Federica Facciotti

Human CD1c+ dendritic cells secrete high levels of IL-12 and potently prime cytotoxic T cell responses

Dendritic cells (DC) have the unique capacities to induce primary T-cell responses. In mice, CD8α(+)DC are specialized to cross-prime CD8(+) T cells and produce interleukin-12 (IL-12) that promotes cytotoxicity. Human BDCA-3(+)DC share several relevant characteristics with CD8α(+)DC, but the capacities of human DC subsets to induce CD8(+) T-cell responses are incompletely understood. Here we compared CD1c(+) myeloid DC (mDC)1, BDCA-3(+)mDC2, and plasmacytoid DC (pDC) in peripheral blood and lymphoid tissues for phenotype, cytokine production, and their capacities to prime cytotoxic T cells. mDC1 were surprisingly the only human DC that secreted high amounts of IL-12p70, but they required combinational Toll-like receptor (TLR) stimulation. mDC2 and pDC produced interferon-λ and interferon-α, respectively. Importantly, mDC1 and mDC2 required different combinations of TLR ligands to cross-present protein antigens to CD8(+) T cells. pDC were inefficient and also expressed lower levels of major histocompatibility complex and co-stimulatory molecules. Nevertheless, all DC induced CD8(+) memory T-cell expansions upon licensing by CD4(+) T cells, and primed naive CD8(+) T cells following appropriate TLR stimulation. However, because mDC1 produced IL-12, they induced the highest levels of cytotoxic molecules. In conclusion, CD1c(+)mDC1 are the relevant source of IL-12 for naive T cells and are fully equipped to cross-prime cytotoxic T-cell responses.

Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus

The development and maturation of semi-invariant natural killer T cells (iNKT cells) rely on the recognition of self antigens presented by CD1d restriction molecules in thymus. The nature of the stimulatory thymic self lipids remains elusive. We isolated lipids from thymocytes and found that ether-bonded mono-alkyl glycerophosphates and the precursors and degradation products of plasmalogens stimulated iNKT cells. Synthetic analogs showed high potency in activating thymic and peripheral iNKT cells. Mice deficient in the peroxisomal enzyme glyceronephosphate O-acyltransferase (GNPAT), essential for the synthesis of ether lipids, had significant alteration of the thymic maturation of iNKT cells and fewer iNKT cells in both thymus and peripheral organs, which confirmed the role of ether-bonded lipids as iNKT cell antigens. Thus, peroxisome-derived lipids are nonredundant self antigens required for the generation of a full iNKT cell repertoire.

Up-regulation of CD1d expression restores the immunoregulatory function of NKT cells and prevents autoimmune diabetes in nonobese diabetic mice.

The immunoregulatory function of NKT cells is crucial for prevention of autoimmunity. The prototypical NKT cell Ag α-galactosylceramide is not present in mammalian cells, and little is known about the mechanism responsible for NKT cell recruitment and activation. Up-regulation of CD1d, the NKT cell restriction molecule, expressed on mononuclear cells infiltrating the target organ, could represent the physiological trigger for NKT cells to self-contain T cell immunity and to prevent autoimmune disease. Recognition of CD1d, either by itself or bound to self-ligands (selfCD1d), could drive NKT cells toward an immunoregulatory phenotype. Hence, ineffective NKT cell-mediated immunoregulation in autoimmune-prone individuals including nonobese diabetic (NOD) mice could be related to defective signals that regulate CD1d expression at time and site of autoimmunity. To test this hypothesis, we transgenically overexpressed CD1d molecules under the control of the insulin promoter within the pancreatic islets of NOD mice (insCD1d). Recognition of overexpressed CD1d molecules rescued NKT cell immunoregulatory function and prevented autoimmune diabetes in insCD1d transgenic NOD mice. Protection from diabetes was associated with a biased IL-4-secreting cytokine phenotype of NKT cells and alteration of the cytokine microenvironment in the pancreatic lymph nodes of transgenic mice. The net effect was a reduced development of the autoimmune T cell repertoire. Our findings suggest that up-regulation of CD1d expression during inflammation is critical to maintain T cell homeostasis and to prevent autoimmunity.

Differential alteration of lipid antigen presentation to NKT cells due to imbalances in lipid metabolism.

