Showing papers in "European Journal of Immunology in 2005"

Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway.

Abstract: Bone marrow mesenchymal progenitor cells (BMSC) are used for regenerating tissues of mesodermal origin, as well as tissues of different embryological derivation. Experimental evidence shows that BMSC are able to suppress the activation of the immune response by mechanisms that are still not completely understood. Thus far, in vitro studies carried using human or mouse cells indicate that autologous or allogeneic BMSC strongly suppress proliferation of T lymphocytes, triggered by cellular stimuli, nonspecific mitogenic stimuli, or antigenic peptides. Using cell proliferation and blocking assays, we demonstrated that BMSC inhibited the activation of murine splenocytes, T, and B lymphocytes. Direct contact of BMSC and target cells in a cognate fashion determined the inhibition of cell proliferation via engagement of the inhibitory molecule programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2, leading the target cells to modulate the expression of different cytokine receptors and transduction molecules for cytokine signaling. Soluble factors present on supernatants of BMSC cultures were effective in suppressing proliferation of B cells to a mitogenic stimulus. Taken together, these results highlight the complexity of the role of BMSC in regulating the immune response, asserting the possibility of their therapeutic application in transplantation and autoimmune diseases.

In vivo quercitrin anti-inflammatory effect involves release of quercetin, which inhibits inflammation through down-regulation of the NF-kappaB pathway.

Abstract: Quercetin is a common antioxidant flavonoid found in vegetables, which is usually present in glycosylated forms, such as quercitrin (3-rhamnosylquercetin). Previous in vitro experiments have shown that quercetin exerts a bigger effect than quercitrin in the down-regulation of the inflammatory response. However, such results have not been reproduced in in vivo experimental models of intestinal inflammation, in which quercetin did not show beneficial effects while its glycosides, quercitrin or rutin, have demonstrated their effectiveness. In this study, we have reported that the in vivo effects of quercitrin in the experimental model of rat colitis induced by dextran sulfate sodium can be mediated by the release of quercetin generated after glycoside's cleavage by the intestinal microbiota. This is supported by the fact that quercetin, but not quercitrin, is able to down-regulate the inflammatory response of bone marrow-derived macrophages in vitro. Moreover, we have demonstrated that quercetin inhibits cytokine and inducible nitric oxide synthase expression through inhibition of the NF-jB pathway without modification of c-Jun N-terminal kinase activity (both in vitro and in vivo). As a conclusion, our report suggests that quercitrin releases quercetin in order to perform its anti-inflammatory effect which is mediated through the inhibition of the NF-jB pathway.

Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level.

Abstract: The transcription factor FOXP3 plays a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4(+)CD25(+) cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti-FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow-cytometric analysis showed that FOXP3 was expressed by the majority of CD4(+)CD25(high) T cells in peripheral blood. By contrast, less than half of the CD4(+)CD25(int) population were FOXP3(+), providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4(+)CD25(high) pool. Although FOXP3 expression was primarily restricted to CD4(+)CD25(+) cells, it was induced following activation of both CD4(+) and CD8(+) T cell clones. These findings indicate that the frequency of FOXP3(+) cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4(+) and CD8(+) T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.

Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis

Abstract: Immunoregulatory T cells of (CD4+)CD25+ phenotype suppress T cell function and protect rodents from organ-specific autoimmune disease The human counterpart of this subset of T cells expresses high levels of CD25 and its role in human autoimmune disorders is currently under intense investigation In multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), the activation of circulating self-reactive T cells with specificity for myelin components is considered to be an important disease initiating event Here, we investigated whether MS is associated with an altered ability of (CD4+)CD25high regulatory T cells (Treg) to confer suppression of myelin-specific immune responses Whereas Treg frequencies were equally distributed in blood and cerebrospinal fluid of MS patients and did not differ compared to healthy controls, the suppressive potency of patient-derived (CD4+)CD25high T lymphocytes was impaired Their inhibitory effect on antigen-specific T cell proliferation induced by human recombinant myelin oligodendrocyte protein as well as on immune responses elicited by polyclonal and allogeneic stimuli was significantly reduced compared to healthy individuals The effect was persistent and not due to responder cell resistance or altered survival of Treg, suggesting that a defective immunoregulation of peripheral T cells mediated by (CD4+)CD25high T lymphocytes promotes CNS autoimmunity in MS

Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-activating agonists.

