Bacterial flagellin elicits innate immune defenses and cardiac dysfunction in vivo

Résumé Les agents pathogènes responsables d'infection entraînent chez l'hôte deux types de réponses immunes, la première, non spécifique, dite immunité innée, la seconde, spécifique à l'agent concerné, dite immunité adaptative. L'immunité innée, qui représente la première ligne de défense contre les pathogènes, est liée à la reconnaissance par les cellules de l'hôte de structures moléculaires propres aux micro-organismes (« Pathogen-Associated Molecular Patterns », PAMPs), grâce à des récepteurs membranaires et cytoplasmiques (« Pattern Recognition Receptors », PRRs) identifiant de manière spécifique ces motifs moléculaires. Les récepteurs membranaires impliqués dans ce processus sont dénommés toll-like récepteurs, ou TLRS. Lorsqu'ils sont activés par leur ligand spécifique, ces récepteurs activent des voies de signalisation intracellulaires initiant la réponse inflammatoire non spécifique et visant à éradiquer l'agent pathogène. Les deux voies de signalisation impliquées dans ce processus sont la voie des « Mitogen-Activated Protein Kinases » (MAPKs) et celle du « Nuclear Factor kappaB » (NF-κB), dont l'activation entraîne in fine l'expression de protéines de l'inflammation dénommées cytokines, ainsi que certaines enzymes produisant divers autres médiateurs inflammatoires. Dans certaines situations, cette réponse immune peut être amplifiée de manière inadéquate, entraînant chez l'hôte une réaction inflammatoire systémique exagérée, appelée sepsis. Le sepsis peut se compliquer de dysfonctions d'organes multiples (sepsis sévère), et dans sa forme la plus grave, d'un collapsus cardiovasculaire, définissant le choc septique. La défaillance circulatoire du choc septique touche les vaisseaux sanguins d'une part, le coeur d'autre part, réalisant un tableau de «dysfonction cardiaque septique », dont on connaît mal les mécanismes pathogéniques. Les bactéries à Gram négatif peuvent déclencher de tels phénomènes, notamment en libérant de l'endotoxine, qui active les voies de l'immunité innée par son interaction avec un toll récepteur, le TLR4. Outre l'endotoxine, la plupart des bactéries à Gram négatif relâchent également dans leur environnement une protéine, la flagelline, qui est le constituant majeur du flagelle bactérien, organelle assurant la mobilité de ces micro-organismes. Des données récentes ont indiqué que la flagelline active, dans certaines cellules, les voies de l'immunité innée en se liant au récepteur TLRS. On ne connaît toutefois pas les conséquences de l'interaction flagelline-TLRS sur le développement de l'inflammation et des dysfonctions d'organes au cours du sepsis. Nous avons par conséquent élaboré le présent travail en formulant l'hypothèse que la flagelline pourrait déclencher une telle inflammation et représenter ainsi un médiateur potentiel de la dysfonction d'organes au cours du sepsis à Gram négatif, en nous intéressant plus particulièrement àl'inflammation et à la dysfonction cardiaque. Dans la première partie de ce travail, nous avons étudié les effets de la flagelline sur l'activation du NF-κB et des MAPKs, et sur l'expression de cytokines inflammatoires au niveau du myocarde in vitro (cardiomyocytes en culture) et in vivo (injection de flagelline recombinante à des souris). Nous avons observé tout d'abord que le récepteur TLRS est fortement exprimé au niveau du myocarde. Nous avons ensuite démontré que la flagelline active la voie du NF-κB et des MAP kinases (p38 et JNK), stimule la production de cytokines et de chemokines inflammatoires in vitro et in vivo, et entraîne l'activation de polynucléaires neutrophiles dans le tissu cardiaque in vivo. Finalement, au plan fonctionnel, nous avons pu montrer que la flagelline entraîne une dilatation et une réduction aiguë de la contractilité du ventricule gauche chez la souris, reproduisant les caractéristiques de la dysfonction cardiaque septique. Dans la deuxième partie, nous avons déterminé la distribution du récepteur TLRS dans les autres organes majeurs de la souris (poumon, foie, intestin et rein}, et avons caractérisé dans ces organes l'effet de la flagelline sur l'activation du NF-κB et des MAPKs, l'expression de cytokines, et l'induction de l'apoptose. Nous avons démontré que le TLRS est exprimé de façon constitutive dans ces organes, et que l'injection de flagelline y déclenche les cascades de l'immunité innée et de processus apoptotiques. Finalement, nous avons également déterminé que la flagelline entraîne une augmentation significative de multiples cytokines dans le plasma une à six heures après son injection. En résumé, nos données démontrent que la flagelline bactérienne (a) entraîne une inflammation et une dysfonction importantes du myocarde et (b) active de manière très significative les mécanismes d'immunité innée dans les principaux organes et entraîne une réponse