Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor.

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-gamma secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-gamma production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND:: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18. METHODS:: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS:: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS:: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations. (Inflamm Bowel Dis 2010).

Mitochondria: Sovereign of inflammation?

NLRP3 inflammasome-dependent inflammatory responses are triggered by a variety of signals of host danger, including infection, tissue damage and metabolic dysregulation. How these diverse activators cause inflammasome activation is poorly understood. Recent data suggest that the mitochondria integrate these distinct signals and relay this information to the NLRP3 inflammasome. Dysfunctional mitochondria generate ROS, which is required for inflammasome activation. On the contrary, the NLRP3 inflammasome is negatively regulated by autophagy, which is a catabolic process that removes damaged or otherwise dysfunctional organelles, including mitochondria. In addition to the processing and secretion of pro-inflammatory cytokines such as IL-1β, NLRP3 inflammasome activation also influences cellular metabolic pathways such as glycolysis and lipogenesis. Mapping the connections between mitochondria, metabolism and inflammation is of great interest, as malfunctioning of this network is associated with many chronic inflammatory diseases.

The NLRP3 inflammasome in health and disease: the good, the bad and the ugly.

While interleukin (IL)-1β plays an important role in combating the invading pathogen as part of the innate immune response, its dysregulation is responsible for a number of autoinflammatory disorders. Large IL-1β activating platforms, known as inflammasomes, can assemble in response to the detection of endogenous host and pathogen-associated danger molecules. Formation of these protein complexes results in the autocatalysis and activation of caspase-1, which processes precursor IL-1β into its secreted biologically active form. Inflammasome and IL-1β activity is required to efficiently control viral, bacterial and fungal pathogen infections. Conversely, excess IL-1β activity contributes to human disease, and its inhibition has proved therapeutically beneficial in the treatment of a spectrum of serious, yet relatively rare, heritable inflammasomopathies. Recently, inflammasome function has been implicated in more common human conditions, such as gout, type II diabetes and cancer. This raises the possibility that anti-IL-1 therapeutics may have broader applications than anticipated previously, and may be utilized across diverse disease states that are linked insidiously through unwanted or heightened inflammasome activity.

Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2(+) melanoma patients. In two of six HLA-A2(+) HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8(+) T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.

Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases.

Defensins and cathelicidins are anti-microbial peptides (AMPs) that act as natural antibiotics and are part of the innate immune defence in many species. We consider human defensins and LL37, the only human member of the cathelicidin family. In particular, we refer to the human alpha-defensins called human neutrophil peptides (HNP1 through 4), which are produced by neutrophils, HD5 and HD6, mainly expressed in Paneth cells of intestine, the human beta-defensins HBD1, HBD2 and HBD3, synthesized by epithelial cells and LL37, which is located in granulocytes, but is also produced by epithelial cells of the skin, lungs, and gut. In the last years, the study of AMPs activity and regulation has allowed to understand the important role of these peptides not only in the innate defence mechanisms against bacteria, viruses, fungi, but also in the regulation of immune cell activation and migration. Complementary studies have disclosed a role for AMPs in modulating many physiological processes that involve non-immune cells, such as activation of wound healing, angiogenesis, cartilage remodeling. Due to the pleiotropic tasks of these peptides, many of them are now being discovered to contribute to immune pathology of chronic diseases that affect skin, gut, joints; this is supported by many examples of immune-mediated pathologies in which their expression is disregulated. In this article we review the current literature that suggests a role for human defensins and LL37 in pathogenic mechanisms of several chronic diseases that are considered of auto-immune or auto-inflammatory origin.

Immuno-monitoring of CD8+ T cells in whole blood versus PBMC samples.

The study of natural T cell responses against pathogens or tumors, as well as the assessment of new immunotherapy strategies aimed at boosting these responses, requires increasingly precise ex vivo analysis of blood samples. For practical reasons, studies are often performed using purified PBMC samples, usually cryopreserved. Here, we report on FACS analyses of peripheral blood T cells, performed by direct antibody staining of non-purified total blood. For comparison, fresh PBMC, purified by Ficoll, were analysed. Our results show that the latter method can induce a bias in subpopulation distribution, in particular of CD8+ T cells, and sometimes lead to inaccurate measurement of antigen specific CD8+ T cell responses. Direct analysis of total blood can be applied to longitudinal immuno-monitoring of T cell-based therapy. While the need to purify and cryopreserve PBMC for subsequent studies is obvious, the use of whole blood has the advantage of providing unbiased results and only small amounts of blood are used.

