Publicação
Defining the quantitative aspects of dominant tolerance induction and maintenance
| Resumo: | Immune tolerance is a state of unresponsiveness towards a set of antigens while remaining fully competent to mount unrelated immune responses. The immune system is usually tolerant to our own antigens (self), to food antigens (oral tolerance), and to several other foreign substances to which we are regularly exposed (such as pollens and other potential allergens). However, under certain conditions, either genetic or environmental breakdown of tolerance towards specific antigens can occur. This will originate the onset of autoimmune and allergic pathologies. On the other hand, the correct functioning of the immune system can lead to rejection of vital transplants as graft antigens are perceived as foreign antigens thus resulting in a poor clinical outcome. Despite recent advances, most current treatments for immune pathologies still aim to downmodulate immunity and inflammation, rather than to reestablish the healthy state of immunological tolerance. Regulatory T cells (Tregs) are key players in the induction and maintenance of peripheral tolerance, having an essential role in preventing transplant rejection and also allergic responses. Nonetheless, the molecular mechanisms by which they mediate suppression are still obscure, and their investigation is a current priority, as it may reveal important targets for immune intervention. Also, several studies point to other mechanisms that are important for tolerance induction, namely activation induced cell death (AICD) and other regulatory cell subtypes. Studies in mouse models show that a short-term therapy with monoclonal antibodies (MAbs) targeting key lymphocyte molecules is capable of producing long‐term tolerance. This concept became known as reprogramming of the immune system or therapeutic tolerance induction. Non-depleting anti-CD4 monoclonal antibodies (MAbs) were shown efficient in inducing long-term dominant tolerance mediated by regulatory T cells in several animal models of transplantation, allergy, and autoimmunity. However, despite many studies on tolerance induction following CD4- blockade, the consequences of this intervention on T cell kinetics and consequently on tolerance induction still remain to be elucidated. X | In this thesis I describe my studies of tolerance induction achieved through the use of non-depleting anti-CD4 MAb. Not only did I study Treg induction but also mechanism that contribute to establishment of the healthy state of tolerance. First of all, we have found that in vitro suboptimal T cell activation results in induction of Foxp3 expression, in the absence of exogenous TGF-β. The frequency of conversion in these cell cultures is similar to that which we found in vivo upon anti- CD4-induced transplantation tolerance. Besides that, we showed that TCR triggering with low-dose anti-CD3 or low-dose agonist peptide promotes TGF-β production by T cells. This effect was also observed with downmodulation of the TCR signal with nondepleting anti-CD4. TGF-β production by T cells is an event that precedes Foxp3 expression and is Foxp3 independent. Moreover, we confirmed that Foxp3 induction led to regulatory function in these cells as they were able to inhibit the proliferation of effector T cells. These findings support the view that sub-immunogenic regimens lead to dominant tolerance as a result of T-cell intrinsic properties. Secondly we took an interdisciplinary approach to analyzing lymphocyte dynamics, taking advantage of mathematical models that have been shown useful to estimate rates of proliferation and cell death following an intervention. Using the same strategy we studied the dynamics of T cell activation under the cover of non-depleting anti-CD4 MAb. We found that non-depleting anti-CD4 MAb prevents CD4+ T cells from undergoing optimal proliferation and lead to a higher frequency of apoptosis, even before any significant induction of Foxp3. Although the dynamic changes we detected are small, during the course of a proliferative response they lead to a very distinct overall outcome in terms of final cell numbers. The importance of these mechanisms predicted from the mathematical models for the overall tolerance was confirmed by showing that lck-driven Bcl-xL transgenic mice, bearing T cells resistant to apoptosis, fail to become tolerant to skin grafts following CD4-blockade. Our data show that in addition to induction of regulatory T cells, CD4 blockade has a marked effect in the kinetics of the effector T cell pool by the combined action of hindering proliferation while favoring apoptosis. Finally, we compared two well-established models of tolerance induction using non-depleting anti-CD4 MAb. We were able to induce tolerance with anti-CD4 preventing allergy and skin graft rejection. Although transplantation tolerance is dependent on Foxp3+ Treg cells that does not seem to be the case with allergic sensitization, as the treatment of mice that cannot produce Foxp3+ Treg cells still leads to tolerance. We found that tolerance induced with non-depleting anti-CD4 MAb in a model of allergy relies both on AICD and IL-10 as this cytokine was proven essential to tolerance induction. Moreover, the prevention of skin graft rejection also relies on IL- 10 and AICD. Nevertheless, tolerance induction with skin transplant and non-depleting anti-CD4 MAb can prevent eosinophilia (a key feature of allergy), but more remarkably tolerance induction with OVA-Alum and non-depleting anti-CD4 MAb can sustain a skin graft long-term, even though it is independent of Treg cells. Altogether, this thesis represents an advance in our understanding of the conditions that promote tolerance induction with non-depleting anti-CD4 MAb. We analyzed the effect of suboptimal stimuli to CD4+ T-cells, the kinetics of CD4+ T-cell activation in the presence of anti-CD4 MAb and compared different antigen delivery methods (local vs. systemic). Our results point to defects of CD4+ T-cell activation, both qualitative and quantitative, as key to the tolerance induction protocol. |
|---|---|
| Autores principais: | Caridade, Marta Ribeiro Lopes, 1982- |
