Effects of cholera toxin on cells of immune system

In questa tesi sono stati analizzati gli effetti della Tossina Colerica (CT) sulle cellule del sistema immunitario. In particolare, sono stati studiati i meccanismi di inibizione della proliferazione dei linfociti T CD4+ e CD8+ umani da parte della CT. E’ stato osservato che la CT è in grado di prevenire l’attivazione dei linfociti T nelle fasi precoci e tale inibizione coinvolge la modulazione dell’espressione delle molecole CTLA-4 e CD28. E’ stato osservato che la CT up-regola l’espressione delle molecole inibitorie CTLA-4 e down-modula le molecole costimolatorie CD28 sui linfociti T CD4+ e CD8+ resting. L’incremento dell’espressione delle molecole CTLA-4 da parte della CT gioca un ruolo nel controllare l’attivazione e la proliferazione dei linfociti T, infatti, abbiamo osservato che anticorpi bloccanti anti-CTLA-4 F(ab’)2 sono in grado di prevenire, anche se parzialmente, tale inibizione. I nostri studi hanno valutato, inoltre, la funzione di linfociti T pre-trattati con la CT e abbiamo osservato che essi sono in grado di inibire la proliferazione di linfociti T autologhi stimolati con anti-CD3. Abbiamo inoltre osservato che questo fenomeno è mediato dal rilascio di cAMP all’esterno delle cellule. Alla luce di questi risultati, abbiamo analizzato gli effetti esercitati dall’cAMP extracellulare come primo messaggero su diversi tipi cellulari. L’cAMP extracellulare è in grado di inibire la proliferazione dei linfociti T ed è in grado di interferire con il differenziamento dei monociti in cellule dendritiche (DCs). Infatti, monociti differenziati in presenza di cAMP esogeno, non esprimono molecole CD1a e mantengono l’espressione di molecole CD14, acquisendo un fenotipo simile ai macrofagi. Tuttavia, le cellule generate in presenza di cAMP esogeno esprimono alti livelli di molecole MHC di classe II e di classe I e molecole costimolatorie CD86, mostrando un fenotipo attivato in grado di stimolare risposte T allogeniche. Inoltre, tali cellule non sono in grado di produrre TNF-α e IL-12, ma rilasciano quantità elevate di IL-6 e di IL-10. Monociti trattati con cAMP extracellulare hanno una capacità ridotta di indurre il differenziamento di linfociti T CD4+ che producono IFN-γ. Infine, è stato investigato il meccanismo attraverso il quale il cAMP extracellulare interagisce con le cellule. Utilizzando diversi antagonisti dei recettori dell’adenosina, abbiamo osservato che gli effetti mediati dal cAMP extracellulare possono essere prevenuti. Tali risultati suggeriscono che l’cAMP sia trasformato in adenosina e che tale molecola, attraverso l’interazione con i suoi recettori, sia responsabile degli effetti mediati dal cAMP esogeno.In this study, we analysed the effects of Cholera Toxin (CT) on cells of immune system. In particular, the mechanisms underling the inhibition of T cell proliferation mediated by CT on human CD4+ and CD8+ T lymphocytes were analysed. We observed that CT prevents the early activation steps of T lymphocytes and that these effects involve the modulation of costimulatory molecules CTLA-4 and CD28. We observed that CT up-regulates the expression of the inhibitory molecule CTLA-4 in resting CD4+ and CD8+ T lymphocytes. The regulation of CTLA-4 expression by CT is at the transcriptional level. Indeed, in cells treated with CT we observed an increase of two mRNA variants coding for the membrane and the soluble CTLA-4 molecules. In parallel with the up-regulation of the inhibitory molecule CTLA-4, CT down-modulates the costimulatory molecule CD28 on CD4+ and CD8+ resting T cells. The increased expression of CTLA-4 plays a role in controlling T cell activation and function as blocking anti-CTLA-4 F(ab’)2 mAbs partially prevents the inhibition mediated by CT. We evaluated the function of CT-pre-treated CD4+ T lymphocytes and we observed that they are able to inhibit the proliferation of autologous T lymphocytes stimulated with anti-CD3 mAbs. It is interesting that this phenomenon is, at least in part, a result of the release of extracellular cAMP. Therefore, by analysing the direct effects exerted by extracellular cAMP as a primary messenger on different cell types, we found that extracellular cAMP inhibits T cell proliferation and that it is able to interfere with the differentiation of monocytes into DCs. Monocytes induced to differentiated into DCs in the presence of extracellular cAMP, do not express CD1a molecules and retain the expression of CD14 acquiring a macrophages-like phenotype. Furthermore, they strongly up-regulate MHC class I and class II and CD86 costimulatory molecules giving rise to an activated population able to stimulate allogeneic T cell response. In addition, they produce a distinct pattern of cytokines upon maturation stimuli, they are unable to produce TNFα and IL-12 and they release high amount of IL-6 and IL-10. Furthermore, monocytes differentiated in the presence of cAMP show a reduced capacity of inducing the differentiation of IFNγ producing CD4+ T lymphocytes. Finally, the mechanisms through which extracellular cAMP can be sensed by the cells were studied. By using different adenosine receptors antagonists, we found that an extracellular cAMP-adenosine pathway is involved in the effects mediated by exogenous cAMP, suggesting that extracellular cAMP acting as primary messenger can be sensed by the cells of immune system and can modulate their functions

