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2008-08-21

What roles do regulatory T cells play in the control of the adaptive immune response?

Cohn, M. — 2008-08-21

The immune system, like many systems responsive to specific stimuli, requires feedback regulation. The key regulatory element determining antigen-specific responsiveness is the effector T helper. As the response tends to overshoot, a feedback control of the magnitude of the response is critical to avoid immunopathology. This is the proposed role of the effector T suppressor (T_s). The reasons for this interpretation of the data are discussed as are the reasons that the competing postulate is ruled out, namely that T_s function in determining the self-non-self-discrimination. The regulatory T cell family consists of two lineages, T helpers and T_s. Differentiated derivatives of the T helper lineage drive the expression and amplification of specific classes of defensive effector cells. T_s feedback to limit the magnitude of the process so that debilitating immunopathology is acceptably infrequent.

LFA-1 decreases the antigen dose for T cell activation in vivo

2008-08-21

Leukocyte adhesion molecule leukocyte function-associated antigen (LFA)-1 not only mediates intercellular binding but also delivers co-stimulatory signals in T cells. LFA-1 has been shown to decrease the threshold of TCR signal and an antigen dose required for T cell activation and proliferation in vitro. However, physiological significance of the role of LFA-1 in TCR signal has remained unclear. We examined whether LFA-1 decreased the antigen dose for T cell activation in vivo. We showed here that, although collagen-induced arthritis (CIA) could not be induced by immunization and challenge with a standard amount of type-II collagen in LFA-1-deficient mice, a higher dose of the antigen did induce CIA in the absence of LFA-1. We also showed that CD4⁺ T cells could be primed by immunization with a high, but not low, dose of ovalbumin antigen in LFA-1-deficient mice. These results suggest that LFA-1 decreases the threshold of TCR signal for T cell activation in vivo as well as in vitro. Further studies using TCR-transgenic LFA-1-deficient mice showed that LFA-1 cooperated with TCR in sustained Erk1/2 phosphorylation. Moreover, TCR could induce sustained Erk1/2 phosphorylation in the absence of LFA-1 when T cells were stimulated with a high, but not low, dose of antigen, suggesting that LFA-1 may cooperate with TCR in sustaining Erk1/2 phosphorylation.

IL-17A is produced by Th17, {gamma}{delta} T cells and other CD4- lymphocytes during infection with Salmonella enterica serovar Enteritidis and has a mild effect in bacterial clearance

Schulz, S. M., Kohler, G., Holscher, C., Iwakura, Y., Alber, G. — 2008-08-21

T_h17 cells represent a new pro-inflammatory T_h cell lineage distinct from T_h1 and T_h2 cells. T_h17 cells have been shown to be involved in extracellular bacterial infection but their role in intracellular infection remains unclear. We found antigen-specific IL-17A production during a systemic infection of mice with the facultative intracellular bacterium Salmonella enterica serovar Enteritidis (S. Enteritidis) and examined the function and cellular source of IL-17A during the adaptive immune response to S. Enteritidis. Infected IL-17A^–/– mice survived completely after inoculation with the highest infection dose found to be sub-lethal for wild-type (WT) C57BL/6 mice. However, at 20 and 80 days post-infection (d.p.i.), we repeatedly found mildly elevated bacterial burden in spleen and liver of IL-17A^–/– mice as compared with WT mice. Overall, IL-17A^–/– mice showed reduced clearance of S. Enteritidis. S. Enteritidis-specific IL-17A production was induced in splenocytes and lymph node cells of infected WT mice at both time points, 20 and 80 d.p.i. Classical CD4⁺ T_h17 cells developed upon infection with Salmonella. CD4^– TCR⁺ and CD4^– TCR^– cells were found to be additional IL-17A-producing cell populations. In infected IL-17A^–/– mice, a normal T_h1 cytokine profile was observed consistent with the overall subtle phenotype. Nevertheless, in the absence of IL-17A, recruitment of neutrophils and delayed-type hypersensitivity (DTH) reactivity was significantly compromised. Our data indicate that IL-17A responses are induced by Salmonella and mildly contribute to protective immunity during S. Enteritidis infection. Thus, IL-17A complements the IL-12/IFN- axis which is essential for protective immunity against salmonellosis in mice and men.