Abstract: Muropeptides are degradation products of bacterial peptidoglycan (PG) sensed by nucleotide-binding oligomerization domain 1 (NOD1) and NOD2, members of a recently discovered family of pattern recognition molecules (PRM). One of these muropeptides, muramyl dipeptide (MDP) mediates cell signaling by NOD2, exerts adjuvant activity and synergizes with lipopolysaccharide (LPS) to induce pro-inflammatory responses in vitro and in vivo. In contrast, few and contradictory results exist about the stimulatory capacity of NOD1 agonists. Thus, the ability of NOD1 (MurNAc-L-Ala--D-Glu-meso-diaminopimelic acid, MtriDAP) and NOD2 (MurNAc-L-Ala-D-isoGln, MDP; MurNAc-L-Ala--D-Glu-L-Lys, MtriLYS) agonists to activate primary human myeloid cells was examined. We show that both CD14+ monocytes and CD1a+ immature dendritic cells (DC) express NOD1 and NOD2 mRNA. Stimulation of primary human monocytes and DC with highly purified muropeptides (MtriDAP, MDP and MtriLYS) induces release of pro-inflammatory cytokines. We reveal here that NOD1 as well as NOD2 agonists act cooperatively with LPS to stimulate the release of both pro- and anti-inflammatory cytokines in these myeloid cell subsets. Finally, we report that NOD1 as well as NOD2 agonists synergize with sub-active doses of LPS to induce DC maturation, demonstrating that NOD agonists act cooperatively with molecules sensed by Toll-like receptor 4 to instruct the onset of adaptive immune responses.

Cell wall glycosphingolipids of Sphingomonas paucimobilis are CD1d-specific ligands for NKT cells.

Abstract: The current consensus on characterization of NKT cells is based on their reactivity to the synthetic glycolipid, α-galactosylceramide (α-GalCer) in a CD1d-dependent manner. Because of the limited availability of α-GalCer, there is a constant search for CD1d-presented ligands that activate NKT cells. The α-anomericity of the carbohydrate is considered to be an important requisite for the CD1d-specific activation of NKT cells. The gram-negative, lipopolysaccharide-free bacterium Sphingomonas paucimobilis is known to contain glycosphingolipids (GSL) with α-anomeric sugars attached to the lipid chain. Here, we report that GSL extracted from this bacterium are able to stimulate NKT cells in a CD1d-specific manner. In addition, soluble CD1d–Ig dimers loaded with this lipid extract specifically bind to NKT cells (but not conventional T cells). Further studies on the S. paucimobilis GSL could potentially lead to other natural sources of CD1d-specific ligands useful for NKT cell analyses and aimed at identifying novel therapies for a variety of disease states.

Tolerization of dendritic cells by HLA-G

Abstract: The expression of HLA-G at the fetal-maternal interface during pregnancy and in transplanted tissue makes this a key molecule in the acceptance of a semiallogeneic fetus and allogeneic transplant. Dendritic cells (DC) play a critical role in the control of innate and adaptive immune responses. DC are present in maternal decidua, but must be kept under tight control. Here we describe the mechanism of tolerization of DC by HLA-G through inhibitory receptor interactions. The HLA-G-ILT (immunoglobulin-like transcript) interaction leads to development of tolerogenic DC with the induction of anergic and immunosuppressive T cells. Using human monocyte-derived DC and ILT4-transgenic mice, we show that (i) HLA-G induces the development of tolerogenic DC with arrest maturation/activation of myeloid DC, (ii) HLA-G-modified DC induce differentiation of anergic and immunosuppressive CD4(+) and CD8(+) effector T cells, and (iii) the gene expression profile provides evidence that HLA-G induces tolerogenic DC by disruption of the MHC class II presentation pathway. Ligation of ILT4 receptor on DC from transgenic mice diminished peptide presentation by MHC class II molecules and significantly prolonged allograft survival. These findings provide support that HLA-G is an important tolerogenic molecule on DC for the acceptance of a semiallogeneic fetus and transplanted tissue/organ.

Lymphocyte homeostasis following therapeutic lymphocyte depletion in multiple sclerosis.