inflammatoire systémique. Par conséquent, la flagelline peut représenter un médiateur puissant de l'inflammation et de la dysfonction d'organes, notamment du coeur, au cours du choc septique déclenché par les bactéries à Gram négatif. Summary Pathogenic microorganisms trigger two kinds of immune responses in the host. The first one is immediate and non-specific and is termed innate immunity, whereas the second one, specifically targeted at the invading agent, is termed adaptative immunity. Innate immunity, which represents the first line of defense against invading pathogens, confers the host the ability to recognize molecular structures common to many microbial pathogens, ("Pathogen-Associated Molecular Patterns", PAMPs), through cytosolic or membrane-associated receptors ("Pattern Recognition Receptors", PRRs), the latter being represented by a family of receptors termed "toll-like receptors or TLRs". Once activated by the binding of their specific ligand, these receptors activate intracellular signaling pathways, which initiate the non-specific inflammatory response aimed at eradicating the pathogens. The two pathways implicated in this process are the mitogen-activated protein kinases (MAPK) and the nuclear factor kappa B (NF-κB) signaling pathways, whose activation elicit in fine the expression of inflammatory proteins termed cytokines, as well as various enzymes producing a wealth of additional inflammatory mediators. In some circumstances, the innate immune response can become amplified and dysregulated, triggering an overwhelming systemic inflammatory response in the host, identified as sepsis. Sepsis can be associated with multiple organ dysfunction (severe sepsis), and in its most severe form, with cardiovascular collapse, defming septic shock. The cardiovascular failure associated with septic shock affects blood vessels as well as the heart, resulting in a particular form of acute heart failure termed "septic cardiac dysfunction ", whose pathogenic mechanisms remain partly undefined. Gram-negative bacteria can initiate such phenomena, notably by releasing lipopolysaccharide (LPS), which activates innate immune signaling by interacting with its specific toll receptor, the TLR4. Besides LPS, most Gram-negative bacteria also release flagellin into their environment, which is the main structural protein of the bacterial flagellum, an appendage extending from the outer bacterial membrane, responsible for the motility of the microorganism. Recent data indicated that flagellin activate immune responses upon binding to its receptor, TLRS, in various cell types. However, the role of flagellin/TLRS interaction in the development of inflammation and organ dysfunction during sepsis is not known. Therefore, we designed the present work to address the hypothesis that flagellin might trigger such inflammatory responses and thus represent a potential mediator of organ dysfunction during Gram-negative sepsis, with a particular emphasis on cardiac inflammation and contractile dysfunction. In the first part of this work, we investigated the effects of flagellin on NF-κB and MAPK activation and the generation of pro-inflammatory mediators within the heart in vitro (cultured cardiomyocytes) and in vivo (injection of recombinant flagellin into mice). We first observed that TLRS protein is strongly expressed by the myocardium. We then demonstrated that flagellin activates NF-κB and MAP kinases (p38 and JNK), upregulates the transcription of pro-inflammatory cytokines and chemokines in vitro and in vivo, and stimulates the activation of polymorphonuclear neutrophils within the heart in vivo. Finally, we demonstrated that flagellin triggers acute cardiac dilation, and a significant reduction of left ventricular contractility, mimicking characteristics of clinical septic cardiac dysfunction. In the second part, we determined the TLRS distribution in other mice major organs (lung, liver, gut and kidney) and we characterized in these organs the effects of flagellin on NF-κB and MAPK activation, on the expression of pro-inflammatory çytokines, and on the induction of apoptosis. We demonstrated that TLRS protein is constitutively expressed and that flagellin activates prototypical innate immune responses and pro-apoptotic pathways in all these organs. Finally, we also observed that flagellin induces a significant increase of multiple cytokines in the plasma from 1 to 6 hours after its intravenous administration. Altogether, these data provide evidence that bacterial flagellin (a) triggers an important inflammatory response and an acute dysfunction of the myocardium, and (b) significantly activates the mechanisms of innate immunity in most major organs and elicits a systemic inflammatory response. In consequence, flagellin may represent a potent mediator of inflammation and multiple organ failure, notably cardiac dysfunction, during Gram-negative septic shock.

