Publicação
Strategies for T-cell reconstruction : insights from human clinical models
| Resumo: | The thymus is essential for both the establishment of the peripheral T cell pool and the generation of the diverse T cell receptor (TCR) repertoire capable of dealing with new pathogens and controlling the escape of persistent infections. In humans, the thymus is almost fully developed at birth, with the rate of T cell production markedly decreasing after puberty. However, it is now clear that this central lymphoid organ plays an essential role in the lifetime “de novo” generation of T cells. The maintenance of naïve T cells is currently thought to depend upon a combination of peripheral T cell proliferation as well as to an age-dependent contribution of recent thymic emigrants. The overall aim of this work was to investigate the relative roles of the thymus and the “periphery” in the maintenance/recovery of the human T cell compartment through the study of specific clinical models. FOXN1 is a transcription factor, expressed by thymic epithelium, crucial for both the development of the thymus and prevention of its involution. Defects in FOXN1 in mice lead to athymia in association with total alopecia, due to its additional role in hair follicle differentiation (“nude-SCID mice”). Human FOXN1 deficiency was first reported by Pignata et al. in two sisters from Italy that, despite the evidence of athymia, exhibited a significant number of circulating T cells. We identified the same homozygous R255X mutation in a Portuguese child, who presented at 5 months of age with alopecia, respiratory failure due to Bacillus Calmette-Guérin (BCG) dissemination following routine neonatal BCG vaccination, and circulating T cells of non-maternal origin at close to normal numbers. The first aim of this work was to investigate the T cell population generated in the presence of FOXN1 deficiency. Circulating T cells were equally distributed between CD4, CD8 and, strikingly, an abnormally increased population of TCR αβ+ cells that expressed neither CD4 nor CD8 (double-negative, DNαβ), which usually represent less than 1% of the peripheral T cell pool. T cells were non-naïve, oligoclonal, activated, and unable to proliferate in vitro. The thymus is known to produce a regulatory CD4 T cell subset (Treg), with suppressive properties, fundamental for preventing autoimmunity. Currently they are best identified by expression of the forkhead box P3 transcription factor FoxP3. Notably, more than 40% of the CD4 subset expressed high levels of FoxP3 and had a clear regulatory-like T cell (Treg) phenotype. Thus, human FOXN1-deficiency due to R255X mutation was associated with significant numbers of oligoclonal T cells suggesting that, to a certain extent, T cell development still occured, albeit with altered positive/negative selection, as illustrated by the aberrant expansion of FoxP3+ and DN subsets. As FOXN1 mutations impact on thymic epithelium rather than hematopoietic precursors, we predicted that thymic transplantation, although never performed before in this setting, could be a curative strategy. This was confirmed by the documentation of the clinical efficacy of HLA-mismatched thymic transplantation, as attested by the temporal association between the clearance of the ongoing BCG adenitis and the development of specific responses against mycobacterium antigens. Our study of the kinetics of establishment of the T cell pool after HLA mismatched thymic transplantation provides unique data regarding the dynamics of replenishment of the immune system. A progressive increase of naive (antigen non-experienced cells) T cells was also observed, resulting in the generation of a fully diverse CD4 T cell repertoire in spite of the HLA-mismatched thymic epithelia. Reconstitution of the Treg pool occurred in parallel to that of the CD4 subset, leading to stable frequencies within the normal range. Thymic Treg development is currently thought to be dependent upon a small developmental niche that tightly controls Treg output. It is thus possible that FOXN1 plays a role in such niches, contributing to the thymic regulation of Treg numbers. In contrast, a significant population of circulating DNαβ persisted, at relatively stable frequencies (17% of αβ T cells) and phenotype, throughout 5yrs of follow-up. Although the possibility that DNαβ T cells are long-lived cells generated pretransplant cannot be excluded, it is also plausible that their persistence may reflect a continuous production of this population by a putative thymic rudiment. The functionality of the allogeneic thymic graft was further estimated by sj/βTREC quantification; a ratio between early and late products of TCR rearrangements that was shown