CD8- dendritic cell activation status plays an integral role in influencing Th2 response development

Whether dendritic cells (DC) play a passive or active role in Th2 response induction is poorly understood. In this study, we show that CD8- DC pulsed with Th2-polarizing Ag (soluble egg Ag (SEA)) from Schistosoma mansoni potently stimulate Th2 responses in vivo and in vitro while failing to undergo a conventional maturation process. Thus, in contrast to DC pulsed with the Th1 response inducing Ag Propionebacterium acnes, SEA-exposed DC exhibit a phenotype that is most similar to that of immature DC, failing to up-regulate expression of CD40, CD54, CD80, CD86, or OX40L; producing no detectable IL-4, IL-10, or IL-12; and displaying only a minor increase in MHC class II expression. Importantly, in vitro derived DC exposed to SEA were phenotypically similar to CD8- DC isolated from active S. mansoni infection. By discriminating between different types of pathogen and responding appropriately, CD8- DC play a major role in the decision process to mount either a Th1 or Th2 response.     

GM-CSF plays a key role in zymosan-stimulated human dendritic cells for activation of Th1 and Th17 cells

In this study, we compared the effects of zymosan and LPS on human monocyte-derived dendritic cells. The specific effects of zymosan on the expression of several key cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins (IL-1α, IL-1β and IL-12 p70) were quite distinct from the effects of LPS. Unlike activation with LPS, DCs activated by zymosan expressed little or no IL-12 p70 due to lack of expression of the p35 subunit. However, treatment with zymosan resulted in a substantial increase in Th1 and Th17 cell-polarizing capacity of DCs. Furthermore, the GM-CSF secreted by zymosan-activated DCs enhanced IL-23 production, resulting in activation of a Th17 response. GM-CSF and IL-27, rather than IL-12 p70, were both major direct contributors to the activation of a Th1 response. This signaling mechanism is distinct and yet complementary to LPS-mediated T-cell activation. We suggest that this novel zymosan-induced GM-CSF-mediated signaling network may play a key role in regulating specific immune cell type activities.     

Raf1 plays a pivotal role in lipopolysaccharide-induced activation of dendritic cells

Activation of extracellular-regulated kinases 1/2 (ERK) is involved in lipopolysaccharide (LPS)-induced cellular responses such as the increased production of proinflammatory cytokines. However, mitogen-activated protein kinases (MAPKs) such as p38 are also activated by LPS and have been postulated to be important in the control of these end points. Therefore, establishing the relative contribution of MAPKs in each cell type is important, as is elucidating the molecular mechanisms by which these MAPKs are activated in LPS-induced signaling cascades. We demonstrated in DC2.4 dendritic cells that ERK regulates tyrosine phosphorylation of phosphatidyl-inositol-3-kinase (PI3-K) and the production of TNF-alpha. We also demonstrated that Raf1 is phosphorylated and involved in the production of TNF-alpha and tyrosine phosphorylation of PI3-K via ERK. Raf1 also regulates the activation of NF-kappaB. We propose that Raf1 plays a pivotal role in LPS-induced activation of the dendritic cells.     

The source of early IFN-gamma that plays a role in Th1 priming

When naive CD4 T cells are primed, they rapidly differentiate into polarized Th1 and/or Th2 phenotypes. A major factor in producing such polarization is the early production of cytokines (IL-12 and IFN-gamma in the case of Th1 cells and IL-4 in the case of Th2 cells). One issue that remains unresolved is the source of the early IFN-gamma that synergizes with IL-12 to fully polarize CD4 T cells into Th1 cells. We have examined this question by injecting mice with anti-CD3 and examining cells from normal and various MHC-knockout mice. We found that IFN-gamma is induced rapidly in a small subset of CD8 T cells. This subset is absent in mice that lack beta2-microglobulin, but not in K(b)D(b)-double-knockout mice, indicating that these CD8 T cells are dependent on nonclassical MHC class Ib molecules. The early burst of IFN-gamma polarizes CD4 T cells toward Th1 cells, in part by stimulating the release of IL-12 from APC. We also use TAP- and CD1-knockout mice to show that such cells are not CD1-restricted NK T cells, nor are they dependent on TAP-1 transport for surface expression of the relevant MHC class Ib molecule. Therefore, they arise on MHC class Ib molecules that do not depend on TAP-1 transporters.     