Presence of JC virus-specific CTL in the cerebrospinal fluid of PML patients: rationale for immune-based therapeutic strategies.

OBJECTIVES: There is urgent need of a treatment for progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). To evaluate the rationale for immunotherapy of PML, we explored whether JCV-specific cytotoxic T lymphocytes (CTL) can penetrate the central nervous system (CNS). In addition, we studied the breadth of their T-cell receptor (TCR) repertoire, and sought to establish a reliable method to expand these cells in vitro. DESIGN AND METHODS: We enrolled 18 patients in this study, including 16 with proven or possible PML (15 HIV-positive and one HIV-negative), and two HIV-positive patients with other neurological diseases. Detection of JCV-specific CTL in the blood and the cerebrospinal fluid was performed by Cr release and tetramer staining assays in 15 patients. RESULTS: Of 11 PML patients with analyzable cerebrospinal fluid (CSF), two had no detectable JCV-specific CTL in the blood and CSF and died 3.7 and 7.2 months later. The nine remaining patients had an inactive course of PML and detectable JCV-specific CTL in the blood. In addition, four of them (44%) also had detectable JCV-specific CTL in the CSF. Both HIV-positive patients with OND had detectable JCV-specific CTL in the blood and one in the CSF. Using tetramer technology, we obtained highly enriched JCV-specific CTL lines that were able to kill target cells presenting JCV peptides. The breadth of the TCR repertoire was CTL epitope dependent. CONCLUSIONS: These results indicate that JCV-specific CTL are present in the CNS of PML patients and pave the way for an immune-based therapeutic approach.

Immune response mechanisms and protection against "Plasmodium falciparum" and "Plasmodium berghei"

