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Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Agency for Science, Technology and Research

During an infection, one of the principal challenges for the host is to detect the pathogen and activate a rapid defensive response. The Toll-like family of receptors (TLRs), among other pattern recognition receptors (PRR), performs this detection process in vertebrate and invertebrate organisms. These type I transmembrane receptors identify microbial conserved structures or pathogen-associated molecular patterns (PAMPs). Recognition of microbial components by TLRs initiates signaling transduction pathways that induce gene expression. These gene products regulate innate immune responses and further develop an antigen-specific acquired immunity. TLR signaling pathways are regulated by intracellular adaptor molecules, such as MyD88, TIRAP/Mal, between others that provide specificity of individual TLR- mediated signaling pathways. TLR-mediated activation of innate immunity is involved not only in host defense against pathogens but also in immune disorders. The involvement of TLR-mediated pathways in auto-immune and inflammatory diseases is described in this review article.

Toll-like receptors are key participants in innate immune responses.

Unlike the ∼1% of γδ TCR-positive T cells being regularly present in blood and secondary lymphoid organs (peripheral γδ T cells), ∼50-60% of small intestinal intraepithelial lymphocytes (iIELs) in the mouse express the γδ TCR (γδ iIELs). In this study, we investigated the overlap and exchange of γδ iIELs and γδ T cells found in peripheral secondary lymphoid organs. Using two-photon laser-scanning microscopy, we found γδ T cells within peripheral lymph nodes to be highly motile, whereas γδ iIELs were characterized by a locally confined scanning behavior. Our results implied a strict separation of peripheral γδ T cells and γδ iIELs. Nevertheless, γδ iIELs could be efficiently regenerated from bone marrow-derived precursors in irradiated or T cell-deficient adult mice. However, outside the intestinal epithelium, survival of γδ iIELs was very poor. In CCR9-deficient mice, homing of γδ iIELs was impaired, but did not lead to an accumulation of γδ iIEL-like cells in the periphery. Conversely, in situations in which specific γδ iIEL niches were empty, adoptive transfer of isolated γδ iIELs led to a sustained engraftment of transferred γδ iIELs in the intestinal epithelium for at least 100 d. Furthermore, we demonstrated by heterotopic intestinal transplantation experiments that an exchange of γδ iIELs only rarely happens in the steady state of adult mice. We therefore conclude that peripheral versus intestinal intraepithelial γδ T cells are exclusive, nonoverlapping populations that virtually do not exchange with each other.

Intra- and intercompartmental movement of gammadelta T cells: intestinal intraepithelial and peripheral gammadelta T cells represent exclusive nonoverlapping populations with distinct migration characteristics.

Intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL) are involved in the maintenance of epithelial integrity. γδ iIEL have an activated phenotype, characterized by CD69 expression and increased cell size compared with systemic T lymphocytes. As an additional activation marker, the majority of γδ iIEL express the CD8αα homodimer. However, our knowledge about cognate ligands for most γδ TCR remains fragmentary and recent advances show that γδ T cells including iIEL may be directly activated by cytokines or through NK-receptors, TLR and other pattern recognition receptors. We therefore asked whether the TCR of γδ iIEL was functional beyond its role during thymic selection. Using TcrdH2BeGFP (Tcrd, T-cell receptor δ locus; H2B, histone 2B) reporter mice to identify γδ T cells, we measured their intracellular free calcium concentration in response to TCR-crosslinking. In contrast to systemic γδ T cells, CD8αα(+) γδ iIEL showed high basal calcium levels and were refractory to TCR-dependent calcium-flux induction; however, they readily produced CC chemokine ligand 4 (CCL4) and IFN-γ upon TCR triggering in vitro. Notably, in vivo blocking of the γδ TCR with specific mAb led to a decrease of basal calcium levels in CD8αα(+) γδ iIEL. This suggests that the γδ TCR of CD8αα(+) γδ iIEL is constantly being triggered and therefore functional in vivo.

Constant TCR triggering suggests that the TCR expressed on intestinal intraepithelial γδ T cells is functional in vivo.

Dendritic cell (DC)-mediated cross-presentation of exogenous antigens acquired in the periphery is critical for the initiation of CD8(+) T cell responses. Several DC subsets are described in human tissues but migratory cross-presenting DCs have not been isolated, despite their potential importance in immunity to pathogens, vaccines, and tumors and tolerance to self. Here, we identified a CD141(hi) DC present in human interstitial dermis, liver, and lung that was distinct from the majority of CD1c(+) and CD14(+) tissue DCs and superior at cross-presenting soluble antigens. Cutaneous CD141(hi) DCs were closely related to blood CD141(+) DCs, and migratory counterparts were found among skin-draining lymph node DCs. Comparative transcriptomic analysis with mouse showed tissue DC subsets to be conserved between species and permitted close alignment of human and mouse DC subsets. These studies inform the rational design of targeted immunotherapies and facilitate translation of mouse functional DC biology to the human setting.

Human tissues contain CD141hi cross-presenting dendritic cells with functional homology to mouse CD103+ nonlymphoid dendritic cells.

Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying metabolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Furthermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of glycolysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher metabolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation programing, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation.

Frano Malinarich

Translational Immunology,
Institute of Molecular and Cell Biology,
Translational Immunology, Institute of Molecular and Cell Biology

Agency for Science, Technology and Research

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Frano Malinarich

Translational Immunology,Institute of Molecular and Cell Biology,Translational Immunology, Institute of Molecular and Cell Biology

Agency for Science, Technology and Research

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Translational Immunology,
Institute of Molecular and Cell Biology,
Translational Immunology, Institute of Molecular and Cell Biology