Effects of cholera toxin on cells of immune system

In questa tesi sono stati analizzati gli effetti della Tossina Colerica (CT) sulle cellule del sistema immunitario. In particolare, sono stati studiati i meccanismi di inibizione della proliferazione dei linfociti T CD4+ e CD8+ umani da parte della CT. E’ stato osservato che la CT è in grado di prevenire l’attivazione dei linfociti T nelle fasi precoci e tale inibizione coinvolge la modulazione dell’espressione delle molecole CTLA-4 e CD28. E’ stato osservato che la CT up-regola l’espressione delle molecole inibitorie CTLA-4 e down-modula le molecole costimolatorie CD28 sui linfociti T CD4+ e CD8+ resting. L’incremento dell’espressione delle molecole CTLA-4 da parte della CT gioca un ruolo nel controllare l’attivazione e la proliferazione dei linfociti T, infatti, abbiamo osservato che anticorpi bloccanti anti-CTLA-4 F(ab’)2 sono in grado di prevenire, anche se parzialmente, tale inibizione. I nostri studi hanno valutato, inoltre, la funzione di linfociti T pre-trattati con la CT e abbiamo osservato che essi sono in grado di inibire la proliferazione di linfociti T autologhi stimolati con anti-CD3. Abbiamo inoltre osservato che questo fenomeno è mediato dal rilascio di cAMP all’esterno delle cellule. Alla luce di questi risultati, abbiamo analizzato gli effetti esercitati dall’cAMP extracellulare come primo messaggero su diversi tipi cellulari. L’cAMP extracellulare è in grado di inibire la proliferazione dei linfociti T ed è in grado di interferire con il differenziamento dei monociti in cellule dendritiche (DCs). Infatti, monociti differenziati in presenza di cAMP esogeno, non esprimono molecole CD1a e mantengono l’espressione di molecole CD14, acquisendo un fenotipo simile ai macrofagi. Tuttavia, le cellule generate in presenza di cAMP esogeno esprimono alti livelli di molecole MHC di classe II e di classe I e molecole costimolatorie CD86, mostrando un fenotipo attivato in grado di stimolare risposte T allogeniche. Inoltre, tali cellule non sono in grado di produrre TNF-α e IL-12, ma rilasciano quantità elevate di IL-6 e di IL-10. Monociti trattati con cAMP extracellulare hanno una capacità ridotta di indurre il differenziamento di linfociti T CD4+ che producono IFN-γ. Infine, è stato investigato il meccanismo attraverso il quale il cAMP extracellulare interagisce con le cellule. Utilizzando diversi antagonisti dei recettori dell’adenosina, abbiamo osservato che gli effetti mediati dal cAMP extracellulare possono essere prevenuti. Tali risultati suggeriscono che l’cAMP sia trasformato in adenosina e che tale molecola, attraverso l’interazione con i suoi recettori, sia responsabile degli effetti mediati dal cAMP esogeno.In this study, we analysed the effects of Cholera Toxin (CT) on cells of immune system. In particular, the mechanisms underling the inhibition of T cell proliferation mediated by CT on human CD4+ and CD8+ T lymphocytes were analysed. We observed that CT prevents the early activation steps of T lymphocytes and that these effects involve the modulation of costimulatory molecules CTLA-4 and CD28. We observed that CT up-regulates the expression of the inhibitory molecule CTLA-4 in resting CD4+ and CD8+ T lymphocytes. The regulation of CTLA-4 expression by CT is at the transcriptional level. Indeed, in cells treated with CT we observed an increase of two mRNA variants coding for the membrane and the soluble CTLA-4 molecules. In parallel with the up-regulation of the inhibitory molecule CTLA-4, CT down-modulates the costimulatory molecule CD28 on CD4+ and CD8+ resting T cells. The increased expression of CTLA-4 plays a role in controlling T cell activation and function as blocking anti-CTLA-4 F(ab’)2 mAbs partially prevents the inhibition mediated by CT. We evaluated the function of CT-pre-treated CD4+ T lymphocytes and we observed that they are able to inhibit the proliferation of autologous T lymphocytes stimulated with anti-CD3 mAbs. It is interesting that this phenomenon is, at least in part, a result of the release of extracellular cAMP. Therefore, by analysing the direct effects exerted by extracellular cAMP as a primary messenger on different cell types, we found that extracellular cAMP inhibits T cell proliferation and that it is able to interfere with the differentiation of monocytes into DCs. Monocytes induced to differentiated into DCs in the presence of extracellular cAMP, do not express CD1a molecules and retain the expression of CD14 acquiring a macrophages-like phenotype. Furthermore, they strongly up-regulate MHC class I and class II and CD86 costimulatory molecules giving rise to an activated population able to stimulate allogeneic T cell response. In addition, they produce a distinct pattern of cytokines upon maturation stimuli, they are unable to produce TNFα and IL-12 and they release high amount of IL-6 and IL-10. Furthermore, monocytes differentiated in the presence of cAMP show a reduced capacity of inducing the differentiation of IFNγ producing CD4+ T lymphocytes. Finally, the mechanisms through which extracellular cAMP can be sensed by the cells were studied. By using different adenosine receptors antagonists, we found that an extracellular cAMP-adenosine pathway is involved in the effects mediated by exogenous cAMP, suggesting that extracellular cAMP acting as primary messenger can be sensed by the cells of immune system and can modulate their functions