to represent an indirect measurement of thymocyte division-rate, and a direct correlate of thymic output. A progressive increase of the sj/βTREC ratio was observed, reaching levels comparable to those found in healthy children. Importantly, a sharp decline of sj/βTREC, accompanied by a decrease in the proportion of naïve cells was observed 4yrs post-transplant. Notably, these values plateaued thereafter, suggesting that steady-state equilibrium could be established after replenishment of the immune system. Importantly, our data showed that immunocompetence can be achieved through HLA-mismatched thymic transplantation, despite the lack of a sustained thymocytedivision rate (as evidenced by sj/βTREC). These novel data regarding the long-term sustainability of allogeneic thymic tissue and the immunological reconstitution achieved have implications for the design of immune-based therapeutic strategies to be used in other clinical settings. Recent studies suggested the possibility of a thymic rebound as a compensatory feedback loop triggered by emptiness of the peripheral T cell pool, particularly in HIV infection. The cytokine IL-7 has been highlighted as a possible factor in this process. IL- 7 has also a central role in peripheral T cell homeostasis. The IL-7 driven peripheral T cell proliferation/survival is thought to be able to compensate any putative thymic impairment resulting either directly from HIV infection, or from the heightened state of immune-activation that characterizes HIV disease. In fact, the persistent immune stimulation, and the consequent T cell anergy and susceptibility to apoptosis, are considered key determinants of CD4 decline and AIDS progression. The second specific aim of this work was to investigate the interplay of these pathways during HIV/AIDS. The originality of this study mainly resulted from the definition of the cohorts under investigation. HIV-1+ patients with discordant responses to ART (ART-Discordants, poor CD4 recovery despite suppression of viremia) were compared with HIV-2+ patients that exhibited a similar degree of CD4- depletion and reduced circulating virus in the absence of ART. Untreated HIV-1+ patients who are expected to have high viremia, as well as HIV-1+ patients under ART with successful virological and immunological responses were studied in parallel. Low CD4-counts were associated with major naïve CD4 and CD8 depletion, irrespective of type of infection or ART-exposure. Several pathways were likely to contribute to the CD4-count stability and low rate of opportunistic infections documented in ART-Discordants in spite of their presumed thymic impairment, namely lower levels of T cell activation and a better ability to use IL-7 as indicated by the expression levels of the IL-7 induced, anti-apoptotic molecule, Bcl-2. Our third specific aim was based on the study of an individual with chronic granulomatous disease (CGD), a primary defect in the phagocytic oxidative burst, who, despite being HIV negative, presented CD4 T cell depletion at levels similar to those found in advanced AIDS patients. CGD represents the most prevalent (1:200000 live births) primary phagocytic defect. This 32-year-old patient, presenting with typical CGD-associated infections, had a CD4 lymphopenia of less than 200 cells/l, for more than 16 years. Although there are previous reports of diminished T cell numbers in CGD patients, these studies did not include phenotypic and functional T cell analysis. We found a generalized immune activation, in conjunction with markers of increased cell-turnover, including a reduced telomere length of both the CD4 and CD8 subsets, and expansion of terminally-differentiated effector CD8 T cells. Additionally a marked loss of naïve T cells was found, with evidence of impaired thymic production as assessed by sj/βTREC ratio, despite the increased IL-7 levels. This immunological profile has been considered a risk for infections, and was shown to be an independent predictor of death in aged subjects. Therefore, it is worth considering this putative exhaustion of immune resources in the evaluation of long-term therapeutic strategies, including stem-cell transplantation, given the increasing life-expectancy of CGD patients. Overall, our data provide evidence that, although immunodeficiency may be associated with profound lymphocyte disturbances, different pathways can be exploited to achieve immunological competence. The characterization of these pathways in human models is of importance for the definition and design of new strategies for immune reconstitution. |
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| Autores principais: | Albuquerque, Adriana Silva de, 1976- |