Malaria is one of the most important tropical and infectious diseases causing many deaths and enormous social and economic consequences, particularly in the developing countries. Despite of widely use of anti-malaria drugs and insecticide, the development of successful vaccines constitutes one of the main strategies to control malaria transmission. Several proteins expressed from blood stage such as merozoite surface proteins (MSP] or liver stage as circumsporozoite protein (CSP) are shown to be the targets of immune responses in humans and in animals. Thus, several studies have illustrated that natural infection and laboratory immunizations of humans and animals with Plasmodium sporozoite (SPZ) and its derivate-proteins (peptides) can elicit protection and control of parasite infection. However, a clear understanding of immune response against defined Plasmodium proteins should be the prerequisite conditions before any development of appropriate vaccines. In this order, our study focused on the immune responses to MSP2 (dimorphic and C-terminal fragments) in human and mice; and the mechanisms by which mouse infected hepatocytes present Plasmodium antigens to CD8+ T-cells to induce protective immunity in mice.¦The first part of this work shows that infected hepatocytes can present Plasmodium antigens to PbCSP-specific CD8+ T-cells and induce a protective immunity in mice. Here, this was addressed in vivo and showed that the infected hepatocytes were able of stimulating of primed-and naive-CD8+ T-cell clones and induced fully protective immunity against SPZ challenge. The role of infected hepatocytes in antigen presentation was illustrated here by their graft into immuno-deficient mice and depletion of cosspresenting dentritic cells (DCs) that are known to have key role in the activation of CD8+ T-cells during the liver cycle stage of Plasmodium.¦The second part of this project concerned the fine specificity of Ab responses regarding D and C regions of the two allelic families of MSP2 (3D7 and FC27). Covering of the two regions by overlapping-20 mers led to delineate the epitopes in the different endemic areas and different age groups of donors. The major epitopes characterizing D or C regions were conserved in different endemic areas (P12/P13 and P15/P16 for the 3D7-D, P23/24 and P25/26 for the FC27-D; P29/P30 for the C region). This offers thus, the possibility of a multi-epitope vaccine design including the major epitopes from the two domains of the two allelic MSP2 families. On the other, the 20 mers, particularly some major epitopes of the 3D7-Dregion (P12, P13 and P16) belonged to the epitopes that presented a high probability to be associated with protection in the children group [1 to 5 year-old). In addition, D and C LSP purified Abs (pAbs) recognized merozoite derived polypeptides and native proteins. A crossreactivity activity of homologous pAbs against the heterologous was also illustrated between the two allelic MSP2 parasites. Finally, the functional analysis of D regions pAbs showed an inhibition of Plasmodium falciparum growth suggesting the functional biological activity of the D region pAbs in the control of malaria.¦The last part of this project aimed the evaluation of the immunogenicity of the D and C region LSPs of the two allelic MSP2 families in the presence of adjuvants for the possible use in clinical trial study in humans. The MSP2 LSP mixture showed that D and C were immunogenic and defined limited epitopes (whose intensity of immune responses) depending on the adjuvants and mouse strain for the D regions. The major epitopes characterizing the C region were usually conserved in different strains of mouse and adjuvants used. Furthermore, the single region (either with D or C) immunization of mice confirmed the immunogenicity and the presence of their limited epitopes. We concluded that the possibility to finely delineate in animals the immune responses to antigens might help to select optimal antigen/adjuvant combinations to be tested later in clinical trials. Thus, formulation of glucopyranosyl-lipid A stable emulsion, GLA-SE (toll like receptor (TLR) 4 agonist) and its different combination (CpG: TLR9 agonist and GDQ: LR7 agonist) with MSP2 LSP was better than with alum, montanide ISA 720 (Mt) and virosome. Immunization of mice with allelic LSP did not show a crossreactivity between the two allelic MSP2 parasites unlike as humans, suggesting that the crossreactivity could be acquired during natural infection of the population who are usually exposed to both allelic parasite forms (3D7 and FC27).¦Nevertheless, similar epitope of D (P12, P13 and P25) and C (P29) regions have been found both in mice and human. This offers an opportunity to compare their epitopes in naïve immunized donors with LSPs and naturally infected populations in the endemic areas.

Liver Transplantation for Neuroendocrine Tumors in Europe-Results and Trends in Patient Selection: A 213-Case European Liver Transplant Registry Study.

OBJECTIVE:: The purpose of this study was to assess outcomes and indications in a large cohort of patients who underwent liver transplantation (LT) for liver metastases (LM) from neuroendocrine tumors (NET) over a 27-year period. BACKGROUND:: LT for NET remains controversial due to the absence of clear selection criteria and the scarcity and heterogeneity of reported cases. METHODS:: This retrospective multicentric study included 213 patients who underwent LT for NET performed in 35 centers in 11 European countries between 1982 and 2009. One hundred seven patients underwent transplantation before 2000 and 106 after 2000. Mean age at the time of LT was 46 years. Half of the patients presented hormone secretion and 55% had hepatomegaly. Before LT, 83% of patients had undergone surgical treatment of the primary tumor and/or LM and 76% had received chemotherapy. The median interval between diagnosis of LM and LT was 25 months (range, 1-149 months). In addition to LT, 24 patients underwent major resection procedures and 30 patients underwent minor resection procedures. RESULTS:: Three-month postoperative mortality was 10%. At 5 years after LT, overall survival (OS) was 52% and disease-free survival was 30%. At 5 years from diagnosis of LM, OS was 73%. Multivariate analysis identified 3 predictors of poor outcome, that is, major resection in addition to LT, poor tumor differentiation, and hepatomegaly. Since 2000, 5-year OS has increased to 59% in relation with fewer patients presenting poor prognostic factors. Multivariate analysis of the 106 cases treated since 2000 identified the following predictors of poor outcome: hepatomegaly, age more than 45 years, and any amount of resection concurrent with LT. CONCLUSIONS:: LT is an effective treatment of unresectable LM from NET. Patient selection based on the aforementioned predictors can achieve a 5-year OS between 60% and 80%. However, use of overly restrictive criteria may deny LT to some patients who could benefit. Optimal timing for LT in patients with stable versus progressive disease remains unclear.