Deficiencies in enzymes of the lysosomal glycosphingolipid degradation pathway or in lysosomal lipid transfer proteins cause an imbalance in lipid metabolism and induce accumulation of certain lipids. A possible impact of such an imbalance on the presentation of lipid antigens to lipid‐reactive T cells has only been hypothesized but not extensively studied so far. Here we demonstrate that presentation of lipid antigens to, and development of, lipid‐reactive CD1d‐restricted NKT cells, are impaired in mice deficient in the lysosomal enzyme β‐galactosidase (βGal) or the lysosomal lipid transfer protein Niemann‐Pick C (NPC) 2. Importantly, the residual populations of NKT cells selected in βGal–/– and NPC2–/– mice showed differential TCR and CD4 repertoire characteristics, suggesting that differential selecting CD1d:lipid antigen complexes are formed. Furthermore, we provide direct evidence that accumulation of lipids impairs lipid antigen presentation in both cases. However, the mechanisms by which imbalanced lipid metabolism affected lipid antigen presentation were different. Based on these results, the impact of lipid accumulation should be generally considered in the interpretation of immunological deficiencies found in mice suffering from lipid metabolic disorders.

The CD4-centered universe of human T cell subsets

Humans are continuously exposed to a high number of diverse pathogens that induce different types of immune responses. Primary pathogen-specific immune responses generate multiple subsets of memory T cells, which provide protection against secondary infections. In recent years, several novel T cell subsets have been identified and have significantly broadened our knowledge about T cell differentiation and the regulation of immune responses. At the same time the rapidly growing number of incompletely characterized T cell subsets has also generated some controversies. We therefore review here the current knowledge on features and functions of human α/β T cell subsets, focusing on CD4(+) T cells classified according to cytokine production and tissue localization. The principal helper and regulatory T cell subsets can be identified by a limited number of relevant surface markers, which are an integral part of the T cell differentiation programs because they are directly induced by the relevant lineage-defining transcription factors. In vivo occurring human T cell subsets can thus be purified directly ex vivo from relevant tissues for molecular and functional studies, and represent not only an ideal model to study T cell differentiation, but they also offer important clinical opportunities.

Fine tuning by human CD1e of lipid-specific immune responses

CD1e is a member of the CD1 family that participates in lipid antigen presentation without interacting with the T-cell receptor. It binds lipids in lysosomes and facilitates processing of complex glycolipids, thus promoting editing of lipid antigens. We find that CD1e may positively or negatively affect lipid presentation by CD1b, CD1c, and CD1d. This effect is caused by the capacity of CD1e to facilitate rapid formation of CD1–lipid complexes, as shown for CD1d, and also to accelerate their turnover. Similar results were obtained with antigen-presenting cells from CD1e transgenic mice in which lipid complexes are assembled more efficiently and show faster turnover than in WT antigen-presenting cells. These effects maximize and temporally narrow CD1-restricted responses, as shown by reactivity to Sphingomonas paucimobilis-derived lipid antigens. CD1e is therefore an important modulator of both group 1 and group 2 CD1-restricted responses influencing the lipid antigen availability as well as the generation and persistence of CD1–lipid complexes.

Absence of a role for interleukin‐13 in inflammatory bowel disease

IL‐13 has been implicated in the pathogenesis of ulcerative colitis (UC), and may have a role in animal models of gut fibrosis. We studied the involvement of IL‐13 in inflammation and fibrosis in UC and Crohns disease (CD). Intestinal biopsies and anti‐CD3/CD28‐ or anti‐CD2/CD28‐stimulated lamina propria mononuclear cells from UC and CD patients and control subjects were cultured, and IL‐13, IL‐4, IL‐5, IL‐17A and IFN‐γ production was measured. Mucosal IL‐13‐producing cells were characterised by flow cytometry. Gut explants from strictured CD, non‐strictured CD and healthy donors were cultured ex vivo, and secreted IL‐13, IL‐1β and collagen were measured. IL‐13 production by mucosal explants and activated lamina propria mononuclear cells did not differ between CD, UC and control subjects, and was at least a log lower than IFN‐γ and IL‐17A. IL‐13‐producing cells, and in particular natural killer T cells, were uniformly low in all groups. IL‐4 and IL‐5 were undetectable in culture supernatants. Explants of CD strictures produced low amounts of IL‐13, whereas IL‐1β and collagen were elevated. We could not confirm that UC or strictured CD are associated with elevated IL‐13 production. These data suggest that an anti‐IL‐13 Ab would not be an appropriate therapeutic strategy in inflammatory bowel disease.

