The alpha v beta 3 integrin as a tumor homing ligand for lymphocytes.

Despite the presence of tumor-specific effector cells in the circulation of cancer patients, the immune response of the majority of these patients is not sufficient to prevent the growth and spread of their tumors. That tumor cells can be killed in vitro by tumor-reactive cytotoxic T cells is testimony to the fact that the tumors are not inherently resistant to T cell killing, but rather that there is a failure in immune recognition and effector cell activation. Many reasons for this failure of the body's defense system have been suggested, including the inability of tumor-reactive lymphocytes to migrate to tumor tissue. Here we designed a strategy to improve homing of primary lymphocytes into vascularized tumors. As a homing molecule we selected the integrin alpha v beta 3 since it is expressed by angiogenic vascular endothelium in tumors. To promote lymphocyte adhesion to alpha v beta 3 we "painted" primary lymphocytes with a recombinant, glycosylphosphatidylinositol-linked high-affinity ligand for alpha v beta 3. These painted lymphocytes specifically bound to alpha v beta 3 in vitro and homed to vascularized, solid tumors in vivo. This novel strategy may provide a significant advance in anti-tumor treatment such as adoptive immune therapy.

Reaching for the Holy Grail: insights from infection/cure models on the prospects for vaccines for Trypanosoma cruziinfection

Prevention of Trypanosoma cruzi infection in mammals likely depends on either prevention of the invading trypomastigotes from infecting host cells or the rapid recognition and killing of the newly infected cells byT. cruzi-specific T cells. We show here that multiple rounds of infection and cure (by drug therapy) fails to protect mice from reinfection, despite the generation of potent T cell responses. This disappointing result is similar to that obtained with many other vaccine protocols used in attempts to protect animals from T. cruziinfection. We have previously shown that immune recognition ofT. cruziinfection is significantly delayed both at the systemic level and at the level of the infected host cell. The systemic delay appears to be the result of a stealth infection process that fails to trigger substantial innate recognition mechanisms while the delay at the cellular level is related to the immunodominance of highly variable gene family proteins, in particular those of the trans-sialidase family. Here we discuss how these previous studies and the new findings herein impact our thoughts on the potential of prophylactic vaccination to serve a productive role in the prevention of T. cruziinfection and Chagas disease.

Plasmacytoid dendritic cells sense skin injury and promote wound healing through type I interferons.

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-α/β)-producing cells that express intracellular toll-like receptor (TLR) 7 and TLR9 and recognize viral nucleic acids in the context of infections. We show that pDCs also have the ability to sense host-derived nucleic acids released in common skin wounds. pDCs were found to rapidly infiltrate both murine and human skin wounds and to transiently produce type I IFNs via TLR7- and TLR9-dependent recognition of nucleic acids. This process was critical for the induction of early inflammatory responses and reepithelization of injured skin. Cathelicidin peptides, which facilitate immune recognition of released nucleic acids by promoting their access to intracellular TLR compartments, were rapidly induced in skin wounds and were sufficient but not necessary to stimulate pDC activation and type I IFN production. These data uncover a new role of pDCs in sensing tissue damage and promoting wound repair at skin surfaces.

Did cholera toxin finally get caught?

To orchestrate immune responses, pathogen-recognition receptors have evolved sophisticated strategies to monitor pathogenic processes. In this issue of Cell Host & Microbe, a study by Cho et al. reveals a mechanism of immune recognition that relies on the sensing of cholera toxin within the endoplasmic reticulum.

The major histocompatibility complex and perfumers' descriptions of human body odors

The MHC (major histocompatibility complex) is a group of genes that play a crucial role in immune recognition and in tolerance of tissue grafting. The MHC has also been found to influence body odors, body odor preferences, and mate choice in mice and humans. Here we test whether verbal descriptions of human body odors can be linked to the MHC. We asked 45 male students to live as odor neutral as possible for two consecutive days and to wear a T-shirt during the nights. The odors of these T-shirts were then described by five evaluators: two professional perfumers and three laymen. One of the perfumers was able to describe the T-shirt odors in such a way that some of the allelic specificity of the MHC was significantly revealed (after Bonferroni correction for multiple testing). This shows that, although difficult, some people are able to describe MHCcorrelated body odor components.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F®I, 21/21) and 212 (G®S, 19/21) or (G®N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G®D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L®F) and 129 (M®I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W®C), and 5/21 specimens showed another novel change at codon 115 (G®A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.

Trends in adjuvant development for vaccines: DAMPs and PAMPs as potential new adjuvants

Aluminum salts have been widely used in vaccine formulations and, after their introduction more than 80 years ago, only few vaccine formulations using new adjuvants were developed in the last two decades. Recent advances in the understanding of how innate mechanisms influence the adaptive immunity opened up the possibility for the development of new adjuvants in a more rational design. The purpose of this review is to discuss the recent advances in this field regarding the attempts to determine the molecular basis and the general mechanisms underlying the development of new adjuvants, with particular emphasis on the activation of receptors of innate immune recognition. One can anticipate that the use of these novel adjuvants will also provide a window of opportunities for the development of new vaccines.