Contribution of IL-1 to resistance to Streptococcus pneumoniae infection

2008-08-21

The role of IL-1 in susceptibility to Streptococcus pneumoniae infection was studied in mice deficient in genes of the IL-1 family [i.e. IL-1^–/–, IL-1β^–/–, IL-1/β^–/– and IL-1R antagonist (IL-1Ra)^–/– mice] following intra-nasal inoculation. Intra-nasal inoculation of S. pneumoniae of IL-1β^–/– and IL-1/β^–/– mice displayed significantly lower survival rates and higher nasopharyngeal and lung bacterial load as compared with control, IL-1^–/– and IL-1Ra^–/– mice. Treatment of IL-1β^–/– mice with rIL-1β significantly improved their survival. A significant increase in blood neutrophils was found in control, IL-1^–/– and IL-1Ra^–/– but not in IL-1β^–/–and IL-1/β^–/– mice. Local infiltrates of neutrophils and relatively preserved organ architecture were observed in the lungs of IL-1^–/– and control mice. However, S. pneumoniae-infected IL-1β^–/–, IL-1/β^–/– and IL-1Ra^–/– mice demonstrated diffuse pneumonia and tissue damage. Altogether, all three isoforms contribute to protection against S. pneumoniae; our results point to differential role of IL-1 and IL-1β in the pathogenesis and control of S. pneumoniae infection and suggest that IL-1β has a major role in resistance to primary pneumococcal infection while the role of IL-1 is less important.

Characterization of a lymphocyte subset displaying a unique regulatory activity in human decidua

Amsalem, H., Gaiger, A., Mizrahi, S., Yagel, S., Rachmilewitz, J. — 2008-08-21

One of the most intriguing mechanisms of early pregnancy is the maternal immune tolerance toward her semi-allogeneic fetus, specifically in face of the accumulation of lymphocytes to high numbers at implantation sites. Here, we propose that a regulatory decidual lymphocyte (dL) population prevent the activation of reactive T cells and by that may maintain immune tolerance in the decidua. dLs were isolated from first trimester decidua and were then co-cultured with PBMC that were stimulated with anti-CD3 mAbs. Cytokine secretion to the media as well as the proliferative response were tested. The data demonstrate that dLs inhibit the production of IFN-, tumor necrosis factor- (TNF-) and IL-5 but not CD25 expression, IL-2 production or proliferation in the responder PBMC. Suppression is mediated by a cell contact-dependent mechanism, was not restricted by the MHC and was not reversed by the addition of exogenous IL-2 although the inhibitory sub-population was identified as CD3⁺CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells (Treg). Interestingly, suppression can also be overcome by the addition the endotoxin LPS, suggesting a mechanism for preterm labor triggered by chorioamnionitis. While these characteristics are in contrast to known peripheral CD4⁺CD25⁺ Treg activity, we identified these cells as the cellular subset responsible for the regulatory activity, suggesting that in decidua a functionally unique regulatory lymphocyte subset exist. These findings suggest the existence of a dynamic regulatory system in human decidua that is highly responsive to environmental factors.

Human NK cells directly recognize Mycobacterium bovis via TLR2 and acquire the ability to kill monocyte-derived DC

Marcenaro, E., Ferranti, B., Falco, M., Moretta, L., Moretta, A. — 2008-08-21

NK cells are important players of the early innate defense against various pathogens. In this study, we investigated the interaction between human NK cells and Mycobacterium bovis [bacille Calmette–Guérin (BCG)] and we determined whether and how such an interaction might impact on NK cell activation, cytokine production and cytotoxicity. We show that highly purified NK cells, upon short-term co-culture with BCG, expressed activation markers including CD69 and CD25. Moreover, these NK cells released IFN-gamma and tumor necrosis factor-alpha and killed more efficiently different targets including monocyte-derived immature dendritic cell. All these functions were strongly up-regulated in the presence of exogenous IL-12. Although more efficient responses were detected in NK cell populations displaying an NCR^bright phenotype, no direct evidence of an involvement of triggering NK receptors in BCG recognition could be obtained. On the other hand, anti-toll-like receptor (TLR)2 mAb inhibited NK cell responses to BCG, suggesting that NK cells may express a functional TLR2, which plays a role in their mechanism of direct BCG recognition. Taken together, these data suggest that BCG, by inducing simultaneous activation of NK and antigen-presenting cells via their ‘shared’ TLR2, can promote efficient bidirectional NK–dendritic cell interactions necessary for subsequent priming of T_h1 responses.

