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TNF/CD80 mice, a [CD8.sup.8] T cell-mediated model for type 1 diabetes, transgenically express tumor necrosis factor [alpha] (TNF-[alpha]) and the costimulatory molecule CD80 in their pancreatic islets. Here we show that these molecules bypass the need for CD40-CD154 costimulatory interactions in activation of [CD8.sup.+] T cells, allowing us to determine the role of CD40-CD154 signals in regulation of autoaggressive [CD8.sup.+] T cells after their in vivo priming. TNF/CD80 CD154-deficient mice rapidly develop diabetes, whereas CD154-sufficient mice do not. This finding correlates with the decreased numbers of [CD4.sup.+][CD25.sup.+] T regulatory ([T.sub.R]) cells in the islets and pancreatic lymph nodes, in comparison to disease-protected CD154-sufficient mice. Administration of a CD40 agonistic antibody induces a systemic and tissue-specific increase in [T.sub.R] cells. However, this increase fails to delay diabetes development in the absence of CD154. Adoptive transfer studies show that [CD8.sup.+] T cells from TNF/CD80 CD154-deficient, but not CD154-sufficient, mice are resistant to regulation in vivo. This study provides evidence that CD40-transduced signals initiate [T.sub.R] cell increase in vivo and that CD154-transduced signals sensitize autoaggressive [CD8.sup.+] T cells to suppression.

CD[4.sup.+]CD[25.sup.+] regulatory T cells are essential in the protection from organ-specific autoimmune diseases. In the pancreas, they inhibit actions of autoreactive T cells and thereby prevent diabetes progression. The signals that control the generation, the maintenance, or the expansion of regulatory T cell pool in vivo remain poorly understood. Here we show that a transient pulse of transforming growth factor [beta] (TGF-[beta]) in the islets during the priming phase of diabetes is sufficient to inhibit disease onset by promoting the expansion of intraislet CD[4.sup.+]CD[25.sup.+] T cell pool. Approximately 40-50% of intraislet CD[4.sup.+] T cells expressed the CD25 marker and exhibited characteristics of regulatory T cells including small size, high level of intracellular CTLA-4, expression of Foxp3, and transfer of protection against diabetes. Results from in vivo incorporation of BrdUrd revealed that the generation of a high frequency of regulatory T cells in the islets is due to in situ expansion upon TGF-[beta] expression. Thus, these findings demonstrate a previously uncharacterized mechanism by which TGF-[beta] inhibits autoimmune diseases via regulation of the size of the CD[4.sup.+]CD[25.sup.+] regulatory T cell pool in vivo.

Pancreatic lymph node-derived [CD4.sup.+][CD25.sup.+] T regulatory (Treg) cells inhibit in situ differentiation of islet-reactive [CD8.sup.+] T cells into cytotoxic T lymphocytes, thereby preventing diabetes progression. The mechanism by which these Treg cells suppress anti-islet [CD8.sup.+] T cells is unknown. Here, we show by using a [CD8.sup.+] T cell-mediated model of type 1 diabetes that transforming growth factor (TGF)-[beta]-TGF-[beta] receptor signals are critical for [CD4.sup.+][CD25.sup.+] Treg cell regulation of autoreactive islet-specific cytotoxic T lymphocytes. Transgenic expression of tumor necrosis factor [alpha] from birth to 25 days of age in the islets of B6 mice that constitutively express CD80 on their [beta] cells results in accumulation of [CD4.sup.+][CD25.sup.+]TGF-[[beta].sup.+] cells exclusively in the islets and pancreatic lymph nodes, which delays diabetes progression. In contrast, expression of tumor necrosis factor [alpha] until 28 days of age prevents islet accumulation of [CD4.sup.+][CD25.sup.+]TGF-[[beta].sup.+] Treg cells, resulting in acceleration to diabetes. Furthermore, adoptive transfer experiments demonstrated that [CD4.sup.+][CD25.sup.+] Treg cells could not control naive or activated isletreactive [CD8.sup.+] T cells bearing a dominant negative TGF-[beta] receptor type II. Our data demonstrate that, in vivo, TGF-[beta] signaling in [CD8.sup.+] T cells is critical for [CD4.sup.+][CD25.sup.+] Treg cell suppression of isletreactive [CD8.sup.+] T cells in type 1 diabetes.

