James Dooley

Developmental regulation of Foxp3 expression during ontogeny

Thymectomy of neonatal mice can result in the development of autoimmune pathology. It has been proposed that thymic output of regulatory T (T reg) cells is delayed during ontogeny and that the development of autoimmune disease in neonatally thymectomized mice is caused by the escape of self-reactive T cells before thymectomy without accompanying T reg cells. However, the kinetics of T reg cell production within the thymus during ontogeny has not been assessed. We demonstrate that the development of Foxp3-expressing T reg cells is substantially delayed relative to nonregulatory thymocytes during ontogeny. Based on our data, we speculate that induction of Foxp3 in developing thymocytes and, thus, commitment to the T reg cell lineage is facilitated by a signal largely associated with the thymic medulla.

Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3 + regulatory T cells

Foxp3+ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4+ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2–dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

Organization of thymic medullary epithelial heterogeneity: implications for mechanisms of epithelial differentiation

Summary: There are accumulating data to show that thymic epithelium expresses a remarkable array of molecules previously considered to be tissue‐specific antigens, such as parathyroid hormone, thyroglobulin, insulin, and C‐reactive protein. From an immunological perspective, this property of thymic epithelium would provide an ideal mechanism to effect central tolerance of epithelial‐restricted antigens. However, from a mechanistic perspective, this phenomenon remains mysterious. Two explanations have been proposed. One invokes promiscuous gene expression by medullary thymic epithelial cells that would allow transient derepression of selected gene expression. The other proposes that the expression of tissue‐restricted genes by thymic epithelium reflects alternate pathways of epithelial development by small numbers of cells to form a mosaic of different epithelial types within the thymus. Here we show thymic expression of lung‐associated gene products by an organized epithelial ‘organoid’ with ultrastructural features of respiratory epithelium and present data suggesting that the thymus also contains structures that ultrastructurally and phenotypically resemble solitary thyroid follicles. Based on these data, it is proposed that some thymic epithelial progenitor cells resemble pharyngeal endoderm in terms of their developmental potential and that alternative differentiation fates taken by these cells serve to maintain the spectrum of epithelial ‘self’ in the thymus.

Genetic predisposition for beta cell fragility underlies type 1 and type 2 diabetes

Type 1 (T1D) and type 2 (T2D) diabetes share pathophysiological characteristics, yet mechanistic links have remained elusive. T1D results from autoimmune destruction of pancreatic beta cells, whereas beta cell failure in T2D is delayed and progressive. Here we find a new genetic component of diabetes susceptibility in T1D non-obese diabetic (NOD) mice, identifying immune-independent beta cell fragility. Genetic variation in Xrcc4 and Glis3 alters the response of NOD beta cells to unfolded protein stress, enhancing the apoptotic and senescent fates. The same transcriptional relationships were observed in human islets, demonstrating the role of beta cell fragility in genetic predisposition to diabetes.

FGFR2IIIb signaling regulates thymic epithelial differentiation.

Heterogeneous epithelial populations comprising the thymic environment influence early and late stages of T‐cell development. The processes that regulate the differentiation of thymic epithelium and that are responsible for this heterogeneity are not well understood, although mesenchymal/epithelial interactions are clearly involved. Here, we show that targeted expression by thymocytes of an fibroblast growth factor receptor‐2IIIb (FGFR2IIIb) ligand, FGF10, profoundly alters the differentiation and function of thymic epithelium (TE). Reconstitution of irradiated lckFGF10 mice with normal bone marrow restores normal thymic organization and function, while wild‐type mice reconstituted with lckFGF10 bone marrow recapitulates some of the thymic alterations seen in lckFGF10 mice. We also demonstrate that interference with FGFR2IIIb signaling in the thymus with a soluble FGFR2IIIb dominant‐negative fusion protein leads to precocious reductions in thymic size and cellularity that resemble age‐related thymic involution. These findings indicate that TE compartments are dynamically maintained and that FGF signals are involved in this process. Developmental Dynamics 236:3459–3471, 2007.