Impaired SLAM-SLAM Homotypic Interaction between Invariant NKT Cells and Dendritic Cells Affects Differentiation of IL-4/IL-10-Secreting NKT2 Cells in Nonobese Diabetic Mice

The regulatory function of invariant NKT (iNKT) cells for tolerance induction and prevention of autoimmunity is linked to a specific cytokine profile that comprises the secretion of type 2 cytokines like IL-4 and IL-10 (NKT2 cytokine profile). The mechanism responsible for iNKT cell differentiation toward a type 2 phenotype is unknown. Herein we show that costimulatory signals provided by the surface receptor signaling lymphocytic activation molecule (SLAM) on myeloid dendritic cells (mDC) to iNKT cells is crucial for NKT2 orientation. Additionally, we demonstrate that the impaired acquisition of an NKT2 cytokine phenotype in nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes is due to defective SLAM-induced signals generated by NOD mDC. Mature mDC of C57BL/6 mice express SLAM and induce C57BL/6 or NOD iNKT cells to acquire a predominant NKT2 cytokine phenotype in response to antigenic stimulation with the iNKT cell-specific Ag, the α-galactosylceramide. In contrast, mature NOD mDC express significantly lower levels of SLAM and are unable to promote GATA-3 (the SLAM-induced intracellular signal) up-regulation and IL-4/IL-10 production in iNKT cells from NOD or C57BL/6 mice. NOD mice carry a genetic defect of the Slamf1 gene that is associated with reduced SLAM expression on double-positive thymocytes and altered iNKT cell development in the thymus. Our data suggest that the genetic Slamf1 defect in NOD mice also affects SLAM expression on other immune cells such as the mDC, thus critically impairing the peripheral differentiation of iNKT cells toward a regulatory NKT2 type.

NOX2-derived reactive oxygen species are crucial for CD29-induced pro-survival signalling in cardiomyocytes

AIMS The highly expressed cell adhesion receptor CD29 (β(1)-integrin) is essential for cardiomyocyte growth and survival, and its loss of function causes severe heart disease. However, CD29-induced signalling in cardiomyocytes is ill defined and may involve reactive oxygen species (ROS). A decisive source of cardiac ROS is the abundant NADPH oxidase (NOX) isoform NOX2. Because understanding of NOX-derived ROS in the heart is still poor, we sought to test the role of ROS and NOX in CD29-induced survival signalling in cardiomyocytes. METHODS AND RESULTS In neonatal rat ventricular myocytes, CD29 activation induced intracellular ROS formation (oxidative burst) as assessed by flow cytometry using the redox-sensitive fluorescent dye dichlorodihydrofluorescein diacetate. This burst was inhibited by apocynin and diphenylene iodonium. Further, activation of CD29 enhanced NOX activity (lucigenin-enhanced chemiluminescence) and activated the MEK/ERK and PI3K/Akt survival pathways. CD29 also induced phosphorylation of the inhibitory Ser9 on the pro-apoptotic kinase glycogen synthase kinase-3β in a PI3K/Akt- and MEK-dependent manner, and improved cardiomyocyte viability under conditions of oxidative stress. The ROS scavenger MnTMPyP or adenoviral co-overexpression of the antioxidant enzymes superoxide dismutase and catalase inhibited CD29-induced pro-survival signalling. Further, CD29-induced protective pathways were lost in mouse cardiomyocytes deficient for NOX2 or functional p47(phox), a regulatory subunit of NOX. CONCLUSION p47(phox)-dependent, NOX2-derived ROS are mandatory for CD29-induced pro-survival signalling in cardiomyocytes. These findings go in line with a growing body of evidence suggesting that ROS can be beneficial to the cell and support a crucial role for NOX2-derived ROS in cell survival in the heart.

Differences in serum and synovial CD4+ T cells and cytokine profiles to stratify patients with inflammatory osteoarthritis and rheumatoid arthritis

BackgroundThe aim was to investigate CD4+T-cell subsets, immune cells and their cytokine profiles in blood and synovial compartments in rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) to define specific immune signatures.MethodsPeripheral blood, synovial fluid (SF) and synovial membranes (SM) of RA and OA patients were analyzed. CD4+T-cell subset frequencies were determined by flow cytometry, and cytokine concentrations in serum and SF were measured by ELISA.ResultsIn peripheral blood, OA patients had altered frequencies of regulatory T-cell subsets, and higher frequencies of Th17 and of Th1/17 cells than RA patients. In the synovial compartment of OA patients, conventional Th17 cells were largely excluded, while Th1/17 cells were enriched and more frequent than in RA patients. Conversely, in the synovial compartment of RA patients, regulatory T cells and Tfh cells were enriched and more frequent then in OA patients. IL-17 and Blys were increased both in serum and SF of RA patients, and correlated with autoantibodies and disease activity. Notably, Blys levels were already significantly elevated in RA patients with low disease activity score in 28 joints (DAS28) and without autoantibody positivity.ConclusionsAlthough patients with inflammatory OA have immune activation in the synovial compartment, they display different T-cell subset frequencies and cytokine profiles. Soluble mediators such as Blys might help to discriminate mild clinical forms of RA from inflammatory OA particularly at the onset of the disease.