A recombinant malaria protein that can induce Th1 and CD8+ T cell responses without antibody formation.

Inbred strains of mice were immunized with p190-3, a 38-kDa recombinant protein derived from p190, a major merozoite surface Ag of the malaria parasite Plasmodium falciparum. Ag-specific proliferative T cell responses were obtained in H-2b, H-2d, and H-2k mouse strains. Surprisingly, mice of the H-2b haplotype (e.g., C57BL/6) did not give a measurable antibody response to the recombinant protein administered in Freund's adjuvant, but CD8+/CD4- as well as CD4+/CD8- T cells specific for p190-3 could be obtained after in vivo priming and in vitro selection with Ag. Distinct epitopes of p190-3 recognized by the CD8+ and CD4+ T cells from C57BL/6 mice were identified. The CD8+ T cells could kill H-2b APC in the presence of the appropriate epitope-containing peptide. The p190-3-specific CD4+ cells isolated from C57BL/6 mice were of the Th1 type. In contrast, Th2 cells, but no CD8+ T cells were present in a p190-3-specific line from BALB/c mice, which give good antibody responses to p190-3.     

Differential requirement for the CD40-CD154 costimulatory pathway during Th cell priming by CD8 alpha+ and CD8 alpha- murine dendritic cell subsets

Dendritic cells (DCs) regulate the development of distinct Th populations and thereby provoke appropriate immune responses to various kinds of Ags. In the present work, we investigated the role CD40-CD154 interactions play during the process of Th cell priming by CD8 alpha(+) and CD8 alpha(-) murine DC subsets, which have been reported to differently regulate the Th response. Adoptive transfer of Ag-pulsed CD8 alpha(+) DCs induced a Th1 response and the production of IgG2a Abs, whereas transfer of CD8 alpha(-) DCs induced Th2 cells and IgE Abs in vivo. Induction of distinct Th populations by each DC subset was also confirmed in vitro. Although interruption of CD80/CD86-CD28 interactions inhibited Th cell priming by both DC subsets, disruption of CD40-CD154 interactions only inhibited the induction of the Th1 response by CD8 alpha(+) DCs in vivo. CD40-CD154 interactions were not required for the proliferation of Ag-specific naive Th cells stimulated by either DC subset, but were indispensable in the production of IL-12 from CD8 alpha(+) DCs and their induction of Th1 cells in vitro. Taken together, in our immunization model of Ag-pulsed DC transfer, CD40-CD154 interactions play an important role in the development of CD8 alpha(+) DC-driven Th1 responses but not CD8 alpha(-) DC-driven Th2 responses to protein Ags.     

Microbial compounds selectively induce Th1 cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse th cell-polarizing signals

Upon microbial infection, specific Th1 or Th2 responses develop depending on the type of microbe. Here, we demonstrate that different microbial compounds polarize the maturation of human myeloid dendritic cells (DCs) into stably committed Th1 cell-promoting (DC1) or Th2 cell-promoting (DC2) effector DCs that polarize Th cells via different mechanisms. Protein extract derived from the helminth Schistosoma mansoni induced the development of DC2s that promote the development of Th2 cells via the enhanced expression of OX40 ligand. Likewise, toxin from the extracellular bacterium Vibrio cholerae induced development of DC2s as well, however, via an OX40 ligand-independent, still unknown mechanism. In contrast, toxin from the intracellular bacterium Bordetella pertussis induced the development of DC1s with enhanced IL-12 production, which promotes a Th1 cell development. Poly(I:C) (dsRNA, mimic for virus) induced the development of extremely potent Th1-inducing DC1, surprisingly, without an enhanced IL-12 production. The obtained DC1s and DC2s are genuine effector cells that stably express Th cell-polarizing factors and are unresponsive to further modulation. The data suggest that the molecular basis of Th1/Th2 polarization via DCs is unexpectedly diverse and is adapted to the nature of the microbial compounds.     

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

Introduction

Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)⁺ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.

Methods

CD1c⁺ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4⁺ T cells was measured.

Results

CD1c⁺ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4⁺ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.

Conclusions

This study suggests that increased numbers of CD1c⁺ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.     