OBJECTIVE--To determine the role of B-cells in promoting CD8[sup +] T-cell--mediated β cell destruction in chronically inflamed islets. RESEARCH DESIGN AND METHODS--RIP-TNFα-NOD mice were crossed to B-cell-deficient NOD mice, and diabetes development was monitored. We used in vitro antigen presentation assays and in vivo administration of bromodeoxyuridine coupled to flow cytometry assays to assess intra-islet T-cell activation in the absence or presence of B-cells. CD4[sup +]Foxp3[sup +] activity in the absence or presence of B-cells was tested using in vivo depletion techniques. Cytokine production and apoptosis assays determined the capacity of CD8[sup +] T-cells transform to cytotoxic T-lymphocytes (CTLs) and survive within inflamed islets in the absence or presence of B-cells. RESULTS--B-cell deficiency significantly delayed diabetes development in chronically inflamed islets. Reintroduction of B-cells incapable of secreting immunoglobulin restored diabetes development. Both CD4[sup +] and CD8[sup +] T-cell activation was unimpaired by B-cell deficiency, and delayed disease was not due to CD4[sup +]Foxp3[sup +] T-cell suppression of T-cell responses. Instead, at the CTL transition stage, B-cell deficiency resulted in apoptosis of intra-islet CTLs. CONCLUSIONS--In inflamed islets, B-cells are central for the efficient intra-islet survival of CTLs, thereby promoting type 1 diabetes development. [ABSTRACT FROM AUTHOR]

Foxp3+ regulatory T cells develop in the thymus and are essential for maintaining peripheral tolerance to self tissues. We report the critical requirement for CD154 up-regulation specifically on, and during the thymic development of, Foxp3+ regulatory T cells for the induction of their clonal expansion within the medulla. In the absence of this signal, there was a severe reduction in their thymic generation and output, leading to decreased peripheral numbers. Importantly, CD40 expression on either thymic dendritic or epithelial cells was sufficient to promote the development of normal numbers of Foxp3+ regulatory T cells. This work suggests that CD154-transduced signals promote Foxp3+ regulatory T cell development and highlights the plasticity of the thymic stroma for supporting their generation. Crucially, this study demonstrates that Foxp3+ regulatory T cells can promiscuously accept a single critical signal necessary for their thymic development from different cellular sources, redefining our understanding of their generation. [ABSTRACT FROM AUTHOR]

TNF/CD80 mice, a CD8+ T cell-mediated model for type 1 diabetes, transgenically express tumor necrosis factor a (TNF-α) and the costimulatory molecule CD80 in their pancreatic islets. Here we show that these molecules bypass the need for CD40-CD154 costimulatory interactions in activation of CD8+ T cells, allowing us to determine the role of CD40-CD154 signals in regulation of autoaggressive CD8+ T cells after their in vivo priming. TNF/CD80 CD154-deficient mice rapidly develop diabetes, whereas CD154- sufficient mice do not. This finding correlates with the decreased numbers of CD4+CD25+ T regulatory (TR) cells in the islets and pancreatic lymph nodes, in comparison to disease-protected CD154-sufficient mite. Administration of a CD40 agonistic antibody induces a systemic and tissue-specific increase in TR cells. However, this increase fails to delay diabetes development in the absence of CD154. Adoptive transfer studies show that CD8+ T cells from TNF/CD80 CD1 54-deficient, but not CD154-sufficient, mice are resistant to regulation in vivo. This study provides evidence that CD40-transduced signals initiate TR cell increase in vivo and that CD154-transduced signals sensitize autoaggressive CD8+ T cells to suppression. [ABSTRACT FROM AUTHOR]

Discusses the regulation of anti-islet Cd8+ T cells by the pancreatic lymph node-derived CD4+ CD25+ T regulatory cells through transforming growth factor-beta-transforming growth factor-beta receptor interactions in type 1 diabetes. Cell and tissue preparation; Expression of transforming growth factor-beta1 by treg cells; Inducement of diabetes and pancreatic fibrosis by CD8+ cells.

