggest that PU.1 overproduction alone is sufficient for both expression of CD11b and F4/80 and for amplification of LPS-induced IL-6 production. However, IFN- stimulation is essential for PU.1-mediated transactivation of CIITA pIV. Reduced expression of mast cell-related molecules and transcription factors GATA-1/2 and up-regulation of C/EBP in PU.1 transfectants indicate that enforced PU.1 suppresses mast cell-specific gene expression through these transcription factors.

Nasal vaccination with troponin reduces troponin specific T-cell responses and improves heart function in myocardial ischemia-reperfusion injury

2009-06-21

Myocardial ischemia with subsequent reperfusion (MI/R) can lead to significant myocardial damage. Ischemia initiates inflammation at the blood–microvascular endothelial cell interface and contributes significantly to both acute injury and repair of the damaged tissue. We have found that MI/R injury in mice is associated with a cellular immune response to troponin. Myocardial cells exclusively synthesize troponin and release the troponin into the bloodstream following injury. Mucosally administered proteins induce T cells that secrete anti-inflammatory cytokines such as IL-10 and transforming growth factor β at the anatomical site where the protein localizes. We found that nasal administration of the three subunits of troponin (C, I and T isoforms), given prior to or 1 h following MI/R, decreased infarct size by 40% measured 24 h later. At 1.5 months following MI/R, there was a 50% reduction in infarct size and improvement in cardiac function as measured by echocardiography. Protection was associated with a reduction of cellular immunity to troponin. Immunohistochemistry demonstrated increased IL-10 and reduced IFN- in the area surrounding the ischemic infarct following nasal troponin. Adoptive transfer of CD4+ T cells to mice from nasally troponin-treated mice 1 h after the MI/R decreased infarct size by 72%, whereas CD4+ T cells from IL-10–/– mice or nasally BSA-treated mice had no effect. Our results demonstrate that IL-10-secreting CD4+ T cells induced by nasal troponin reduce injury following MI/R. Modulation of cardiac inflammation by nasal troponin provides a novel treatment to decrease myocardial damage and enhance recovery after myocardial ischemia.

Foxo3-/- mice demonstrate reduced numbers of pre-B and recirculating B cells but normal splenic B cell sub-population distribution

2009-06-21

B cell antigen receptor (BCR) cross-linking promotes proliferation and survival of mature B cells. Phosphoinositide-3-kinase-mediated down-regulation of pro-apoptotic and anti-mitogenic genes such as the Foxo family of transcription factors is an important component of this process. Previously, we demonstrated that BCR signaling decreases expression of transcripts for Foxo1, Foxo3 and Foxo4. We now show that BCR-induced down-regulation of Foxo3 and Foxo4 mRNA expression occurs via distinct mechanisms from those established for Foxo1. While Foxo1, Foxo3 and Foxo4 bind the same DNA sequence, the differential control of their expression upon B cell activation suggests that they may have unique functions in the B lineage. To begin to address this issue, we evaluated B cell development and function in Foxo3–/– mice. No effect of Foxo3 deficiency was observed with respect to the following parameters in the splenic B cell compartment: sub-population distribution, proliferation, in vitro differentiation and expression of the Foxo target genes cyclin G2 and B cell translocation gene 1. However, Foxo3–/– mice demonstrated increased basal levels of IgG2a, IgG3 and IgA. A significant reduction in pre-B cell numbers was also observed in Foxo3–/– bone marrow. Finally, recirculating B cells in the bone marrow and peripheral blood were decreased in Foxo3–/– mice, perhaps due to lower than normal expression of receptor for sphingosine-1 phosphate, which mediates egress from lymphoid organs. Thus, Foxo3 makes a unique contribution to B cell development, B cell localization and control of Ig levels.

The analysis of the functions of human B and T cells in humanized NOD/shi-scid/{gamma}cnull (NOG) mice (hu-HSC NOG mice)

2009-06-21

‘Humanized mice’ are anticipated to be a valuable tool for studying the human immune system, but the reconstituted human immune cells have not yet been well characterized. Here, we extensively investigated the differentiation and functions of human B and T cells in a supra-immunodeficient mouse strain, NOD/shi-scid/c^null (NOG) reconstituted with CD34⁺ hematopoietic stem cells obtained from umbilical cord blood. In these hu-HSC NOG mice, the development of human B cells was partially blocked, and a significant number of B-cell progenitors accumulated in the spleen. The mature CD19⁺IgM⁺IgD⁺ human B cells of the hu-HSC NOG mice could produce IgG in vivo and in vitro by antigenic stimulation. In contrast, although human T cells with an apparently normal phenotype developed, most of them could neither proliferate nor produce IL-2 in response to antigenic stimulation by anti-CD3 and anti-CD28 antibodies in vitro. The positive selection of human T cells in the thymus was sufficiently functional, if not complete, and mainly mediated by mouse class II, suggesting that the human T cells lost their function in the periphery. We found that multiple mechanisms were involved in the T-cell abnormalities. Collectively, our results demonstrate that further improvements are necessary before humanized mice with a functional human immune system are achieved.