Strength and limitations of regulatory T Cells for immunotherapy in transplantation.

In many experimental models, CD4+CD25+Foxp3+ regulatory T cells (nTreg) have been identifi ed as key players in promoting peripheral transplantation (Tx) tolerance. We have been focusing on therapies based on antigen-specifi c nTreg that can control effector T cells (Teff) and prevent allograft rejection. The use of nTreg in immunotherapeutic protocols for solid organ Tx is however limited by their overall low numbers as well as the low precursor frequency of alloantigen cross-reactive nTreg expected to be found in a normal individual. Moreover, although we previously described robust protocols to generate and expand antigen-specifi c nTreg in vitro, the process requires careful selection of highly pure nTreg and cumbersome ex-vivo manipulations, rendering this strategy not easily applicable in clinical solid organ Tx. In this study, we aimed to expand Treg directly in vivo and determine their suppressive function, effi cacy and stability in promoting donor-specifi c tolerance in a stringent murine Tx model. Our data suggest that IL-2-based therapies lead to a signifi cant increase of Treg in vivo. The expanded Treg suppressed Teff proliferation (albeit slightly less effi ciently than nTreg isolated from control mice) and allowed prolonged graft survival of major MHC-mismatched skin grafts in wild-type non-lymphopenic recipients. The expanded Treg alone were however not suffi cient to induce tolerance in stringent experimental conditions. Rapamycin reduced the frequency of Teff but did not impede expansion of Treg. Pro-infl ammatory stimuli hindered the expansion of Treg and resulted in an increase in the frequency of CD4+IFN-γ+ and CD4+IL17+ T cells. We propose that IL-2-based treatments would be an effi cient method for expanding functional Treg in vivo without affecting other immune cell populations, thereby favorably shifting the pool of alloreactive T cells towards regulation in response to an allograft. However, we also highlight some potential limitations of Treg expansion such as concomitant infl ammatory events.

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity.

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.

Clonotype selection and composition of human CD8 T cells specific for persistent herpes viruses varies with differentiation but is stable over time.

Protection from reactivation of persistent herpes virus infection is mediated by Ag-specific CD8 T cell responses, which are highly regulated by still poorly understood mechanisms. In this study, we analyzed differentiation and clonotypic dynamics of EBV- and CMV-specific T cells from healthy adults. Although these T lymphocytes included all subsets, from early-differentiated (EM/CD28(pos)) to late-differentiated (EMRA/CD28(neg)) stages, they varied in the sizes/proportions of these subsets. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28(neg) subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, because some clonotypes were selected with differentiation while others were not. Late-differentiated CMV-specific clonotypes were mostly characterized by TCR with lower dependency on CD8 coreceptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of 4 years. Remarkably, clonotypic distribution was stable not only in late-differentiated but also in less-differentiated T cell subsets. Thus, T cell clonotypes segregate with differentiation, but the clonal composition once established is kept constant for at least several years. These findings reveal novel features of the highly sophisticated control of steady state protective T cell activity in healthy adults.

