Differential T and B cell responses against Mycobacterium tuberculosis heparin-binding hemagglutinin adhesin in infected healthy individuals and patients with tuberculosis.

Because only 10% of individuals infected with Mycobacterium tuberculosis will eventually develop disease, antigens that are recognized differently by the immune systems of infected healthy and diseased subjects may constitute potential vaccine candidates. Here, the heparin-binding hemagglutinin adhesin (HBHA) is identified as such an antigen. Lymphocytes from 60% of healthy infected individuals (n=25) produced interferon (IFN)-gamma after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n=24). In the responders, both CD4(+) and CD8(+) cells secreted HBHA-specific IFN-gamma, and the antigen was presented by both major histocompatibility complex class I and II molecules. In contrast to the reduced ability of patients with tuberculosis to produce HBHA-specific IFN-gamma, most of them (82%) produced anti-HBHA antibodies, compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and infected healthy individuals and might provide a marker for protection against tuberculosis.

Bordetella pertussis infection in 2-month-old infants promotes type 1 T cell responses.

Neonatal immaturity of the immune system is currently believed to generally limit the induction of immune responses to vaccine Ags and to skew them toward type 2 responses. We demonstrated here that Bordetella pertussis infection in very young infants (median, 2 mo old) as well as the first administration of whole-cell pertussis vaccine induces B. pertussis Ag-specific IFN-gamma secretion by the PBMC of these infants. IFN-gamma was secreted by both CD4(+) and CD8(+) T lymphocytes, and the levels of Ag-induced IFN-gamma secretion did not correlate with the age of the infants. Appearance of the specific Th-1 cell-mediated immunity was accompanied by a general shift of the cytokine secretion profile of these infants toward a stronger Th1 profile, as evidenced by the response to a polyclonal stimulation. We conclude that the immune system of 2-mo-old infants is developmentally mature enough to develop Th1 responses in vivo upon infection by B. pertussis or vaccination with whole-cell pertussis vaccines.

Repertoire of HLA-DR1-restricted CD4 T cell responses to capsular Caf1 antigen of Y. pestis HLA-transgenic mice

Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T cell responses for Caf1. We characterized CD4 T cell epitopes of Caf1 in 'humanized'-HLA-DR1 transgenic mice lacking endogenous MHC class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T cell immunity was measured with respect to proliferative and IFNgamma T cell responses and recognition by a panel of T cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased towards a single immunodominant epitope near the C-terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

Skin langerin+ dendritic cells transport intradermally injected anti-DEC-205 antibodies but are not essential for subsequent cytotoxic CD8+ T cell responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8a(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8a(+) DCs are sufficient to subsequent CD8(+) T cell responses.

The soluble isoform of CTLA-4 as a regulator of T-cell responses

CTLA-4 is a crucial immune regulator that mediates both negative co-stimulation signals to T cells, and regulatory T (Treg) cell extrinsic control of effector responses. Here we present evidence supporting a novel mechanism for this extrinsic suppression, executed by the alternatively spliced soluble CTLA-4 isoform (sCTLA-4). Analyses of human T cells in vitro show that sCTLA-4 secretion can be increased during responses, and has potent inhibitory properties, since isoform-specific blockade of its activity significantly increased antigen-driven proliferation and cytokine (interferon-?, IL-17) secretion. Treg cells were demonstrated to be a prominent source of sCTLA-4, which contributed to suppression in vitro when their numbers were limiting. The soluble isoform was also produced by, and inhibited, murine T cells responding to antigen in vitro, and blockade of its activity in vivo protected against metastatic spread of melanoma in mice. We conclude that sCTLA-4 is an important immune regulator, responsible for at least some of the inhibitory effects previously ascribed to the membrane-bound isoform. These results suggest that the immune system exploits the different CTLA-4 isoforms for either intrinsic or extrinsic regulation of T-cell activity.

In vitro mesenchymal stem cell responses on laser-welded NiTi alloy

The biocompatibility of NiTi after laser welding was studied by examining the in vitro (mesenchymal stem cell) MSC responses at different sets of time varying from early (4 to 12 h) to intermediate phases (1 and 4 days) of cell culture. The effects of physical (surface roughness and topography) and chemical (surface Ti/Ni ratio) changes as a consequence of laser welding in different regions (WZ, HAZ, and BM) on the cell morphology and cell coverage were studied. The results in this research indicated that the morphology of MSCs was affected primarily by the topographical factors in the WZ: the well-defined and directional dendritic pattern and the presence of deeper grooves. The morphology of MSCs was not significantly modulated by surface roughness. Despite the possible initial Ni release in the medium during the cell culture, no toxic effect seemed to cause to MSCs as evidenced by the success of adhesion and spreading of the cells onto different regions in the laser weldment. The good biocompatibility of the NiTi laser weldment has been firstly reported in this study.

Secreted Proteins from the Helminth Fasciola hepatica Inhibit the Initiation of Autoreactive T Cell Responses and Prevent Diabetes in the NOD Mouse

Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-γ secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFβ and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.

Effect of laser treatment on the attachment and viability of mesenchymal stem cell responses on shape memory NiTi alloy

The objectives of this study were to investigate the effect of laser-induced surface features on the morphology, attachment and viability of mesenchymal stem cells (MSCs) at different periods of time, and to evaluate the biocompatibility of different zones: laser-melted zone (MZ), heat-affected zone (HAZ) and base metal (BM) in laser-treated NiTi alloy. The surface morphology and composition were studied by scanning electron microscope (SEM) and X-ray photoemission spectroscopy (XPS), respectively. The cell morphology was examined by SEM while the cell counting and viability measurements were done by haemocytometer and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The results indicated that the laser-induced surface features, such as surface roughening, presence of anisotropic dendritic pattern and complete surface Ni oxidation were beneficial to improve the biocompatibility of NiTi as evidenced by the highest cell attachment (4 days of culture) and viability (7 days of culture) found in the MZ. The biocompatibility of the MZ was the best, followed by the BM with the HAZ being the worst. The defective and porous oxide layer as well as the coarse grained structure might attribute to the inferior cell attachment (4 days of culture) and viability (7 days of culture) on the HAZ compared with the BM which has similar surface morphology.