Abstract: Following lymphocyte depletion, homeostatic mechanisms drive the reconstitution of lymphocytes. We prospectively studied this process in 16 patients for 1 year after a single pulse of treatment with Campath-1H, a humanised anti-CD52 monoclonal antibody. We observed two phases of lymphocyte reconstitution. In the first 6 months after treatment the precursor frequency and proliferation index of the patients' autologous mixed lymphocyte reaction increased; the depleted T cell pool was dominated by memory T cells, especially (CD4+)CD25high T cells, a putative regulatory phenotype; and there was a non-significant rise in peripheral mononuclear cell FoxP3 mRNA expression and fall in constitutive cytokine mRNA expression. In the later phase, from 6-to-12 months after Campath-1H, these changes reversed and there was a rise in ROG mRNA expression. However, total CD4+ numbers remained below 50% of pre-treatment levels at 12 months, perhaps reflecting a failure in homeostasis. This was not due to an impaired IL-7 response, as in rheumatoid arthritis, nor to a lack of IL-7 receptors, which are found on fewer human (CD4+)CD25high than naive cells. We speculate that CCL21 and IL-15 responses to lymphopaenia may be suboptimal in multiple sclerosis.

An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions.

Abstract: Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method. The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.

A therapeutic vaccine for nicotine dependence: preclinical efficacy, and phase I safety and immunogenicity

Abstract: Nicotine is the principal addictive component in tobacco, and following uptake acts in the central nervous system. The smoking-cessation efforts of most smokers fail because a single slip often delivers sufficient nicotine to the brain to reinstate the drug-seeking behaviour. Blocking nicotine from entering the brain by induction of specific antibodies may be an effective means to prevent such relapses. The hapten nicotine was coupled to virus-like particles (VLP) formed by the coat protein of the bacteriophage Qb. In preclinical experiments, this Nicotine-Qb VLP (NicQb) vaccine induced strong antibody responses. After intravenous nicotine challenge, vaccinated mice exhibited strongly reduced nicotine levels in the brain compared with control mice. In a phase I study, 32 healthy non-smokers were immunized with NicQb. The vaccine was safe and well-tolerated. All volunteers who received NicQb showed nicotine-specific IgM antibodies at day 7 and nicotine-specific IgG antibodies at day 14. Antibody levels could be boosted by a second injection or the addition of Alum as an adjuvant and the antibodies had a high affinity for nicotine. These data suggest that antibodies induced by NicQb may prevent relapses by sequestering nicotine in the blood of immunized smokers.

Systematic microanatomical analysis of CXCL13 and CCL21 in situ production and progressive lymphoid organization in rheumatoid synovitis.

Abstract: CXCL13 and CCL21 have been functionally implicated in lymphoid tissue organization both in the upstream phases of lymphoid tissue embryogenesis and in ectopic lymphoid neogenesis in transgenic mice. Here, we analyzed the relationship between CXCL13 and CCL21 production and lymphoid tissue organization in rheumatoid synovitis as a model of a naturally occurring ectopic lymphoneogenesis. Through systematic analysis of mRNA and protein expression, we defined the microanatomical relationship between CXCL13 and CCL21 in progressive aggregational and structural phases of synovial inflammatory infiltrate. We provide the first direct in situ evidence that production of CXCL13 and CCL21 (rather than simply protein binding) is associated with inflammatory lymphoid tissue formation and development with the demonstration, in organized aggregates, of a secondary lymphoid organ-like compartmentalization and vascular association. Notably, the presence of CXCL13 and CCL21 (protein and mRNA) was also demonstrated in non-organized clusters and minor aggregational stages, providing evidence that their induction can take place independently and possibly upstream of T-B compartmentalization, CD21(+) follicular dendritic cell network differentiation and germinal center formation. Our data support the concept that, under inflammatory conditions, CXCL13 and CCL21 participate in lymphoid tissue microanatomical organization, attempting to recapitulate, in an aberrant lymphoid neogenetic process, their homeostatic and morphogenetic physiologic functions.