Impact of Sirtuin 2 knockout on innate immune responses

Purpose/Objective: Histone deacetylases (HDACs) deacetylate histones and transcriptional regulators thereby affecting numerous biological functions. Seven mammalian sirtuins (SIRT1-7) constitute the NAD-dependent class III subfamily of HDACs. Sirtuins are the center of great interest due to their regulatory role in the control of metabolism, ageing and age-related diseases. Up to now, little is known about the influence of sirtuins on immune responses, and nothing about the role of SIRT2. The aim of the study was to analyze the influence of SIRT2 knockout on immune cell development and innate immune responses in vitro and in vivo. Materials and methods: SIRT2 germline knockout were produced on a C57BL/6J background. The cellularity of thymus and spleen was assessed by flow cytometry (n = 3). Bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) and splenocytes were stimulated with LPS, Pam3CSK4 lipopeptide, CpG ODN, E. coli, S. aureus, TSST-1, SEB, anti-CD3+ CD28 and concanavalin A (n = 3_8). TNF, IL-2, IL-6, IL-12p40 and IFNc production, SIRT1_7 and CD40 expression, and proliferation were quantified by real time-PCR, ELISA, flow cytometry and H3-thymidine incorporation. Mice (n = 6_16) were challenged with LPS, TNF/D-galactosamine, E. coli and K. pneumonia titrated to cause either mild or severe infections or shock. Blood was collected to quantify cytokines and bacteria. Mortality was checked regularly. Results: SIRT2 is the most expressed sirtuin in macrophages and myeloid DCs. To test whether SIRT2 impacts on innate immune responses, we generated SIRT2 germline knockout mice. SIRT2-/- mice born at the expected Mendelian ratio and develop normally. The proportions and absolute numbers of DN1-4, DP and SP thymocytes, and of T-cells (DN and SP, naı¨ve and memory), B-cells (immature and mature), DCs (cDCs and pDCs) and granulocytes in the spleen are similar in SIRT2+/+ and SIRT2-/- mice. SIRT2+/+ and SIRT2-/- BMDMs, BMDCs and splenocytes produce cytokines (RNA and protein), upregulate CD40, and proliferate to the same extent. SIRT2+/+ and SIRT2-/- mice respond similarly (cytokine blood levels, bacterial counts and mortality) to non-severe and lethal endotoxemia, E. coli peritonitis, K. pneumonia pneumonia and TNF-induced shock. Conclusions: SIRT2 knockout has no dramatic impact on the development of immune cells and on innate immune responses in vitro and in vivo. Considering that SIRT2 may participate to control metabolic homeostasis, we are currently assessing the impact of SIRT2 deficiency on innate immune responses under metabolic stress.

The robustness of the weak selection approximation for the evolution of altruism against strong selection.

The weak selection approximation of population genetics has made possible the analysis of social evolution under a considerable variety of biological scenarios. Despite its extensive usage, the accuracy of weak selection in predicting the emergence of altruism under limited dispersal when selection intensity increases remains unclear. Here, we derive the condition for the spread of an altruistic mutant in the infinite island model of dispersal under a Moran reproductive process and arbitrary strength of selection. The simplicity of the model allows us to compare weak and strong selection regimes analytically. Our results demonstrate that the weak selection approximation is robust to moderate increases in selection intensity and therefore provides a good approximation to understand the invasion of altruism in spatially structured population. In particular, we find that the weak selection approximation is excellent even if selection is very strong, when either migration is much stronger than selection or when patches are large. Importantly, we emphasize that the weak selection approximation provides the ideal condition for the invasion of altruism, and increasing selection intensity will impede the emergence of altruism. We discuss that this should also hold for more complicated life cycles and for culturally transmitted altruism. Using the weak selection approximation is therefore unlikely to miss out on any demographic scenario that lead to the evolution of altruism under limited dispersal.

Targeted expression of human CD1d in transgenic mice reveals independent roles for thymocytes and thymic APCs in positive and negative selection of Valpha14i NKT cells.

CD1d-dependent invariant Valpha14 (Valpha14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Valpha14-Jalpha18 TCR alpha-chain paired mostly with Vbeta8.2 and Vbeta7. The cellular requirements for thymic positive and negative selection of Valpha14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Valpha14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Valpha14i NKT cells that proved functional when activated ex vivo with the Ag alpha-galactosyl ceramide. Valpha14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vbeta8.2. The low number of Vbeta8.2+ Valpha14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vbeta7+ Valpha14i NKT cells. Vbeta8.2+, but not Vbeta7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Valpha14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.

Expression pattern of sirtuins in innate immune cells and influence of Sirtuin 1/2 inhibition on innate immune responses to Toll-like receptor ligands and to infection