CD38 plays a role in effective containment of mycobacteria within granulomata and polarization of Th1 immune responses against Mycobacterium avium

CD38 is a multifunctional ectoenzyme that behaves either as an enzyme, a cell adhesion molecule or as a cell surface receptor involved in cell signalling. It is expressed in cells of several lineages, including B and T lymphocytes, and macrophages. CD38 was shown to be important for the development of T-cell dependent humoral immune responses against extracellular pathogens. It also appears to be functionally important in macrophages, which are the host cells of Mycobacterium avium, an intracellular parasite that survives within these cells by avoiding a number of their microbicidal strategies. The present work aimed at investigating whether CD38 had any role on the immune response against mycobacterial infection. After intraperitoneal M. avium infection, the immune response of CD38KO mice was compared to that of their parental strain, C57Bl.6 mice. Absence of CD38 rendered mice more susceptible to mycobacterial infection. This susceptibility seems to be due to ineffective Th1 differentiation and polarization, which is essential for the control of M. avium infection. In addition, absence of CD38 seems to compromise the maintenance of the granulomatous barrier, leading to dissemination and unrestrained growth of mycobacteria.     

Effector function of diabetogenic CD4 Th1 T cell clones: a central role for TNF-alpha

Effector function of T cells in autoimmune diabetes has been widely studied with mixed populations of lymphoid T cells stimulated ex vivo, but this approach does not permit evaluation of the contribution by a single T cell clone in the inflammatory site during pathogenesis. We have investigated cytokine production both in vitro and in vivo in a panel of diabetogenic CD4 Th1 T cell clones derived from the NOD mouse. SuperArray analysis showed a common pattern of mRNA expression for inflammatory cytokines and receptors in vitro after TCR stimulation. Ex vivo intracellular cytokine staining demonstrated that two important inflammatory cytokines, IFN-gamma and TNF-alpha, were being made by these T cells recovered from the pancreas 6 days following adoptive transfer. TNF-alpha produced in the pancreas by pathogenic T cell clones and recruited macrophages was not the membrane-bound form. Secreted TNF-alpha can lead to production of multiple inflammatory chemokines, as were observed in the pathogenic clones by intracellular cytokine staining. Our results not only define the nature of an inflammatory cytokine response critical to development of diabetes, but also suggest its role in the regulation of other events during pathogenesis induced by CD4 T cells. Similar analyses in other models demonstrated that disease induced by CD4 T cell clones closely resembles spontaneous autoimmune diabetes in which both CD4 and CD8 T cells are required. Thus, cloned T cells in effect amplify effector function of T cells which otherwise may be difficult to detect without ex vivo stimulation.     

Synchronization of dendritic cell activation and antigen exposure is required for the induction of Th1/Th17 responses

The dendritic cell (DC) targeting/activation patterns required to elicit Th1/Th17 responses remain undefined. One postulated requirement was that of a physical linkage between Ags and immunomodulators. Accordingly, the separate same-site administration of Ag85B-ESAT-6 (hybrid-1 protein; H1), a mycobacterial fusion Ag, and the CAF01 liposome-based adjuvant induced similar Ab and weak Th2 responses as those of coformulated H1/CAF01 but failed to elicit Th1/Th17 responses. Yet, this separate same-site injection generated the same type and number of activated Ag(+)/adjuvant(+) DCs in the draining lymph nodes (LN) as that of protective H1/CAF01 immunization. Thus, targeting/activating the same DC population by Ag and adjuvant is not sufficient to elicit Th1/Th17 responses. To identify the determinants of Th1/Th17 adjuvanticity, in vivo tracking experiments using fluorescently labeled Ag and adjuvant identified that a separate same-site administration elicits an additional early Ag(+)/adjuvant(-) DC population with a nonactivated phenotype, resulting from the earlier targeting of LN DCs by H1 than by CAF01 molecules. This asynchronous targeting pattern was mimicked by the injection of free H1 prior to or with, but not after, H1/CAF01 or H1/CpG/ aluminum hydroxide immunization. The injection of soluble OVA similarly prevented the induction of Th1 responses by OVA/CAF01. Using adoptively transferred OT-2 cells, we show that the Ag targeting of LN DCs prior to their activation generates nonactivated Ag-pulsed DCs that recruit Ag-specific T cells, trigger their initial proliferation, but interfere with Th1 induction in a dose-dependent manner. Thus, the synchronization of DC targeting and activation is a critical determinant for Th1/Th17 adjuvanticity.     