Treatment-dependent loss of polyfunctional CD8+ T-cell responses in HIV-infected kidney transplant recipients is associated with herpesvirus reactivation.

Antiretroviral-therapy has dramatically changed the course of HIV infection and HIV-infected (HIV(+)) individuals are becoming more frequently eligible for solid-organ transplantation. However, only scarce data are available on how immunosuppressive (IS) strategies relate to transplantation outcome and immune function. We determined the impact of transplantation and immune-depleting treatment on CD4+ T-cell counts, HIV-, EBV-, and Cytomegalovirus (CMV)-viral loads and virus-specific T-cell immunity in a 1-year prospective cohort of 27 HIV(+) kidney transplant recipients. While the results show an increasing breadth and magnitude of the herpesvirus-specific cytotoxic T-cell (CTL) response over-time, they also revealed a significant depletion of polyfunctional virus-specific CTL in individuals receiving thymoglobulin as a lymphocyte-depleting treatment. The disappearance of polyfunctional CTL was accompanied by virologic EBV-reactivation events, directly linking the absence of specific polyfunctional CTL to viral reactivation. The data provide first insights into the immune-reserve in HIV+ infected transplant recipients and highlight new immunological effects of thymoglobulin treatment. Long-term studies will be needed to assess the clinical risk associated with thymoglobulin treatment, in particular with regards to EBV-associated lymphoproliferative diseases.

Monitoring of human CD4 T cell responses in metastatic melanoma and arthritic patients

SUMMARY : Detailed knowledge of the different components of the immune system is required for the development of new immunotherapeutic strategies. CD4 T lymphocytes represent a highly heterogeneous group of cells characterized by various profiles of cytokine production and effector vs. regulatory functions. They are central players in orchestrating adaptive immune responses: unbalances between the different subtypes can lead either to aggressive autoimmune disorders or can favour the uncontrolled growth of malignancies. In this study we focused on the characterization of human CD4 T cells in advanced stage melanoma patients as well as in patients affected by various forms of autoimmune inflammatory spondyloarthropathies. In melanoma patients we report that a population of FOXP3 CD4 T cells, known as regulatory T cells, is overrepresented in peripheral blood, and even more in tumor-infitrated lymph nodes as well as at tumor sites, as compared to healthy donors. In tumor-infiltrated lymph nodes, but not in normal lymph nodes or in peripheral blood, FOXP3 CD4 T cells feature a highly differentiated phenotype (CD45RA-CCR7+/-), which suggests for a recent encounter with their cognate antigen. FOXP3 CD4 T cells have been described to be an important component of the several known immune escape mechanisms. We demonstrated that FOXP3 CD4 T cells isolated from melanoma patients exert an in vitro suppressive action on autologous CD4 T cells, thus possibly inhibiting an efficient anti-tumor response. Next, we aimed to analyse CD4 T cells at antigen-specific level. In advanced stage melanoma patients, we identified for the first time, using pMHCII multimers, circulating CD4 T cells specific for the melanoma antigen Melan-A, presented by HLA-DQB1 *0602. Interestingly, in a cohort of melanoma patients enrolled in an immunotherapy trails consisting of injection of a Melan-A derived peptide, we did not observe signif cant variations in the ex vivo frequencies of Melan-A specific CD4 T cells, but important differences in the quality of the specific CD4 T cells. In fact, up to 50% of the ex vivo Melan-A/DQ6 specific CD4 T cells displayed a regulatory phenotype and were hypoproliferative before vaccination, while more effector, cytokine-secreting Melan-A/DQ6 specific CD4 T cells were observed after immunization. These observations suggest that peptide vaccination may favourably modify the balance between regulatory and effector tumor-specific CD4 T cells. Finally, we identified another subset of CD4 T cells as possible mediator of pathology in a group of human autoimmune spondyloarthropathies, namely Th17 cells. These cells were recently described to play a critical role in the pathogenesis of some marine models of autommunity. We document an elevated presence of circulating Th17 cells in two members of seronegative spondyloarthropathies, e.g. psoriatic arthritis and ankylosing spondylitis, while we do not observe increased frequencies of Th17 cells in peripheral blood of rheumatoid arthritic patients. In addition, Th17 cells with a more advanced differentiation state (CD45RA-CCR7-CD27-) and polyfunctionality (concomitant secretion of IL-17, IL-2 and TNFα) were observed exclusively in patients with seronegative spondylarthropathies. Together, our observations emphasize the importance of CD4 T cells in various diseases and suggest that immunotherapeutic approaches considering CD4 T cells as targets should be evaluated in the future.

Quantitative multiparameter assays to measure the effect of adjuvants on human antigen-specific CD8 T-cell responses.

Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based vaccines have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic vaccines aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic vaccines to activate T cells, but more research is necessary to identify optimal compositions of potent vaccine formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigen-specific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during the last 15 years, and monitoring techniques become more and more standardized.

Increased Degranulation of Natural Killer Cells during Acute HCV Correlates with the Magnitude of Virus-specific T cell Responses

Ce manuscrit est une pré-publication d'un article paru dans Journal of Hepatology 2010; 53(5): 805-816.