Inflammation can activate self-reactive CD8+ T cells and induce autoimmunity. Here we show in a CD8+ T cell-mediated model of type 1 diabetes that CD4+CD25+ Treg cells prevent β cell destruction following localized inflammation in the islets of Langerhans. These Treg cells accumulate preferentially in the pancreatic lymph nodes and islets but not other lymph nodes or spleen. PLN-derived Treg cells are extremely potent; only 2 × 103 cells are needed to prevent diabetes development, and their capacity to regulate is dependent on TNF-related activation induced cytokine-receptor activator of NFκB signals. Indeed, blockade of this pathway results in decreased frequency of CD4+CD25+ Treg cells in the PLN, resulting in intra-islet differentiation of CD8+ T cells into CTLs and rapid progression to diabetes. [Copyright &y& Elsevier]

Effective therapies that prevent chronic inflammation from developing into type 1 diabetes remain elusive. In this study, we show that expression of TGF-β for just 1 wk in inflamed islets of NOD mice significantly delays diabetes development. Time course studies demonstrated that the brief TGF-β pulse protects only if administered when extensive β cell destruction has occurred. Surprisingly, TGF-β-mediated protection is not linked to enhanced Foxp3+ regulatory T cell activity or to decreased intrapancreatic presentation of islet Ags. Instead, TGF-β disables the transition of primed autoreactive CD8+ T cells to cytotoxic effectors and decreases generation, or maintenance, of CD8+ memory T cells within the pancreas, significantly impairing their diabetogenic capacity. [ABSTRACT FROM AUTHOR]

Identification of candidate genes and their immunological mechanisms that control autoaggressive T cells in inflamed environments, may lead to novel therapies for autoimmune diseases, like type 1 diabetes (T1D). In this study, we used transgenic NOD mice that constitutively express TNF-alpha in their islets from neonatal life (TNF-alpha-NOD) to identify protective alleles that control T1D in the presence of a proinflammatory environment. We show that TNF-alpha-mediated breakdown in T cell tolerance requires recessive NOD alleles. To identify some of these recessive alleles, we crossed TNF-alpha-NOD mice to diabetes-resistant congenic NOD mice having protective alleles at insulin-dependent diabetes (Idd) loci that control spontaneous T1D at either the preinsulitis (Idd3.Idd5) or postinsulitis (Idd9) phases. No protection from TNF-alpha-accelerated T1D was afforded by resistance alleles at Idd3.Idd5. Lack of protection was not at the level of T cell priming, the efficacy of islet-infiltrating APCs to present islet peptides, nor the ability of high levels of CD4+ Foxp3+ T cells to accumulate in the islets. In contrast, protective alleles at Idd9 significantly increased the age at which TNF-alpha-NOD mice developed T1D. Disease delay was associated with a decreased ability of CD8+ T cells to respond to islet Ags presented by islet-infiltrating APCs. Finally, we demonstrate that the protective region on chromosome 4 that controls T1D in TNF-alpha-Idd9 mice is restricted to the Idd9.1 region. These data provide new evidence of the mechanisms by which selective genetic loci control autoimmune diseases in the presence of a strong inflammatory assault.

Inflammation can activate self-reactive CD8(+) T cells and induce autoimmunity. Here we show in a CD8(+) T cell-mediated model of type 1 diabetes that CD4(+)CD25(+) Treg cells prevent beta cell destruction following localized inflammation in the islets of Langerhans. These Treg cells accumulate preferentially in the pancreatic lymph nodes and islets but not other lymph nodes or spleen. PLN-derived Treg cells are extremely potent; only 2 x 10(3) cells are needed to prevent diabetes development, and their capacity to regulate is dependent on TNF-related activation induced cytokine-receptor activator of NFkappaB signals. Indeed, blockade of this pathway results in decreased frequency of CD4(+)CD25(+) Treg cells in the PLN, resulting in intra-islet differentiation of CD8(+) T cells into CTLs and rapid progression to diabetes.