Purpose/Objective: The family of histone deacetylases comprises 18 members in mammals, among which seven sirtuins (SIRT1-7). Sirtuins are NADP-dependent enzymes that have been involved in the control of cell metabolism, proliferation and survival. The expression pattern of sirtuins and their influence on host response to microbial infection remain largely unknown. The aim of the study was to analyze the expression of SIRT1-7 and to address the effects of SIRT1/2 inhibition on innate immune responses in vitro and in vivo.. Materials and methods: in vitro: Bone marrow (BM), BM-derived macrophages (BMDMs) and dendritic cells (BMDCs) and RAW 264.7 and J774.1 macrophage cell lines were stimulated for 0, 2, 6 and 18 h with LPS, Pam3CSK4 and CpG ODN. SIRT1-7 mRNA was quantified by real time-PCR. TNF was measured by ELISA. In vivo: BALB/c mice were challenged with LPS (350 lg i.p.) with or without a SIRT1/2 inhibitor. Blood and organs were collected after 0, 1, 4, 8 and 24 h to quantify SIRT1-7 and TNF. Mortality was assessed daily. Results: Bone marrow, macrophages and DCs express, in order of abundance, SIRT2 > > SIRT1, SIRT3 and SIRT6 > SIRT4, SIRT5 and SIRT7. Microbial products decrease the expression of all sirtuins except SIRT6 in a time dependent manner in BMDMs (0_24 h). SIRT2 is the most expressed sirtuin also in the liver, kidney (together with SIRT3) and spleen. Upon LPS challenge, SIRT1, SIRT3, SIRT4 and SIRT7 mRNA levels decrease in the liver (from 4 h to 24 h), whereas SIRT1-7 mRNA levels decrease within 1 h in both kidney and spleen. Pharmacological inhibition of SIRT1/2 decreases TNF production by macrophages stimulated with LPS, Pam3CSK4 and CpG ODN (n = 6; P < 0.001). In agreement, prophylactic treatment with a SIRT1/2 inhibitor decreases TNF production (n = 8; P = 0.04) and increases survival (n = 13, P = 0.03) of mice challenged with LPS. Conclusions: Sirtuins are expressed in innate immune cells. Inhibition of SIRT1/2 activity decreases cytokine production by macrophages and protects from endotoxemia, suggesting that sirtuin inhibitors may represent novel adjunctive therapy for treating inflammatory disorders such as sepsis.

A role for T-bet-mediated tumour immune surveillance in anti-IL-17A treatment of lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide. The cytokine interleukin-17A supports tumour vascularization and growth, however, its role in lung cancer is unknown. Here we show, in the lungs of patients with lung adenocarcinoma, an increase in interleukin-17A that is inversely correlated with the expression of T-bet and correlated with the T regulatory cell transcription factor Foxp3. Local targeting of interleukin-17A in experimental lung adenocarcinoma results in a reduction in tumour load, local expansion of interferon-γ-producing CD4(+) T cells and a reduction in lung CD4(+)CD25(+)Foxp3(+) regulatory T cells. T-bet((-/-)) mice have a significantly higher tumour load compared with wild-type mice. This is associated with the local upregulation of interleukin-23 and induction of interleukin-17A/interleukin-17R-expressing T cells infiltrating the tumour. Local anti-interleukin-17A antibody treatment partially improves the survival of T-bet((-/-)) mice. These results suggest that local anti-interleukin-17A antibody therapy could be considered for the treatment of lung tumours.

Selection of peptides and synthesis of pentameric peptabody molecules reacting specifically with ErbB-2 receptor.

The HER-2/ErbB-2 oncoprotein is overexpressed in human breast and ovarian adenocarcinomas and is clearly associated with the malignant phenotype. Although no specific ligand for this receptor has been positively identified, ErbB-2 was shown to play a central role in a network of interactions with the related ErbB-1, ErbB-3 and ErbB-4 receptors. We have selected new peptides binding to ErbB-2 extracellular domain protein (ECD) by screening 2 newly developed constrained and unconstrained random hexapeptide phage libraries. Out of 37 phage clones, which bound specifically to ErbB-2 ECD, we found 6 constrained and 10 linear different hexapeptide sequences. Among the latter, 5 consensus motifs, all with a common methionine and a positively charged residue at positions 1 and 3, respectively, were identified. Furthermore, 3 representative hexapeptides were fused to a coiled-coil pentameric recombinant protein to form the so-called peptabodies recently developed in our laboratory. The 3 peptabodies bound specifically to the ErbB-2 ECD, as determined by enzyme-linked immunosorbent assay and BIAcore analysis and to tumor cells overexpressing ErbB-2, as shown by flow cytometry. Interestingly, one of the free selected linear peptides and all 3 peptabodies inhibited the proliferation of tumor cells overexpressing ErbB-2. In conclusion, a novel type of ErbB-2-specific ligand is described that might complement presently available monoclonal antibodies.

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.