87 research outputs found
Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis
Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning
Antigen Discovery for the Identification of Vaccine Candidates and Biomarkers Using a T Cell Driven Approach in Combination with Positional Scanning Peptide Libraries
The prevention and treatment of infectious diseases is highly dependent on the availability of reliable diagnostic tests and protective or therapeutic vaccines. There also exists an urgent need to develop reliable biomarkers to monitor treatment success and to predict disease progression from asymptomatic to symptomatic disease in several disease scenarios. The elucidation of the disease-relevant antigens that elicit the protective immune responses is critical and required for the development of biomarkers, diagnostics, and vaccines. However; one of the main obstacles to the study of antigen specificity in human T cells is their low frequency in PBMC samples. To overcome this problem we have implemented strategies to generate memory T cell libraries and clones specific to the pathogen of interest. Due to the fact that memory T cells represent a repository of the human T cell response to infection, examination of their antigen specificity can efficiently reveal immunogenic and relevant antigens involved in the in vivo response to infection or vaccines. To examine the specificity of the memory T cells we use an unbiased collection of antigens together with an in silico analysis, namely positional scanning based biometrical analysis. Here we present a summary of our approach and ongoing work on the development of strategies for the culture of memory T cells from patients with Chagas disease. While most studies focus on the identification of vaccine candidates using preselected immunogenic proteins derived from animal models or by or bioinformatics prediction, here we present an innovative approach that directly examines the specificity of the memory response following infection or immunization in humans
Methodological approach to the ex vivo expansion and detection of T. cruzi-specific T cells from chronic Chagas disease patients
The discovery of T cell epitopes is essential not only for gaining knowledge about host response to infectious disease but also for the development of immune-intervention strategies. In Chagas disease, given the size and complexity of the Trypanosoma cruzi proteome and its interaction with the hostβs immune system, the fine specificity of T cells has not been extensively studied yet, and this is particularly true for the CD4+ T cell compartment. The aim of the present work was to optimize a protocol for the generation of parasite-specific memory T cell lines, representative of their in vivo precursor populations and capable of responding to parasite antigens after long-term culture. Accordingly, peripheral blood mononuclear cells (PBMC) from both chronic asymptomatic and cardiac patients, and from non-infected individuals, underwent different in vitro culture and stimulation conditions. Subsequently, cells were tested for their capacity to respond against T. cruzi lysate by measuring [3H]-thymidine incorporation and interferon-Ξ³ and GM-CSF secretion. Results allowed us to adjust initial T. cruzi lysate incubation time as well as the number of expansions with phytohemagglutinin (PHA) and irradiated allogeneic PBMC prior to specificity evaluation. Moreover, our data demonstrated that parasite specific T cells displayed a clear and strong activation by using T. cruzi lysate pulsed, Epstein-Barr virus (EBV)-transformed human B lymphocytes (B-LCL), as autologous antigen presenting cells. Under these culture conditions, we generated a clone from an asymptomatic patientβs memory CD4+ T cells which responded against epimastigote and trypomastigote protein lysate. Our results describe a culture method for isolating T. cruzispecific T cell clones from patients with Chagas disease, which enable the acquisition of information on functionality and specificity of individual T cells
Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins
Background:Trypanosoma cruzi ribosomal P proteins, P2Ξ² and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with Ξ²1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals.Methodology/Principal findings:We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2Ξ², the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2Ξ² or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-Ξ± and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells.Conclusions/Significance:Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-Ξ± and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones CientΓficas y TΓ©cnicas. Instituto de Investigaciones en IngenierΓa GenΓ©tica y BiologΓa Molecular "Dr. HΓ©ctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquΓmica; ArgentinaFil: Atienza, Augusto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos MejΓa"; ArgentinaFil: Perez Prados, Graciela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. FernΓ‘ndez"; ArgentinaFil: Buying, Alcinette. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Balouz, Virginia. Consejo Nacional de Investigaciones CientΓficas y TΓ©cnicas. Centro CientΓfico TecnolΓ³gico Conicet - La Plata. Instituto de Investigaciones BiotecnolΓ³gicas. Universidad Nacional de San MartΓn. Instituto de Investigaciones BiotecnolΓ³gicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones CientΓficas y TΓ©cnicas. Centro CientΓfico TecnolΓ³gico Conicet - La Plata. Instituto de Investigaciones BiotecnolΓ³gicas. Universidad Nacional de San MartΓn. Instituto de Investigaciones BiotecnolΓ³gicas; ArgentinaFil: Santos, Radleigh. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Tasso, Laura MΓ³nica. Consejo Nacional de Investigaciones CientΓficas y TΓ©cnicas. Instituto de Investigaciones en IngenierΓa GenΓ©tica y BiologΓa Molecular "Dr. HΓ©ctor N. Torres"; ArgentinaFil: Bonato, Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos MejΓa"; ArgentinaFil: Chiale, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos MejΓa"; ArgentinaFil: Pinilla, Clemencia. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Judkowski, Valeria A.. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones CientΓficas y TΓ©cnicas. Instituto de Investigaciones en IngenierΓa GenΓ©tica y BiologΓa Molecular "Dr. HΓ©ctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquΓmica; Argentin