The human β‐glucan receptor is widely expressed and functionally equivalent to murine Dectin‐1 on primary cells

Abstract: We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal β-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the β-glucan receptor (βGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional βGR isoforms – βGR-A and βGR-B – were expressed by these cell populations in peripheral blood; however, only βGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other β-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major β-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

Humoral immune response to citrullinated collagen type II determinants in early rheumatoid arthritis

Abstract: Collagen type II (CII) is a relevant joint-specific autoantigen in the pathogenesis of rheumatoid arthritis (RA). Whereas the reasons for the breakage of self tolerance to this major cartilage component are still enigmatic, T cell responses to glycosylated CII determinants in RA patients indicate that post-translational modifications play a role. Since the conversion of arginine into citrulline by peptidylarginine deiminases (PAD) in some non-joint-specific antigens such as filaggrin or fibrin has been shown to give rise to RA-specific humoral immune responses, we investigated whether PAD modification of cartilage-specific CII might affect its recognition by circulating autoantibodies in early RA. In vitro treatment with purified PAD led to arginine deimination of native CII or of synthetic CII peptides as evidenced by amino acid analysis. The citrullination resulted in modified recognition of the immunodominant CII epitope C1(III) (amino acid residues 359-369) by murine and human antibodies. In a cohort of early RA patients (n=286), IgG antibodies directed toward a synthetic citrullinated C1(III) peptide (citC1(III)-P) were detectable with a prevalence of 40.4%. The partial autoantibody cross-reactivity between citC1(III)-P and citrullinated peptides mimicking epitopes of the cytoskeletal autoantigen filaggrin suggests that autoimmunity to cartilage-specific modified self might be a critical intermediate bridging recognition of PAD-modified extra-articular autoantigens with the disruption of tolerance to native cartilage constituents.

Calcium and 1α,25-dihydroxyvitamin D3 target the TNF-α pathway to suppress experimental inflammatory bowel disease

Abstract: Vitamin D-deficient IL-10 knockout (KO) mice develop accelerated inflammatory bowel disease (IBD). Removing dietary calcium from the diets of vitamin D-deficient IL-10 KO mice increased the severity of IBD. The mice fed either calcium or active vitamin D (1 alpha,25-dihydroxyvitamin D3, 1,25D3), developed an intermediate form of IBD, while the mice fed both calcium and 1,25D3 had the mildest form of IBD. TNF-alpha secretion from Con A-stimulated splenocytes was reduced by dietary calcium or 1,25D3 treatment. The IL-10 KO mice that received both high calcium diets and 1,25D3 treatments had the lowest TNF-alpha production. In the colons, a TNF-alpha-inducing transcription factor, LPS-induced TNF-alpha factor (LITAF), was inhibited by 1,25D3, but not by calcium. The inhibition of several TNF-alpha-related genes was associated with the decreased colitis in 1,25D3-treated IL-10 KO mice. Furthermore, fulminating IBD in vitamin D receptor/IL-10 double-KO mice corresponded with the increased expression of TNF-alpha and LITAF in the colon. Our results suggest that dietary calcium has independent effects on IBD severity and that 1,25D3 and high calcium together result in the maximal suppression of experimental IBD. The data support a model where dietary calcium and 1,25D3 treatment directly and indirectly inhibit the TNF-alpha pathway and suppress IBD.

Immunomodulatory dendritic cells in intestinal lamina propria

Abstract: The lamina propria (LP) of the small intestine contains many dendritic cells (DC), which are likely to be in close contact with luminal antigens, but their role in intestinal immune responses has been overlooked. Here we show that after feeding mice ovalbumin (OVA), the majority of antigen uptake is associated with DC in the small intestinal LP, and we describe the isolation, purification and initial characterization of theses DC. We obtained >90% CD11c+ DC using magnetic cell sorting, of which the majority were CD11b+CD8–, with smaller numbers of CD11b–CD8+ and CD11b–CD8– DC as well as a distinct population of CD11cintclass II MHClo B220+ DC. Freshly isolated LP DC expressed variable but generally low levels of CD40, CD80 and CD86, which were up-regulated by activation with LPS. LP DC were endocytic in vivo and in vitro and could present antigen to OVA-specific CD4+ T cells in vitro. Antigen-loaded LP DC from OVA-fed mice also primed specific CD4+ T cells in vivo and in vitro, but adoptive transfer of these DC into naive recipients induced hyporesponsiveness to subsequent challenge. LP DC also expressed significant levels of mRNA for IL-10 and type I IFN, but not IL-12, suggesting they may play a central and unique role in immune homeostasis in the gut.

Changes in systemic type 1 and type 2 immunity in normal pregnancy and pre-eclampsia may be mediated by natural killer cells.