| Assunto: | Linfócitos T Timo Síndrome de imunodeficiência adquirida HIV Doença granulomatosa crónica Factor 3-alfa nuclear de hepatócito Teses de doutoramento - 2010 |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | The thymus is essential for both the establishment of the peripheral T cell pool and the generation of the diverse T cell receptor (TCR) repertoire capable of dealing with new pathogens and controlling the escape of persistent infections. In humans, the thymus is almost fully developed at birth, with the rate of T cell production markedly decreasing after puberty. However, it is now clear that this central lymphoid organ plays an essential role in the lifetime “de novo” generation of T cells. The maintenance of naïve T cells is currently thought to depend upon a combination of peripheral T cell proliferation as well as to an age-dependent contribution of recent thymic emigrants. The overall aim of this work was to investigate the relative roles of the thymus and the “periphery” in the maintenance/recovery of the human T cell compartment through the study of specific clinical models. FOXN1 is a transcription factor, expressed by thymic epithelium, crucial for both the development of the thymus and prevention of its involution. Defects in FOXN1 in mice lead to athymia in association with total alopecia, due to its additional role in hair follicle differentiation (“nude-SCID mice”). Human FOXN1 deficiency was first reported by Pignata et al. in two sisters from Italy that, despite the evidence of athymia, exhibited a significant number of circulating T cells. We identified the same homozygous R255X mutation in a Portuguese child, who presented at 5 months of age with alopecia, respiratory failure due to Bacillus Calmette-Guérin (BCG) dissemination following routine neonatal BCG vaccination, and circulating T cells of non-maternal origin at close to normal numbers. The first aim of this work was to investigate the T cell population generated in the presence of FOXN1 deficiency. Circulating T cells were equally distributed between CD4, CD8 and, strikingly, an abnormally increased population of TCR αβ+ cells that expressed neither CD4 nor CD8 (double-negative, DNαβ), which usually represent less than 1% of the peripheral T cell pool. T cells were non-naïve, oligoclonal, activated, and unable to proliferate in vitro. The thymus is known to produce a regulatory CD4 T cell subset (Treg), with suppressive properties, fundamental for preventing autoimmunity. Currently they are best identified by expression of the forkhead box P3 transcription factor FoxP3. Notably, more than 40% of the CD4 subset expressed high levels of FoxP3 and had a clear regulatory-like T cell (Treg) phenotype. Thus, human FOXN1-deficiency due to R255X mutation was associated with significant numbers of oligoclonal T cells suggesting that, to a certain extent, T cell development still occured, albeit with altered positive/negative selection, as illustrated by the aberrant expansion of FoxP3+ and DN subsets. As FOXN1 mutations impact on thymic epithelium rather than hematopoietic precursors, we predicted that thymic transplantation, although never performed before in this setting, could be a curative strategy. This was confirmed by the documentation of the clinical efficacy of HLA-mismatched thymic transplantation, as attested by the temporal association between the clearance of the ongoing BCG adenitis and the development of specific responses against mycobacterium antigens. Our study of the kinetics of establishment of the T cell pool after HLA mismatched thymic transplantation provides unique data regarding the dynamics of replenishment of the immune system. A progressive increase of naive (antigen non-experienced cells) T cells was also observed, resulting in the generation of a fully diverse CD4 T cell repertoire in spite of the HLA-mismatched thymic epithelia. Reconstitution of the Treg pool occurred in parallel to that of the CD4 subset, leading to stable frequencies within the normal range. Thymic Treg development is currently thought to be dependent upon a small developmental niche that tightly controls Treg output. It is thus possible that FOXN1 plays a role in such niches, contributing to the thymic regulation of Treg numbers. In contrast, a significant population of circulating DNαβ persisted, at relatively stable frequencies (17% of αβ T cells) and phenotype, throughout 5yrs of follow-up. Although the possibility that DNαβ T cells are long-lived cells generated pretransplant cannot be excluded, it is also plausible that their persistence may reflect a continuous production of this population by a putative thymic rudiment. The functionality of the allogeneic thymic graft was further estimated by sj/βTREC