T-Cell Promiscuity in Autoimmune Diabetes
OBJECTIVEβIt is well established that the primary mediators of Ξ²-cell destruction in type 1 diabetes are T-cells. Nevertheless, the molecular basis for recognition of Ξ²-cellβspecific epitopes by pathogenic T-cells remains ill defined; we seek to further explore this issue
CD28/CD154 Blockade Prevents Autoimmune Diabetes by Inducing Nondeletional Tolerance After Effector T-Cell Inhibition and Regulatory T-Cell Expansion
OBJECTIVEβBlocking T-cell signaling is an effective means to prevent autoimmunity and allograft rejection in many animal models, yet the clinical translation of many of these approaches has not resulted in the success witnessed in experimental systems. Improved understanding of these approaches may assist in developing safe and effective means to treat disorders such as autoimmune diabetes
Viral infection prevents diabetes by inducing regulatory T cells through NKT cellβplasmacytoid dendritic cell interplay
iNKT cell and pDC cross talk prevents type 1 diabetes by inducing T reg cells in the pancreatic lymph node during viral infection
Therapy of experimental type 1 diabetes by isolated Sertoli cell xenografts alone
Type I diabetes mellitus is caused by autoimmune destruction of pancreatic Ξ² cells, and effective treatment of the disease might require rescuing Ξ² cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional Ξ² cell or insulin therapy. The effect was associated with restoration of systemic immune tolerance and detection of functional pancreatic islets that consisted of glucose-responsive and insulin-secreting cells. Curative effects by SC were strictly dependent on efficient tryptophan metabolism in the xenografts, leading to TGF-Ξ²βdependent emergence of autoantigen-specific regulatory T cells and recovery of Ξ² cell function in the diabetic recipients
Key Role of the GITR/GITRLigand Pathway in the Development of Murine Autoimmune Diabetes: A Potential Therapeutic Target
BACKGROUND: The cross-talk between pathogenic T lymphocytes and regulatory T cells (Tregs) plays a major role in the progression of autoimmune diseases. Our objective is to identify molecules and/or pathways involved in this interaction and representing potential targets for innovative therapies. Glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand are key players in the T effector/Treg interaction. GITR is expressed at low levels on resting T cells and is significantly up-regulated upon activation. Constitutive high expression of GITR is detected only on Tregs. GITR interacts with its ligand mainly expressed on antigen presenting cells and endothelial cells. It has been suggested that GITR triggering activates effector T lymphocytes while inhibiting Tregs thus contributing to the amplification of immune responses. In this study, we examined the role of GITR/GITRLigand interaction in the progression of autoimmune diabetes. METHODS AND FINDINGS: Treatment of 10-day-old non-obese diabetic (NOD) mice, which spontaneously develop diabetes, with an agonistic GITR-specific antibody induced a significant acceleration of disease onset (80% at 12 weeks of age). This activity was not due to a decline in the numbers or functional capacity of CD4(+)CD25(+)Foxp3(+) Tregs but rather to a major activation of 'diabetogenic' T cells. This conclusion was supported by results showing that anti-GITR antibody exacerbates diabetes also in CD28(-/-) NOD mice, which lack Tregs. In addition, treatment of NOD mice, infused with the diabetogenic CD4(+)BDC2.5 T cell clone, with GITR-specific antibody substantially increased their migration, proliferation and activation within the pancreatic islets and draining lymph nodes. As a mirror image, blockade of the GITR/GITRLigand pathway using a neutralizing GITRLigand-specific antibody significantly protected from diabetes even at late stages of disease progression. Experiments using the BDC2.5 T cell transfer model suggested that the GITRLigand antibody acted by limiting the homing and proliferation of pathogenic T cells in pancreatic lymph nodes. CONCLUSION: GITR triggering plays an important costimulatory role on diabetogenic T cells contributing to the development of autoimmune responses. Therefore, blockade of the GITR/GITRLigand pathway appears as a novel promising clinically oriented strategy as GITRLigand-specific antibody applied at an advanced stage of disease progression can prevent overt diabetes
GM-CSF Production Allows the Identification of Immunoprevalent Antigens Recognized by Human CD4+ T Cells Following Smallpox Vaccination
The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-Ξ³ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-Ξ± is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a βT cellβdrivenβ methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens
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