Abstract: A bias of T cell immunity towards type 2 (Th2) is thought to be critical for normal pregnancy Pathological pregnancies, such as pre-eclampsia, are characterised by cell-mediated (Th1) immune dominance The Th1/Th2 paradigm, however, is too simplistic Normal pregnancy is associated with a systemic inflammatory response which increases throughout gestation This inflammatory response is exaggerated in pre-eclampsia, a syndrome of the third trimester T helper (Th) cells are considered the primary mediators of these altered immune responses, and other T cells, ie T cytotoxic (Tc) cells, and lymphocytes of the innate immune system, ie natural killer (NK) and NKT cells, have been largely disregarded In this study, we have used novel pan type 1 (IL-18 receptor) and pan type 2 (ST2L) lymphocyte function markers in four-colour flow cytometry to broadly characterise peripheral blood lymphocyte populations from non-pregnant, normal pregnant and pre-eclamptic women There were no changes in the Th1/Th2 or Tc1/Tc2 cell ratios between the three groups; however, the NK1/NK2 and NKT1/NKT2 cell ratios were significantly decreased in normal pregnancy compared with non-pregnant (p <0001 and p <001, respectively) and pre-eclamptic women (p <005) These results confirm that immunoregulation occurs in pregnancy, but suggest a dominant role of the innate rather than the adaptive immune system

Toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mRNA.

Abstract: We reported that RNA condensed on protamine is protected from RNase-mediated degradation and can be used for vaccination. Here, we show that such complexes are also danger signals that activate mouse cells through a MyD88-dependent pathway. Moreover, mRNA-protamine complexes stimulate human blood cells. They strongly activate DC and monocytes, leading to TNF-alpha and IFN-alpha secretion. In addition, protamine-RNA complexes directly activate B cells, NK cells and granulocytes. The detailed analysis of the activated cell types, the study of the cytokines released from PBMC cultured with protamine-RNA complexes and recently published results suggest that TLR-7 and TLR-8 may be involved in the recognition of protamine-stabilized RNA. Our data indicate that protamine-stabilized RNA, which may be similar to RNA condensed in the nucleocapsids of RNA viruses, is a strong danger signal. Thus, similarly to plasmid DNA, protamine-RNA combines antigen production and non-specific immunostimulation. The studies presented here explain the capacity of protamine-RNA to act as a vaccine, and pave the way towards the development of safe and efficient mRNA-based immunotherapies.

Multifocal structure of the T cell – dendritic cell synapse

Abstract: The structure of immunological synapses formed between murine naive T cells and mature dendritic cells has been subjected to a quantitative analysis. Immunofluorescence images of synapses formed in the absence of antigen show a diffuse synaptic accumulation of CD3 and LFA-1. In electron microscopy, these antigen-free synapses present a number of tight appositions (cleft size ∼15 nm), all along the synapse. These tight appositions cover a significantly larger surface fraction of antigen-dependent synapses. In immunofluorescence, antigen-dependent synapses show multiple patches of CD3 and LFA-1 with a variable overlap. A similar distribution is observed for PKCθ and talin. A concentric organization characteristic of prototypical synapses is rarely observed, even when dendritic cells are paralyzed by cytoskeletal poisons. In T–DC synapses, the interaction surface is composed of several tens of submicronic contact spots, with no large-scale segregation of CD3 and LFA-1. As a comparison, in T–B synapses, a central cluster of CD3 is frequently observed by immunofluorescence, and electron microscopy reveals a central tight apposition. Our data show that it is inappropriate to consider the concentric structure as a “mature synapse” and multifocal structures as immature.

Toll-like receptor 6-independent signaling by diacylated lipopeptides.

Abstract: Bacterial lipopeptides are strong immune modulators that activate early host responses after infection as well as initiating adjuvant effects on the adaptive immune system. These lipopeptides induce signaling in cells of the immune system through Toll-like receptor 2 (TLR2)–TLR1 or TLR2–TLR6 heteromers. So far it has been thought that triacylated lipopeptides, such as the synthetic N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3)-CSK4, signal through TLR2–TLR1 heteromers, whereas diacylated lipopeptides, like the macrophage-activating lipopeptide from Mycoplasma fermentans (MALP2) or S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam2)-CGNNDESNISFKEK, induce signaling through TLR2–TLR6 heteromers. Using new synthetic lipopeptide derivatives we addressed the contribution of the lipid and, in particular, the peptide moieties with respect to TLR2 heteromer usage. In contrast to the current model of receptor usage, not only triacylated lipopeptides, but also diacylated lipopeptides like Pam2CSK4 and the elongated MALP2 analog Pam2CGNNDESNISFKEK-SK4 (MALP2-SK4) induced B lymphocyte proliferation and TNF-α secretion in macrophages in a TLR6-independent manner as determined with cells from TLR6-deficient mice. Our results indicate that both the lipid and the N-terminal peptides of lipoproteins contribute to the specificity of recognition by TLR2 heteromers and are responsible for the ligand–receptor interaction on host cells.