quantification; a ratio between early and late products of TCR rearrangements that was shown to represent an indirect measurement of thymocyte division-rate, and a direct correlate of thymic output. A progressive increase of the sj/βTREC ratio was observed, reaching levels comparable to those found in healthy children. Importantly, a sharp decline of sj/βTREC, accompanied by a decrease in the proportion of naïve cells was observed 4yrs post-transplant. Notably, these values plateaued thereafter, suggesting that steady-state equilibrium could be established after replenishment of the immune system. Importantly, our data showed that immunocompetence can be achieved through HLA-mismatched thymic transplantation, despite the lack of a sustained thymocytedivision rate (as evidenced by sj/βTREC). These novel data regarding the long-term sustainability of allogeneic thymic tissue and the immunological reconstitution achieved have implications for the design of immune-based therapeutic strategies to be used in other clinical settings. Recent studies suggested the possibility of a thymic rebound as a compensatory feedback loop triggered by emptiness of the peripheral T cell pool, particularly in HIV infection. The cytokine IL-7 has been highlighted as a possible factor in this process. IL- 7 has also a central role in peripheral T cell homeostasis. The IL-7 driven peripheral T cell proliferation/survival is thought to be able to compensate any putative thymic impairment resulting either directly from HIV infection, or from the heightened state of immune-activation that characterizes HIV disease. In fact, the persistent immune stimulation, and the consequent T cell anergy and susceptibility to apoptosis, are considered key determinants of CD4 decline and AIDS progression. The second specific aim of this work was to investigate the interplay of these pathways during HIV/AIDS. The originality of this study mainly resulted from the definition of the cohorts under investigation. HIV-1+ patients with discordant responses to ART (ART-Discordants, poor CD4 recovery despite suppression of viremia) were compared with HIV-2+ patients that exhibited a similar degree of CD4- depletion and reduced circulating virus in the absence of ART. Untreated HIV-1+ patients who are expected to have high viremia, as well as HIV-1+ patients under ART with successful virological and immunological responses were studied in parallel. Low CD4-counts were associated with major naïve CD4 and CD8 depletion, irrespective of type of infection or ART-exposure. Several pathways were likely to contribute to the CD4-count stability and low rate of opportunistic infections documented in ART-Discordants in spite of their presumed thymic impairment, namely lower levels of T cell activation and a better ability to use IL-7 as indicated by the expression levels of the IL-7 induced, anti-apoptotic molecule, Bcl-2. Our third specific aim was based on the study of an individual with chronic granulomatous disease (CGD), a primary defect in the phagocytic oxidative burst, who, despite being HIV negative, presented CD4 T cell depletion at levels similar to those found in advanced AIDS patients. CGD represents the most prevalent (1:200000 live births) primary phagocytic defect. This 32-year-old patient, presenting with typical CGD-associated infections, had a CD4 lymphopenia of less than 200 cells/l, for more than 16 years. Although there are previous reports of diminished T cell numbers in CGD patients, these studies did not include phenotypic and functional T cell analysis. We found a generalized immune activation, in conjunction with markers of increased cell-turnover, including a reduced telomere length of both the CD4 and CD8 subsets, and expansion of terminally-differentiated effector CD8 T cells. Additionally a marked loss of naïve T cells was found, with evidence of impaired thymic production as assessed by sj/βTREC ratio, despite the increased IL-7 levels. This immunological profile has been considered a risk for infections, and was shown to be an independent predictor of death in aged subjects. Therefore, it is worth considering this putative exhaustion of immune resources in the evaluation of long-term therapeutic strategies, including stem-cell transplantation, given the increasing life-expectancy of CGD patients. Overall, our data provide evidence that, although immunodeficiency may be associated with profound lymphocyte disturbances, different pathways can be exploited to achieve immunological competence. The characterization of these pathways in human models is of importance for the definition and design of new strategies for immune reconstitution. |
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