L'expression de la protéine de l'hémochromatose HFE est modulée par les lymphocytes T activés et inhibe la présentation antigénique par MHC I

La présentation antigénique par le complexe majeur d’histocompatibilité (MHC) I est un processus ubiquitaire permettant la présentation de protéines endogènes qui reflètent l'état de la cellule à la surface cellulaire aux lymphocytes T CD8+ dans le contexte de la surveillance et la réponse immunitaires. Ainsi, l'expression des molécules du MHC I classiques est induite en réponse aux stimuli inflammatoires afin de favoriser la reconnaissance immunitaire et l'élimination des pathogènes. HFE est une molécule du MHC Ib non-classique qui sert de régulateur négatif de l'absorption du fer. HFE est associé au développement de l'hémochromatose héréditaire (HH), maladie associée au métabolisme du fer mais souvent accompagnée de défauts immunitaires. Ainsi, nous avons en premier lieu étudié l'impact de HFE sur la présentation antigénique par MHC I, afin d'expliquer en partie les défauts immunitaires liés à l'HH associée à HFEC282Y. Puis, compte tenu de l'impact de l'inflammation sur l'expression des molécules du MHC I classiques, nous avons étudié la régulation de l'expression de HFE en réponse aux stimuli inflammatoires induits par les cellules du sang périphérique mononucléées (PBMC). Nous avons mis au point un système d’expression antigénique dans lequel nous contrôlons l’expression de MHC I, de HFE et d’un antigène pour lequel nous avons généré des lymphocytes T CD8+ spécifiques. Nos résultats démontrent que la forme sauvage de HFE (HFEWT), contrairement à sa forme mutée (HFEC282Y), inhibe la reconnaissance de complexes MHC I/peptide (pMHC). Nous avons également démontré que l'inhibition de la reconnaissance est maintenue, indépendamment des niveaux d'expression de MHC I à la surface, d'une compétition pour la β2-microglobuline, de la capacité de HFE d'interagir avec le récepteur de la transferrine, de l'origine de l'antigène ou de l'affinité de celui-ci. Par ailleurs, nous avons identifié les domaines α1-2 de HFEWT comme étant responsables de l'inhibition de la reconnaissance antigénique. Par contre, la reconnaissance de peptides chargés de manière externe sur les molécules du MHC I présentes à la surface n'a démontré aucune inhibition en présence de HFEWT, suggérant que HFEWT pourrait affecter la reconnaissance en interférant avec le processus d'apprêtement antigénique intracellulaire. À l’inverse, nous avons souhaité déterminer si les lymphocytes T activés pouvaient influencer les niveaux d'expression de HFE. En termes de régulation de l'expression de HFE, nous avons établi que HFE est exprimé dans les tissus sains chez l'humain et induit chez les lignées de cancers du colon, du sein, du poumon, du rein et du mélanome. Par ailleurs, en co-cultivant des lymphocytes T activés avec ces lignées tumorales, nous avons démontré que l'expression de HFE est fortement inhibée dans toutes ces lignées tumorales lorsqu'exposées à des lymphocytes T activés. Finalement, la modulation de l'expression de HFE est indépendante du contact cellulaire et semble médiée en partie par le GM-CSF, l'IFN-γ et le TNF. En somme, ces résultats suggèrent que les lymphocytes T de l'hôte modulent l'expression de HFE dans le microenvironnement inflammatoire, ce qui pourrait promouvoir la reconnaissance des antigènes présentés sur les molécules du MHC I présentées aux lymphocytes T CD8+ antigène-spécifiques. De plus, ces études soulèvent la possibilité d'un nouveau rôle physiologique de HFEWT dans la voie de présentation antigénique par MHC I, qui pourrait moduler l'immunogénicité des antigènes et la réponse immunitaire cellulaire chez l'hôte.

A Novel Pathway for Inducible Nitric-oxide Synthase Activation through Inflammasomes

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1 beta/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1 beta and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

Avaliação ex vivo da expressão de TLR-2 e TLR-4 em leucócitos de equinos e sua relação com a tolerância à endotoxina

Pós-graduação em Ciência Animal - FMVA

Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-alpha production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1

Background: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. Objective: To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of beta-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Methods: Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. Results: PBMCs from patients with APECED syndrome had reduced TNF-alpha responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-a release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane. Conclusion: AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome. (J Allergy Clin Immunol 2012;129:464-72.)