Immunogenicity of a Recombinant Influenza Virus Bearing Both the CD4+ and CD8+ T Cell Epitopes of Ovalbumin

Recombinant influenza viruses that bear the single immunodominant CD8+ T cell epitope OVA257−264 or the CD4+ T cell epitope OVA323−339 of the model antigen ovalbumin (OVA) have been useful tools in immunology. Here, we generated a recombinant influenza virus, WSN-OVAI/II, that bears both OVA-specific CD8+ and CD4+ epitopes on its hemagglutinin molecule. Live and heat-inactivated WSN-OVAI/II viruses were efficiently presented by dendritic cells in vitro to OT-I TCR transgenic CD8+ T cells and OT-II TCR transgenic CD4+ T cells. In vivo, WSN-OVAI/II virus was attenuated in virulence, highly immunogenic, and protected mice from B16-OVA tumor challenge in a prophylactic model of vaccination. Thus, WSN-OVAI/II virus represents an additional tool, along with OVA TCR transgenic mice, for further studies on T cell responses and may be of value in vaccine design

Enhancement of T cell-mediated immune responses to whole inactivated influenza virus by chloroquine treatment in vivo

Current influenza vaccines induce poor cross-reactive CD8+ T cell responses. Cellular immunity is generally specific for epitopes that are remarkably conserved among different subtypes, suggesting that strategies to improve the cross-presentation of viral antigens by dendritic cells (DC) could elicit a broadly protective immune response. Previous studies have shown that limited proteolysis within the endocytic pathway can favorably influence antigen processing and thus immune responses. Herein, we demonstrate that chloroquine improves the cross-presentation of non-replicating influenza virus in vitro and T cell responses in mice following a single administration of inactivated HI-X31 virus. CD8+ T cells were also recruited to lymph nodes draining the site of infection and able to reduce viral load following pulmonary challenge with the heterologous PR8 virus. These findings may have implications for vaccination strategies aimed at improving the cross-presentation capacity of DCs and thus the size of effector and memory CD8+ T cells against influenza vaccines

Cholera Toxin Impairs the Differentiation of Monocytes into Dendritic Cells, Inducing Professional Antigen-Presenting Myeloid Cells ▿

Cholera toxin (CT) is a potent adjuvant for mucosal vaccination; however, its mechanism of action has not been clarified completely. It is well established that peripheral monocytes differentiate into dendritic cells (DCs) both in vitro and in vivo and that monocytes are the in vivo precursors of mucosal CD103− proinflammatory DCs. In this study, we asked whether CT had any effects on the differentiation of monocytes into DCs. We found that CT-treated monocytes, in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), failed to differentiate into classical DCs (CD14low CD1ahigh) and acquired a macrophage-like phenotype (CD14high CD1alow). Cells differentiated in the presence of CT expressed high levels of major histocompatibility complex class I (MHC-I) and MHC-II and CD80 and CD86 costimulatory molecules and produced larger amounts of IL-1β, IL-6, and IL-10 but smaller amounts of tumor necrosis factor alpha (TNF-α) and IL-12 than did monocytes differentiated into DCs in the absence of CT. The enzymatic activity of CT was found to be important for the skewing of monocytes toward a macrophage-like phenotype (Ma-DCs) with enhanced antigen-presenting functions. Indeed, treatment of monocytes with scalar doses of forskolin (FSK), an activator of adenylate cyclase, induced them to differentiate in a dose-dependent manner into a population with phenotype and functions similar to those found after CT treatment. Monocytes differentiated in the presence of CT induced the differentiation of naïve T lymphocytes toward a Th2 phenotype. Interestingly, we found that CT interferes with the differentiation of monocytes into DCs in vivo and promotes the induction of activated antigen-presenting cells (APCs) following systemic immunization