Activation of invariant NKT cells confers protection against Chlamydia trachomatis-induced arthritis

Bharhani, M. S., Chiu, B., Na, K.-S., Inman, R. D. — 2009-06-21

The role of invariant NKT (iNKT) cells in reactive arthritis is unknown. We explored the functional role of NKT cells in reactive arthritis using an established murine model of Chlamydia trachomatis-induced arthritis (CtIA). CtIA in wild-type and CD1d knockout (KO) mice was induced by intra-articular injection of C. trachomatis. The effect of -galactosylceramide (-GalCer) activation of iNKT cells was investigated by intra-peritoneal administration of -GalCer. Histopathological and phenotypic changes, chlamydial clearance and cytokine and chemokine production in synovial tissue of the knee joint were investigated after onset of the arthritis. The severity of CtIA was significantly increased in CD1d KO mice, which was associated with decrease in bactericidal cytokine IFN-, regulatory cytokines IL-4 and IL-10 and increase in pro-inflammatory chemokines macrophage inflammatory protein-2 (MIP-2) and IFN--inducible protein-10 (IP-10). Local clearance of the pathogen from the joint was also decreased. Prior treatment of mice with -GalCer, a potent activator of iNKT cells, significantly reduced the severity of CtIA in mice. The amelioration of CtIA was associated with decrease in chlamydial load and induction of cytokines IFN-, IL-4 and IL-10 and significant suppression of MIP-2 and IP-10. Treatment of established CtIA with -GalCer also demonstrated modulation of CtIA and decrease in chlamydial load. These results suggest that iNKT cells are protective against CtIA and -GalCer-activated iNKT cells have an immunoregulatory role not only in preventing the induction of reactive arthritis but also in modulating established disease.

PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation

2009-06-21

Dendritic cells (DC) are key players in the initiation and modulation of adaptive immune responses due to their ability to acquire and present antigen and stimulate T cells. For the induction of effector T cell functions, antigen must be presented by activated DC. In this study, we have compared uptake of antigen by mouse DC in the presence of different Toll-like receptor (TLR) agonists, which are potent inducers of DC activation. Here we show that the reduction in uptake of soluble antigen in the presence of the viral double-stranded RNA (dsRNA) analogues polyinosinic–polycytidylic acid and Ampligen is independent of TLR-mediated DC activation. A reduction in antigen uptake by bone marrow-derived and splenic DC was also observed in response to other RNA homopolymers such as polyinosinic and polyguanylic acids, which are known inhibitors of scavenger receptor-mediated endocytosis. Pinocytosis and mannose receptor-mediated uptake of soluble antigen were not affected by any of the tested nucleic acids. The reduction in antigen uptake by dsRNA did not negatively influence the T cell stimulating properties of the DC. In summary, we conclude that the decrease in antigen endocytosis observed in the presence of a variety of TLR agonists is independent of TLR signalling and is caused by competition for specific surface receptors that are involved in the uptake of these TLR agonists and the antigen.

Gfi1 negatively regulates Th17 differentiation by inhibiting ROR{gamma}t activity

2009-06-21

T_h cells have long been divided into two subsets, T_h1 and T_h2; however, recently, T_h17 and inducible regulatory T (iTreg) cells were identified as new T_h cell subsets. Although T_h1- and T_h2-polarizing cytokines have been shown to suppress T_h17 and iTreg development, transcriptional regulation of T_h17 and iTreg differentiation by cytokines remains to be clarified. In this study, we found that expression of the growth factor independent 1 (Gfi1) gene, which has been implicated in T_h2 development, was repressed in T_h17 and iTreg cells compared with T_h1 and T_h2 lineages. Gfi1 expression was enhanced by the IFN-/STAT1 and IL-4/STAT6 pathways, whereas it was repressed by the transforming growth factor-β1 stimulation at the promoter level. Over-expression of Gfi1 strongly reduced IL-17A transcription in the EL4 T cell line, as well as in primary T cells. This was due to the blockade of recruitment of retinoid-related orphan receptor t to the IL-17A promoter. In contrast, IL-17A expression was significantly enhanced in Gfi1-deficient T cells under T_h17-promoting differentiation conditions as compared with wild-type T cells. In contrast, the impacts of Gfi1 in iTregs were not as strong as in T_h17 cells. Taken together, these data strongly suggest that Gfi1 is a negative regulator of T_h17 differentiation, which represents a novel mechanism for the regulation of T_h17 development by cytokines.