Joint NOD2/RIPK2 Signaling Regulates IL-17 Axis and Contributes to the Development of Experimental Arthritis

Intracellular pattern recognition receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors family members are key for innate immune recognition of microbial infection and may play important roles in the development of inflammatory diseases, including rheumatic diseases. In this study, we evaluated the role of NOD1 and NOD2 on development of experimental arthritis. Ag-induced arthritis was generated in wild-type, NOD1(-/-)!, NOD2(-/-), or receptor-interacting serine-threonine kinase 2(-/-) (RIPK2(-/-)) immunized mice challenged intra-articularly with methylated BSA. Nociception was determined by electronic Von Frey test. Neutrophil recruitment and histopathological analysis of proteoglycan lost was evaluated in inflamed joints. Joint levels of inflammatory cytokine/chemokine were measured by ELISA. Cytokine (IL-6 and IL-23) and NOD2 expressions were determined in mice synovial tissue by RT-PCR. The NOD2(-/-) and RIPK2(-/-), but not NOD1(-/-), mice are protected from Ag-induced arthritis, which was characterized by a reduction in neutrophil recruitment, nociception, and cartilage degradation. NOD2/RIPK2 signaling impairment was associated with a reduction in proinflammatory cytokines and chemokines (TNF, IL-1 beta, and CXCL1/KC). IL-17 and IL-17 triggering cytokines (IL-6 and IL-23) were also reduced in the joint, but there is no difference in the percentage of CD4(+) IL-17(+) cells in the lymph node between arthritic wild-type and NOD2(-/-) mice. Altogether, these findings point to a pivotal role of the NOD2/RIPK2 signaling in the onset of experimental arthritis by triggering an IL-17-dependent joint immune response. Therefore, we could propose that NOD2 signaling is a target for the development of new therapies for the control of rheumatoid arthritis. The Journal of Immunology, 2012, 188: 5116-5122.

Genome of the host-cell transforming parasite Theileria annulata compared with T. parva

Theileria annulata and T. parva are closely related protozoan parasites that cause lymphoproliferative diseases of cattle. We sequenced the genome of T. annulata and compared it with that of T. parva to understand the mechanisms underlying transformation and tropism. Despite high conservation of gene sequences and synteny, the analysis reveals unequally expanded gene families and species-specific genes. We also identify divergent families of putative secreted polypeptides that may reduce immune recognition, candidate regulators of host-cell transformation, and a Theileria-specific protein domain [frequently associated in Theileria (FAINT)] present in a large number of secreted proteins.

Molecular survival strategies of Echinococcus multilocularis in the murine host

Larval infection with Echinococcus multilocularis starts with the intrahepatic postoncospheral development of a metacestode that-at its mature stage-consists of an inner germinal and an outer laminated layer (GL ; LL). In certain cases, an appropriate host immune response may inhibit parasite proliferation. Several lines of evidence obtained in vivo and in vitro indicate the important bio-protective role of the LL. For instance, the LL has been proposed to protect the GL from nitric oxide produced by periparasitic macrophages and dendritic cells, and also to prevent immune recognition by surrounding T cells. On the other hand, the high periparasitic NO production by peritoneal exsudate cells contributes to periparasitic immunosuppression, explaining why iNOS deficienct mice exhibit a significantly lower susceptibility towards experimental infection. The intense periparasitic granulomatous infiltration indicates a strong host-parasite interaction, and the involvement of cellular immunity in control of the metacestode growth kinetics is strongly suggested by experiments carried out in T cell deficient mouse strains. Carbohydrate components of the LL, such as Em2(G11) and Em492, as well as other parasite metabolites yield immunomodulatory effects that allow the parasite to survive in the host. I.e., the IgG response to the Em2(G11)-antigen takes place independently of alpha-beta+CD4+T cells, and in the absence of interactions between CD40 and CD40 ligand. Such parasite molecules also interfere with antigen presentation and cell activation, leading to a mixed Th1/Th2-type response at the later stage of infection. Furthermore, Em492 and other (not yet published) purified parasite metabolites suppress ConA and antigen-stimulated splenocyte proliferation. Infected mouse macrophages (AE-MØ) as antigen presenting cells (APC) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells when compared to normal MØ. As AE-MØ fully maintain their capacity to appropriately process antigens, a failure in T cell receptor occupancy by antigen-Ia complex or/and altered co-stimulatory signals can be excluded. Studying the status of accessory molecules implicated in T cell stimulation by MØ, it could be shown that B7-1 (CD80) and B7-2 (CD86) remained unchanged, whereas CD40 was down-regulated and CD54 (=ICAM-1) slightly up-regulated. FACS analysis of peritoneal cells revealed a decrease in the percentage of CD4+ and CD8+T cells in AE-infected mice. Taken together the obstructed presenting-activity of AE-MØ appeared to trigger an unresponsiveness of T cells leading to the suppression of their clonal expansion during the chronic phase of AE infection. Interesting information on the parasite survival strategy and potential can be obtained upon in vitro and in vivo treatment. Hence, we provided very innovative results by showing that nitazoxanide, and now also, respectively, new modified compounds may represent a useful alternative to albendazole. In the context of chemotherapeutical repression of parasite growth, we searched also for parasite molecules, whose expression levels correlate with the viability and growth activity of E. multilocularis metacestode. Expression levels of 14-3-3 and II/3-10, relatively quantified by realtime reverse transcription-PCR using a housekeeping gene beta-actin, were studied in permissive nu/nu and in low-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro-treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide for a period of 8 days resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels,

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.