Nude thymic rudiment lacking functional foxn1 resembles respiratory epithelium.

The epithelial compartment of the thymus arises from endoderm of the 3rd pharyngeal pouch. As it moves from a cervical to a mediastinal position during development, this epithelium becomes populated by lymphoid progenitor cells from the blood and begins to support their differentiation along the T cell lineage. Productive differentiation of thymic epithelium is strictly dependent on the foxn1 transcription factor, as evidenced by the lack of functional thymic tissue in nude mice that carry a spontaneous loss‐of‐function mutation of foxn1. Evaluation of the thymic rudiment epithelium from nude mice revealed phenotypic properties and tissue organization that was strongly reminiscent of respiratory epithelium. These data suggest that foxn1 may be involved in directing lineage choices of multi‐potential progenitor epithelial cells rather than simply affecting the terminal differentiation program of epithelial cells specified to a thymic fate. Developmental Dynamics 233:1605–1612, 2005.

Promiscuous Foxp3-cre activity reveals a differential requirement for CD28 in Foxp3 + and Foxp3 − T cells

Costimulatory signals by CD28 are critical for thymic regulatory T‐cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)‐CreYFP mice to mice bearing a conditional Cd28 allele. Treg‐specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28‐deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3‐CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3‐CreYFP allele in non‐Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26‐RFP). This hypomorphism and ‘leaky’ expression of the Foxp3‐CreYFP allele should be considered when analysing the conditionally mutated Treg.

Immunologic profiles of multiple sclerosis treatments reveal shared early B cell alterations

Objective: We undertook a systems immunology approach of the adaptive immune system in multiple sclerosis (MS), overcoming tradeoffs between scale and level of detail, in order to identify the immunologic signature of MS and the changes wrought by current immunomodulatory treatments. Methods: We developed a comprehensive flow cytometry platform measuring 38 immunologic cell types in the peripheral blood of 245 individuals in a routine clinical setting. These include patients with MS, untreated or receiving any of 4 current immunomodulatory treatments (interferon-β, glatiramer acetate, natalizumab, or fingolimod), patients with autoimmune thyroid disease, and healthy controls. Results: An increase in memory CD8+ T cells and B cells was observed in untreated patients with MS. Interferon-β and fingolimod induce significant changes upon multiple aspects of the peripheral immune system, with an unexpectedly prominent alteration of B cells. Overall, both treatments push the immune system in different directions, with only 2 significant effects shared across these treatments—an increase in transitional B cells and a decrease in class-switched B cells. We further identified heightened B cell-activating factor (BAFF) levels as regulating this shared B cell pathway. Conclusions: A systems immunology approach established different immunologic profiles induced by current immunomodulatory MS treatments, offering perspectives for personalized medicine. Pathways shared between the immunologic architecture of existing efficacious treatments identify targets for future treatment design.

Immunological Ignorance Allows Long-Term Gene Expression After Perinatal Recombinant Adeno-Associated Virus-Mediated Gene Transfer to Murine Airways

Gene therapy of the lung has the potential to treat life-threatening diseases such as cystic fibrosis and α(1)-antitrypsin or surfactant deficiencies. A major hurdle for successful gene therapy is the development of an immune response against the transgene and/or viral vector. We hypothesized that by targeting the airways in the perinatal period, induction of an immune response against the vector particle could be prevented because of immaturity of the immune system, in turn allowing repeated gene transfer later in adult life to ensure long-term gene expression. Therefore, we readministered recombinant adeno-associated viral vector serotype 5 (rAAV2/5) to mouse airways 3 and 6 months after initial perinatal gene transfer. Our findings demonstrate that perinatal rAAV2/5-mediated gene transfer to the airways avoids a strong immune response. This immunological ignorance allows the readministration of an autologous vector later in adult life, resulting in efficient and stable gene transfer up to 7 months, without evidence of a decrease in transgene expression. Together, these data provide a basis to further explore perinatal gene therapy for pulmonary conditions with adequate gene expression up to 7 months.