Soluble G protein of respiratory syncytial virus inhibits Toll-like receptor 3/4-mediated IFN-beta induction

2008-08-21

Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR) 3 on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early (~4 h) induction of IFN-β usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-β induction, and this early IFN-β-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) reporter analysis in HEK293 cells, polyI:C- or LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR3/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-I/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-β in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV.

ADAM10 is essential for proteolytic activation of Notch during thymocyte development

Tian, L., Wu, X., Chi, C., Han, M., Xu, T., Zhuang, Y. — 2008-08-21

Notch signaling pathway has been shown to play essential roles in T lymphocyte development. Activation of Notch requires a sequential proteolytic cleavage, which converts Notch from the full-length membrane-bound form to a transcriptionally active intracellular fragment. Studies in Drosophila showed that Kuzbanian (Kuz) is responsible for the enzymatic cleavage of extracellular S2 site upon Notch binding to its ligand Delta. Both a disintegrin and metalloprotease (ADAM) 10 and ADAM17, members of the ADAM family metalloproteases, have been indicated as the mammalian counterpart of Kuz in activating Notch in mammals. Here, we investigated functions of ADAM10 in Notch signaling during thymocyte development. We show that conditional disruption of the Adam10 gene in mouse thymocytes results in a developmental defect similar to the phenotypes previously described for T lineage-specific disruption of Notch1. We further show that the activation of Notch1 and its downstream target genes Deltex-1 and Pre-Ta are impaired in Adam10-deficient thymocytes. Our study demonstrates a T cell intrinsic role for Adam10 in activation of Notch1 during thymocyte development.

Phenotypic classification of human CD4+ T cell subsets and their differentiation

Okada, R., Kondo, T., Matsuki, F., Takata, H., Takiguchi, M. — 2008-08-21

CD4⁺ T cells have T_h cell function and include two major functional subsets, T_h1 and T_h2. However, there are a restricted number of studies concerning phenotypic classification of human CD4⁺ T cells. Here by using seven- and eight-color flow cytometric analysis, we investigated the function of the subsets classified by four markers, CD27, CD28, CD45RA and CCR7. Five major subsets were identified by using these markers. These subsets showed different patterns of cytokine production after they were stimulated with phorbol myristate acetate and ionomycin. The analyses of cytokine production suggested that CCR7⁺CD45RA⁺CD27⁺CD28⁺, CCR7⁺CD45RA^–CD27⁺CD28⁺ and CCR7^–CD45RA^–CD27⁺CD28⁺ subsets were naive, central memory and effector memory T cells, respectively, whereas CCR7^–CD45RA^–CD27^–CD28⁺ and CCR7^–CD45RA^–CD27^–CD28^– subsets included T_h1 and T_h2 cells. The analysis of cytokine production by these subsets stimulated with anti-CD3 and anti-CD28 mAbs or with human cytomegalovirus antigens showed that IFN- production was significantly higher in the CCR7^–CD45RA^–CD27^–CD28^– subset than in other subsets and that both CCR7^–CD45RA^–CD27^–CD28⁺ and CCR7^–CD45RA^–CD27^–CD28^– subsets produced a higher level of IL-4 than did other subsets. Our analyses demonstrated that the CCR7^–CD45RA^–CD27^–CD28^– subset predominantly included T_h1 effector cells and that CCR7^–CD45RA^–CD27^–CD28⁺ subsets included T_h1 and T_h2 effector memory/effector cells as well as unclassified cells. The analysis of classification by using these four markers also suggested the differentiation pathway of human CD4⁺ T cells.