Expression of lymphocyte activation gene 3 (LAG-3) on B cells is induced by T cells

Abstract: Lymphocyte activation gene 3 (LAG-3/CD223) is a CD4 homolog known to be selectively expressed in activated T and NK cells. It is thought to have a negative regulatory function in T cells. With the help of new monoclonal antibodies against mouse LAG-3, we show that LAG-3 surface expression is not limited to activated T and NK cells but is also found on activated B cells. Induction of B cell surface expression is T cell dependent and mediated by a soluble factor. The majority of LAG-3 on B cell surface is endogenously produced, even though soluble LAG-3 is present in the culture supernatants and can be passively absorbed. As B cells express LAG-3 in a T cell dependent manner and not when activated by Toll-like-receptor agonists alone, we propose LAG-3 as a new marker of T cell induced B cell activation.

Requirement of HMGB1 and RAGE for the maturation of human plasmacytoid dendritic cells.

Abstract: Dendritic cells (DC) are key components of innate and adaptive immune responses. Plasmacytoid DC (PDC) are a specialized DC subset that produce high amounts of type I interferons in response to microbes. High mobility group box 1 protein (HMGB1) is an abundant nuclear protein, which acts as a potent pro-inflammatory factor when released extracellularly. We show that HMGB1 leaves the nucleus of maturing PDC following TLR9 activation, and that PDC express on the plasma membrane the best-characterized receptor for HMGB1, RAGE. Maturation and type I IFN secretion of PDC is hindered when the HMGB1/RAGE pathway is disrupted. These results reveal HMGB1 and RAGE as the first known autocrine loop modulating the maturation of PDC, and suggest that antagonists of HMGB1/RAGE might have therapeutic potential for the treatment of systemic human diseases.

Human mitochondria‐derived N‐formylated peptides are novel agonists equally active on FPR and FPRL1, while Listeria monocytogenes‐derived peptides preferentially activate FPR

Abstract: N-formyl peptides are cleavage products of bacterial and mitochondrial proteins, and can attract leukocytes to sites of infection or tissue damage. In this study, HL-60 cell lines expressing the human N-formyl peptide receptor FPR or its two homologues (FPRL1, FPRL2) were used to determine the receptor selectivity of N-formylated peptides derived from Listeria monocytogenes or from human mitochondrial proteins. Bacterial peptides were 100-fold more potent on FPR than on FPRL1, whereas none of them could trigger intracellular signaling through FPRL2. In contrast, N-formylated hexapeptides corresponding to the N terminus of mitochondrial NADH dehydrogenase subunits 4 (fMLKLIV) and 6 (fMMYALF), and cytochrome c oxidase subunit I (fMFADRW) were equally potent on FPR and FPRL1. They triggered cellular responses with the following order of potency: fMMYALF > fMLKLIV > fMFADRW, with an EC50, in a Fura-2 calcium mobilization assay, of 10 nM, 44 nM, and 160 nM on FPR-expressing cells, and 15 nM, 55 nM and 120 nM on FPRL1-expressing cells. fMMYALF was also a low-affinity agonist of FPRL2 (EC50 of 1 microM) and was chemotactic for both FPRL1- and FPRL2-expressing cells. We identified novel mitochondrial host-derived agonists for human N-formyl-peptide receptors that might play a role in inflammatory or degenerative processes linked to their stimulation.

Inverse correlation between IL-7 receptor expression and CD8 T cell exhaustion during persistent antigen stimulation.

Abstract: Persistence is a hallmark of infection by viruses such as HIV, hepatitis B virus, hepatitis C virus and LCMV. In the case of LCMV, persistence may often be associated with exhaustion of CD8(+) T cells. We demonstrate here that persistent antigen suppressed IL-7Ralpha expression and this correlated with T cell exhaustion and reduced expression of the anti-apoptotic molecule B cell leukemia/lymphoma 2 (Bcl-2). In contrast, exposure to short-lived antigen only temporarily suppressed IL-7Ralpha expression, failed to induce T cell exhaustion, and primed T cells. Persistent antigen also suppressed IL-7Ralpha expression on primed T cells and this correlated with exhaustion of a previously stable primed T cell population. These findings suggest that antigen longevity regulates T cell fate.

Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes

Abstract: In comparison to secondary lymphoid organs, gut-associated lymphoid tissues such as isolated lymphoid follicles (ILF) and cryptopatches (CP) have been less intensively studied. To gain a better insight into processes regulating organization and function of these structures, which are believed to participate in immune responses and extrathymic T cell development, we characterized the lymphoid structures of the murine small intestine in more detail. The size and cellular composition of small intestinal lymphoid aggregations were analyzed in C57BL/6 and BALB/c wild-type and lymphotoxin (LT)-deficient mice, by flow cytometry, histology and automated multi-color immunofluorescence microscopy evaluating large coherent areas of the intestine. These evaluations demonstrate that aggregated lymphoid structures in the small intestine vary in size and cellular composition, with a majority of structures not matching the current definitions of CP or ILF. Accordingly, significant variations depending on species, age and mouse strain were observed. Furthermore, small bowel transplantation revealed a rapid exchange of B but not T cells between host and grafted tissue. Moreover, LT-deficient animals lack any intestinal lymphoid aggregations yet possess the complete panel of intraepithelial lymphocytes (IEL). In summary, our observations disclose intestinal lymphoid aggregations as dynamic structures with a great deal of inborn plasticity and demonstrate their dispensability for the generation of IEL.

Macrophage migration inhibitory factor promotes innate immune responses by suppressing glucocorticoid-induced expression of mitogen-activated protein kinase phosphatase-1.

Abstract: The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) acts as a physiological counter-regulator of the immuno-suppressive effects of glucocorticoids. However, the mechanisms whereby MIF exerts its counter-balancing effect remain largely unknown. Here we report that MAPK phosphatase 1 (MKP-1), an archetypal member of dual specificity phosphatase that inactivates MAPK activity in response to pro-inflammatory stimuli, is a critical target of MIF-glucocorticoid crosstalk. Recombinant MIF counter-regulated in a dose-dependent fashion dexamethasone inhibition of TNF and IL-8 production by RAW 264.7 macrophages and U-937 promonocytes stimulated with lipoplysaccharides (LPS) or with LPS plus phorbol 12-myristate 13-acetate. Stimulation of RAW 264.7 macrophages with dexamethasone or dexamethasone plus LPS led to a robust up-regulation of MKP-1 mRNA and protein expressions that were counter-regulated by addition of recombinant MIF. Antisense MIF macrophages expressing reduced levels of endogenous MIF produced higher amount of MKP-1 and lower amount of TNF after exposure to dexamethasone and dexamethasone plus LPS, indicating that endogenous MIF acts in an autocrine fashion to override glucocorticoid-induced MKP-1 expression and inhibition of cytokine production. Taken together, these data identify MKP-1 as a molecular target of MIF-glucocorticoid crosstalk and provide a molecular basis for the control of macrophage responses by a pair of physiological regulators of innate immunity. See accompanying commentary: http://dx.doi.org/10.1002/eji.200535556

ABCB1 transporter discriminates human resting naive B cells from cycling transitional and memory B cells.

Abstract: The exact identification of B cell subsets is instrumental to understand their dynamics under physiological and pathological conditions. Human memory B cells are currently identified according to the expression of CD27, which is absent on naive B cells. We report here that the ATP-binding cassette (ABC)B1 transporter is exclusively present on mature CD27– naive B cells, while it is absent in CD27+ memory B cells and in a heterogeneous subset of CD27– cells that comprise both switch memory and transitional B cells. Thus, ABCB1 activity precisely discriminates naive from transitional and all memory B cells. Using this improved method to discriminate human B cell subsets, and Ki67 staining to identify recently divided cells, we show that in both cord blood and adult peripheral blood, mature naive B cells are quiescent while transitional B cells and memory B cells have a high in vivo turnover.

GITR: a multifaceted regulator of immunity belonging to the tumor necrosis factor receptor superfamily.