SLG1 plays a role during G1 in the decision to enter or exit the cell cycle

Saccharomyces cerevisiae cells decide to divide during G1. If nutrients are abundant, cells pass through START and coordinately undergo DNA replication, bud emergence, and spindle pole body duplication. Phenotypic analysis of the slg1delta mutant revealed that this mutation uncouples post-START events. At the nonpermissive temperature, slg1delta cells that have undergone bud emergence but not DNA replication or SPB duplication accumulate. Furthermore, while wild-type cells arrest in GO when starved, the slg1delta mutant fails to arrest at this point; instead, cells with small buds accumulate. The slg1delta mutation displayed genetic interactions with cdc34, which encodes a regulator of exit from G1. This is consistent with a role of SLG1 in G1 regulation. Epitope-tagged Slg1p cofractionated with the plasma membrane, suggesting that Slglp may function by integrating external cues and relaying them to the interior of the cell. We propose that SLG1 plays a regulatory role in bud emergence or stationary phase.     

TLR-dependent activation stimuli associated with Th1 responses confer NK cell stimulatory capacity to mouse dendritic cells

Dendritic cells (DCs) have an important role in the activation of NK cells that exert direct antitumor and antimicrobial effects and can influence the development of adaptive T cell responses. DCs acquire NK cell stimulatory capacity after exposure to various stimuli. In this study we investigated the nature of the stimuli that confer to DCs the NK cell-activating capacity. After exposure of DCs to TLR-dependent and -independent microbial stimuli and to nonmicrobial stimuli, we evaluated the ability of activated DCs to elicit IFN-gamma production from NK cells in vitro and to promote NK cell activation in vivo. We show in this study that only TLR-dependent microbial stimuli typically associated with Th1 responses confer to DCs the ability to activate NK cells, whereas stimuli associated with Th2 responses do not have this property.     

Borrelia burgdorferi bb0426 encodes a 2'-deoxyribosyltransferase that plays a central role in purine salvage

Borrelia burgdorferi is an obligate parasite with a limited genome that severely narrows its metabolic and biosynthetic capabilities. Thus survival of this spirochaete in an arthropod vector and mammalian host requires that it can scavenge amino acids, fatty acids and nucleosides from a blood meal or various host tissues. Additionally, the utilization of ribonucleotides for DNA synthesis is further complicated by the lack of a ribonucleotide reductase for the conversion of nucleoside-5'-diphosphates to deoxynucleosides-5'-diphosphates. The data presented here demonstrate that B. burgdorferi must rely on host-derived sources of purine bases, deoxypurines and deoxypyrimidines for the synthesis of DNA. However, if deoxyguanosine (dGuo) is limited in host tissue, the enzymatic activities of a 2'-deoxyribosyltransferase (DRTase, encoded by bb0426 ), IMP dehydrogenase (GuaB) and GMP synthase (GuaA) catalyse the multistep conversion of hypoxanthine (Hyp) to dGMP for DNA synthesis. This pathway provides additional biochemical flexibility for B. burgdorferi when it colonizes and infects different host tissues.     

A role for the chemokine RANTES in regulating CD8 T cell responses during chronic viral infection

RANTES (CCL5) is a chemokine expressed by many hematopoietic and non-hematopoietic cell types that plays an important role in homing and migration of effector and memory T cells during acute infections. The RANTES receptor, CCR5, is a major target of anti-HIV drugs based on blocking viral entry. However, defects in RANTES or RANTES receptors including CCR5 can compromise immunity to acute infections in animal models and lead to more severe disease in humans infected with west Nile virus (WNV). In contrast, the role of the RANTES pathway in regulating T cell responses and immunity during chronic infection remains unclear. In this study, we demonstrate a crucial role for RANTES in the control of systemic chronic LCMV infection. In RANTES^−/− mice, virus-specific CD8 T cells had poor cytokine production. These RANTES^−/− CD8 T cells also expressed higher amounts of inhibitory receptors consistent with more severe exhaustion. Moreover, the cytotoxic ability of CD8 T cells from RANTES^−/− mice was reduced. Consequently, viral load was higher in the absence of RANTES. The dysfunction of T cells in the absence of RANTES was as severe as CD8 T cell responses generated in the absence of CD4 T cell help. Our results demonstrate an important role for RANTES in sustaining CD8 T cell responses during a systemic chronic viral infection.