T cell-derived IL-3 plays key role in parasite infection-induced basophil production but is dispensable for in vivo basophil survival

2008-08-21

Enhanced basophil production is often associated with T_h2-related conditions such as parasite infections or allergic inflammations. Our previous study demonstrated that T cell activation is necessary to promote basophil production in Nippostrongylus brasiliensis (Nb)-infected mice. Yet, mechanisms underlying how T cells aid infection-induced basophil production are not clear. In this report, we show that IL-3 produced by T cells activated by the infection enhances basophil production in Nb-infected mice. IL-3-deficient mice or Rag2–/– recipients of IL-3-deficient T cells but not of wild-type T cells failed to support basophil production following the Nb infection. Interestingly, although IL-3 was critical for preventing basophil apoptosis in vitro, IL-3 had little contribution to basophil survival and proliferation in vivo. Collectively, these results highlight a novel mechanism by which activation of adaptive immune components induces basophil production but not basophil survival via IL-3 production.

Manipulation of immune system via immortal bone marrow stem cells

Ruedl, C., Khameneh, H. J., Karjalainen, K. — 2008-08-21

Extensive amplification of hematopoietic stem cells (HSCs) and their multipotent primitive progenitors (MPPs) in culture would greatly benefit not only clinical transplantation but also provide a potential tool to manipulate all cellular lineages derived from these cells for gene therapy and experimental purposes. Here, we demonstrate that mouse bone marrow cultures containing cells engineered to over-express NUP98–HOXB4 fusion protein support self-renewal of physiologically normal HSC and MPP for several weeks leading practically to their unlimited expansion. This allows time consuming and cumulative in vitro experimental manipulations without sacrificing their ability to differentiate in vivo or in vitro to any hematopoietic lineage.

Prostaglandin E2 is a major soluble factor produced by stromal cells for preventing inflammatory cytokine production from dendritic cells

2008-08-21

Dendritic cells (DCs) are specialized antigen-presenting cells that play pivotal roles in initiating immune responses. However, DC maturation is usually strongly restricted by the stromal microenvironment, especially in non-lymphoid tissues, such as skin and mucosa. Although suppression of DC maturation by stromal cells has been well documented, the molecular basis of this suppression has not been established. In this study, we examined the role of fibroblasts for DC maturation in vitro. The mouse embryonic fibroblasts (MEFs) strongly suppressed LPS-induced DC maturation. Although suppression of class II MHC and CD40 required DC–MEF contact, soluble factors in the culture supernatant of MEFs were sufficient for the suppression of IL-12 and tumor necrosis factor- production. Using molecular-size selection and HPLC, we determined that prostaglandin E₂ (PGE₂) is a major soluble inhibitory factor secreted by MEFs. This was confirmed by the fact that cyclooxygenase inhibitors inhibited the production of the suppressive factor by MEFs. These results suggest that PGE₂ is a major soluble factor produced by MEFs for the suppression of inflammatory cytokine production from DCs, while a contact mechanism between MEFs and DCs is required for the suppression to induce T cell-stimulating molecules.

Protection of IFN-{gamma} signaling-deficient NOD mice from diabetes by cyclophosphamide

Mori, Y., Kato, T., Kodaka, T., Kanagawa, E. M., Hori, S., Kanagawa, O. — 2008-08-21

Non-obese diabetic (NOD) mice that are genetically deficient in either IFN- or β chain of the IFN-R develop diabetes with similar kinetics to wild-type NOD mice. In the current study, we demonstrated that treatment of IFN- signaling-deficient NOD mice with cyclophosphamide (CY) not only fails to induce acute diabetes but also confers permanent protection from diabetes. Protection was mediated by the preferential generation of regulatory T cells (Treg cells) that are capable of suppressing the diabetogenic process, with no change in the total number and function of Treg cells. Moreover, CY treatment of IFN- signaling-deficient NOD mice reversed the ongoing pathogenic process and eliminated cellular infiltrates of pancreatic islets. While these results have been derived using a genetically modified mouse model of diabetes, they indicate that knowledge of host genetic factors and environmental factors influencing the development of Type I diabetes mellitus may provide a rational approach to develop a means to reverse the development of Type I diabetes in human.