Abstract: Glucocorticoid-induced TNFR-related gene (GITR; TNFRSF18), a receptor belonging to the TNFR superfamily (TNFRSF), is activated by GITRL. GITR is expressed at low levels on resting responder T lymphocytes and is up-regulated in T regulatory cells (Treg cells) and in activated T cells. GITRL is expressed in endothelial and antigen-presenting cells. The cytoplasmic region of GITR has a striking homology with other TNFRSF members (4-1BB, CD27, OX40) and binds TRAF molecules and Siva. Over recent years, the role of GITR in the development and in the pathophysiology of the immune system has been actively explored by several groups. GITR triggering induces both pro- and anti-apoptotic effects, abrogates the suppressive activity of Treg cells and co-stimulates responder T cells, with the latter activities over-stimulating the immune system. In vivo, GITR activation causes development of autoimmune diseases and restores immune responses in a persistent retroviral infection model and in a tumor model. Intriguingly, GITR knockout mice demonstrate lower mortality in an ischemia model. The GITR-GITRL system appears crucial in regulating immunity and warrants further study.

Regulation of CD4+CD25+ regulatory T cell activity: it takes (IL-)two to tango.

Abstract: Although CD4+CD25+ regulatory T cells (Treg) represent a well-characterized population of T cells with in vitro and in vivo suppressive capacity, the basic mechanisms of suppression are still not understood. The constitutive expression of the high-affinity receptor for IL-2 has raised the question about the role of IL-2 in Treg function. Here, we review recent data indicating that IL-2 is not only necessary for the homeostasis of Treg but is also critical for the activation of Treg function. Since Treg do not produce IL-2 by themselves, their capacity to utilize IL-2 secreted by other T cells appears to be an essential component of Treg biology. This indicates that Treg suppressive activity is controlled by interaction with activated target cells via the soluble mediator IL-2. In Treg, IL-2 has been identified as a potent inducer of the immunosuppressive cytokine IL-10, an important mediator of Treg suppression in vivo. The efficient capture of IL-2 by Treg may, under conditions of limited IL-2 supply, cause IL-2 deprivation of responder T cells. This competition can explain some of the currently discussed discrepancies between in vivo and in vitro activity of Treg.

Adenosine and cAMP are potent inhibitors of the NF‐κB pathway downstream of immunoreceptors

Abstract: Anergic B lymphocytes exert compromised signal transduction towards the activation of NF-kappa B in response to B cell antigen receptor (BCR) triggering, whereas activation of the ERK pathway appears normal. How this differential down-regulation of the NF-kappa B pathway is regulated remains still elusive. Here, we demonstrate that stimuli known to enhance 3',5'-cyclic adenosine monophosphate (cAMP) are capable of selectively suppressing the activation both of NF-kappa B downstream of the BCR and Toll-like receptor 4 in splenic B lymphocytes and of the high-affinity receptor for IgE in BM-derived mast cells. This suppression is accomplished by blocking phosphorylation and subsequent degradation of the inhibitor of NF-kappa B. A cAMP-dependent protein kinase (PKA) inhibitor reverses this suppressive effect, indicating that PKA is a downstream effector of cAMP in this process. Importantly, not only drugs that artificially elevate intracellular cAMP levels, but also the nucleoside adenosine, which is known to be a mediator of cellular distress, inhibit the NF-kappa B pathway. This suggests that adenosine-mediated signals represent an important step in the molecular decision process controlling inflammation versus anergic immune responses.

Dendritic cells govern induction and reprogramming of polarized tissue‐selective homing receptor patterns of T cells: important roles for soluble factors and tissue microenvironments

Abstract: Tissue-selective homing is established during naive T cell activation by the tissue microenvironment and tissue-specific dendritic cells (DC). The factors driving induction and maintenance of T cell homing patterns are still largely unknown. Here we show that soluble factors produced during the interaction of T cells with CD11c(+) DC isolated from skin- or small intestine-associated tissues differentially modulate expression of the corresponding tissue-selective homing receptors (E-selectin ligands and alpha4beta7 integrin/CCR9, respectively) on murine CD8(+) T cells. Injection of tissue-specific DC via different routes induces T cells with homing receptors characteristic of the corresponding local tissue microenvironment, independent of the origin of the DC. These data indicate an important role for signals delivered in trans. Moreover, DC can reprogram the homing receptor expression on T cells previously polarized in vitro for homing to skin or small intestine. Importantly, skin-homing memory T cells stimulated directly ex vivo can also be reprogrammed by intestinal DC to a gut-homing phenotype. Our results show that tissue-selective homing receptor expression on effector and memory T cells is governed by inductive as well as suppressive signals from both DC and tissue microenvironments.