| Assunto: | Tolerância imunológica Linfócitos T reguladores Antígenios CD4 Alergia e imunologia Teses de doutoramento - 2013 |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso a metadados |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Immune tolerance is a state of unresponsiveness towards a set of antigens while remaining fully competent to mount unrelated immune responses. The immune system is usually tolerant to our own antigens (self), to food antigens (oral tolerance), and to several other foreign substances to which we are regularly exposed (such as pollens and other potential allergens). However, under certain conditions, either genetic or environmental breakdown of tolerance towards specific antigens can occur. This will originate the onset of autoimmune and allergic pathologies. On the other hand, the correct functioning of the immune system can lead to rejection of vital transplants as graft antigens are perceived as foreign antigens thus resulting in a poor clinical outcome. Despite recent advances, most current treatments for immune pathologies still aim to downmodulate immunity and inflammation, rather than to reestablish the healthy state of immunological tolerance. Regulatory T cells (Tregs) are key players in the induction and maintenance of peripheral tolerance, having an essential role in preventing transplant rejection and also allergic responses. Nonetheless, the molecular mechanisms by which they mediate suppression are still obscure, and their investigation is a current priority, as it may reveal important targets for immune intervention. Also, several studies point to other mechanisms that are important for tolerance induction, namely activation induced cell death (AICD) and other regulatory cell subtypes. Studies in mouse models show that a short-term therapy with monoclonal antibodies (MAbs) targeting key lymphocyte molecules is capable of producing long‐term tolerance. This concept became known as reprogramming of the immune system or therapeutic tolerance induction. Non-depleting anti-CD4 monoclonal antibodies (MAbs) were shown efficient in inducing long-term dominant tolerance mediated by regulatory T cells in several animal models of transplantation, allergy, and autoimmunity. However, despite many studies on tolerance induction following CD4- blockade, the consequences of this intervention on T cell kinetics and consequently on tolerance induction still remain to be elucidated. X | In this thesis I describe my studies of tolerance induction achieved through the use of non-depleting anti-CD4 MAb. Not only did I study Treg induction but also mechanism that contribute to establishment of the healthy state of tolerance. First of all, we have found that in vitro suboptimal T cell activation results in induction of Foxp3 expression, in the absence of exogenous TGF-β. The frequency of conversion in these cell cultures is similar to that which we found in vivo upon anti- CD4-induced transplantation tolerance. Besides that, we showed that TCR triggering with low-dose anti-CD3 or low-dose agonist peptide promotes TGF-β production by T cells. This effect was also observed with downmodulation of the TCR signal with nondepleting anti-CD4. TGF-β production by T cells is an event that precedes Foxp3 expression and is Foxp3 independent. Moreover, we confirmed that Foxp3 induction led to regulatory function in these cells as they were able to inhibit the proliferation of effector T cells. These findings support the view that sub-immunogenic regimens lead to dominant tolerance as a result of T-cell intrinsic properties. Secondly we took an interdisciplinary approach to analyzing lymphocyte dynamics, taking advantage of mathematical models that have been shown useful to estimate rates of proliferation and cell death following an intervention. Using the same strategy we studied the dynamics of T cell activation under the cover of non-depleting anti-CD4 MAb. We found that non-depleting anti-CD4 MAb prevents CD4+ T cells from undergoing optimal proliferation and lead to a higher frequency of apoptosis, even before any significant induction of Foxp3. Although the dynamic changes we detected are small, during the course of a proliferative response they lead to a very distinct overall outcome in terms of final cell numbers. The importance of these mechanisms predicted from the mathematical models for the overall tolerance was confirmed by showing that lck-driven Bcl-xL transgenic mice, bearing T cells resistant to apoptosis, fail to become tolerant to skin grafts following CD4-blockade. Our data show that in addition to induction of regulatory T cells, CD4 blockade has a marked effect in the kinetics of the effector T cell pool by the combined action of hindering proliferation while favoring apoptosis. Finally, we compared two well-established models of tolerance induction using non-depleting anti-CD4 MAb. We were able to induce tolerance with anti-CD4 preventing allergy and skin graft rejection. Although transplantation tolerance is dependent on Foxp3+ Treg cells that does not seem to be the case with allergic sensitization, as the treatment of mice that cannot produce Foxp3+ Treg cells still leads to tolerance. We found that tolerance induced with non-depleting anti-CD4 MAb in a model of allergy relies both on AICD and IL-10 as this cytokine was proven essential to tolerance induction. Moreover, the prevention of skin graft rejection also relies on IL- 10 and AICD. Nevertheless, tolerance induction with skin transplant and non-depleting anti-CD4 MAb can prevent eosinophilia (a key feature of allergy), but more remarkably tolerance induction with OVA-Alum and non-depleting anti-CD4 MAb can sustain a skin graft long-term, even though it is independent of Treg cells. Altogether, this thesis represents an advance in our understanding of the conditions that promote tolerance induction with non-depleting anti-CD4 MAb. We analyzed the effect of suboptimal stimuli to CD4+ T-cells, the kinetics of CD4+ T-cell activation in the presence of anti-CD4 MAb and compared different antigen delivery methods (local vs. systemic). Our results point to defects of CD4+ T-cell activation, both qualitative and quantitative, as key to the tolerance induction protocol. |
|---|