Measuring the inflammasome.

Inflammasomes are multiprotein complexes whose activity has been implicated in physiological and pathological inflammation. The hallmarks of inflammasome activation are the secretion of the mature forms of Caspase-1 and IL-1β from cells of the innate immune system. This protocol covers the methods required to study inflammasome activation using mouse bone marrow-derived dendritic cells (BMDCs) as a model system. The protocol includes the generation and handling of BMDCs, the stimulation of BMDCs with established Nlrp3 inflammasome activators, and the measurement of activation by both ELISA and western blot. These methods can be useful for the study of potential inflammasome activators, and of the signaling pathways involved in inflammasome activation. General considerations are provided that may help in the design and optimization of modified methods for the study of other types of inflammasomes and in other cell types.

Fas ligand-induced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis.

Mycobacterium tuberculosis-specific cytolytic activity is mediated mostly by CD4+CTL in humans. CD4+CTL kill infected target cells by inducing Fas (APO-1/CD95)-mediated apoptosis. We have examined the effect of Fas ligand (FasL)-induced apoptosis of human macrophages infected in vitro with M. tuberculosis on the viability of the intracellular bacilli. Human macrophages expressed Fas and underwent apoptosis after incubation with soluble recombinant FasL. In macrophages infected either with an attenuated (H37Ra) or with a virulent (H37Rv) strain of M. tuberculosis, the apoptotic death of macrophages was associated with a substantial reduction in bacillary viability. TNF-induced apoptosis of infected macrophages was coupled with a similar reduction in mycobacterial viability, while the induction of nonapoptotic complement-induced cell death had no effect on bacterial viable counts. Infected macrophages also showed a reduced susceptibility to FasL-induced apoptosis correlating with a reduced level of Fas expression. These data suggest that apoptosis of infected macrophages induced through receptors of the TNF family could be an immune effector mechanism not only depriving mycobacteria from their growth environment but also reducing viable bacterial counts by an unknown mechanism. On the other hand, interference by M. tuberculosis with the FasL system might represent an escape mechanism of the bacteria attempting to evade the effect of apoptosis.

Leishmania major-specific B cells are necessary for Th2 cell development and susceptibility to L. major LV39 in BALB/c mice.

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.

PPARs as drug targets to modulate inflammatory responses?

The three peroxisome proliferator-activated receptors (PPARs) isotypes (PPAR alpha, beta/delta and gamma) belong to the nuclear hormone receptor family. During the last decade, they have been identified as anti-inflammatory transcription factors. Part of this regulation antiinflammatory is mediated through negative interference between PPARs and other nuclear factors such as NFkB, AP-1 and C/EBP, which regulate innate as well as adaptative immunity. In addition, the PPARs control the functions of macrophages, B cells and T cells. In this review, we summarise the pathways through which the PPARs control inflammatory responses. We also discuss the potential utilisation of PPAR specific ligands in the treatment of inflammatory diseases, such as inflammatory bowel diseases, atherosclerosis, Parkinson's and Alzheimer's diseases.

Danger signaling through the inflammasome acts as a master switch between tolerance and sensitization.

Efficient priming of adaptive immunity depends on danger signals provided by innate immune pathways. As an example, inflammasome-mediated activation of caspase-1 and IL-1beta is crucial for the development of reactive T cells targeting sensitizers like dinitrofluorobenzene (DNFB). Surprisingly, DNFB and dinitrothiocyanobenzene provide cross-reactive Ags yet drive opposing, sensitizing vs tolerizing, T cell responses. In this study, we show that, in mice, inflammasome-signaling levels can be modulated to turn dinitrothiocyanobenzene into a sensitizer and DNFB into a tolerizer, and that it correlates with the IL-6 and IL-12 secretion levels, affecting Th1, Th17, and regulatory T cell development. Hence, our data provide the first evidence that the inflammasome can define the type of adaptive immune response elicited by an Ag, and hint at new strategies to modulate T cell responses in vivo.

IL-4 rapidly produced by V beta 4 V alpha 8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice.

BALB/c mice develop aberrant T helper 2 (Th2) responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of interleukin-4 (IL-4) early after infection. Here we demonstrate that the burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hr after infection, occurs within CD4+ T cells that express V beta 4 V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient BALB/c mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and T helper 1 responses occurred following infection. Recombinant LACK antigen from L. major induced comparable IL-4 production in V beta 4 V alpha 8 CD4+ cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4 V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex organism.