T lymphocytes bearing the αβ T cell receptor (TCR) but lacking CD4, CD8, and markers of natural killer (NK) cell differentiation are known as 'double-negative' (DN) T cells and have been described in both humans and rodent models. We and others have shown that DN T cells can act as regulatory T cells (Tregs) that are able to prevent allograft rejection, graft-versus-host disease, and autoimmune diabetes. In the last few years, new data have revealed evidence of DN Treg function in vivo in rodents and humans. Moreover, significant advances have been made in the mechanisms by which DN Tregs target antigen-specific T cells. One major limitation of the field is the lack of a specific marker that can be used to distinguish truly regulatory DN T cells (DN Tregs) from non-regulatory ones, and this is the central challenge in the coming years. Here, we review recent progress on the role of DN Tregs in transplantation and autoimmunity, and their mechanisms of action. We also provide some perspectives on how DN Tregs compare with Foxp3(+) Tregs.

Lymphangioleiomyomatosis (LAM) is a rare progressive cystic lung disease affecting young women. The pivotal observation that LAM occurs both spontaneously and as part of the tuberous sclerosis complex (TSC) led to the hypothesis that these disorders share common genetic and pathogenetic mechanisms. In this review we describe the evolution of our understanding of the molecular and cellular basis of LAM and TSC, beginning with the discovery of the TSC1 and TSC2 genes and the demonstration of their involvement in sporadic (non-TSC) LAM. This was followed by rapid delineation of the signaling pathways in Drosophila melanogaster with confirmation in mice and humans. This knowledge served as the foundation for novel therapeutic approaches that are currently being used in human clinical trials.Keywords: tuberous sclerosis; TSC1; TSC2; mTOR; signal transduction; estrogen Lymphangioleiomyomatosis (LAM) is a rare progressive cystic lung disease of uncertain etiology that affects young women. LAM cysts are formed as a result of the proliferation of an abnormal smooth muscle-like cell, the LAM cell. The mechanism by which LAM cells cause this architectural distortion of the lung is unknown. The clinical course of LAM is frequently inexorable, leading to death or lung transplantation in 10 to 15 years, although recent studies indicate that there is much variability in the natural history of the disorder (1, 2). The most common clinical manifestation is the insidious onset of exertional dyspnea; patients may also experience a nonproductive cough. Other common features include spontaneous pneumothorax, which results from cyst rupture, and chylothorax, which results from obstruction of pulmonary lymphatics and hilar lymph nodes by the slowly proliferating LAM cells. Less frequently, hemoptysis or chyloptysis may occur (1, 2).Most of the current therapies for LAM are supportive in nature. Bronchodilators are offered because many patients have obstructive physiology, often with some degree of reversibility, on pulmonary function testing. Oxygen is provided to patients with significant hypoxia. Current recommendations stipulate

Owing to the adverse effects of immunosuppression and an inability to prevent chronic rejection, there is a pressing need for alternative strategies to control alloimmunity. In three decades, regulatory T cells (Tregs) have evolved from a hypothetical mediator of adoptively transferred tolerance to a well-defined population that can be expanded ex vivo and returned safely to patients in clinical trials. Herein, we review the historical developments that have permitted these advances and the current status of clinical trials examining Tregs as a cellular therapy in transplantation. We conclude by discussing the critical unanswered questions that face this field in the coming years.

Ex vivo normothermic lung perfusion (EVLP) is a novel platform and method developed to facilitate functional assessment and implementation of advanced therapies for donor lungs prior to transplantation. This study aimed to determine the safety and immunological and functional benefits of ex vivo adenoviral human interleukin-10 (AdhIL-10) gene delivery to prevent the development of primary graft dysfunction in a large animal survival model. Pig donor lungs were retrieved, preserved for 6 h at 4°C, and then randomly assigned to four groups: (1) AdhIL-10 gene therapy: 12 h EVLP + AdhIL-10 intra-bronchial delivery; (2) EVLP-control: 12 h EVLP; (3) Vector-control: 12 h EVLP + adenoviral vector intra-bronchial delivery; and (4) prolonged hypothermic preservation: additional 12 h of cold ischemia. The left lung was then transplanted and evaluated. The recipients were recovered and kept alive until day 7 post-transplant under standard triple immunosuppression. Plasma levels of hIL-10 were detected in the treatment group throughout the 7 days. Analysis of peripheral blood obtained after transplant showed no signs of hematological, renal, or hepatic toxicity in the AdhIL-10 group. The immediate post-transplant lung function was significantly better in the EVLP-control and AdhIL-10 groups. Gas exchange at day 7 was superior in allografts from the AdhIL-10 group, and the histologic inflammation score was significantly lower. Lymphocytes from AdhIL-10 group harvested from mediastinal lymph nodes at day 7 post-transplantation and co-cultured with donor lymphocytes showed significantly less interferon gamma production in an Enzyme-Linked ImmunoSpot assay when compared with non-treatment groups. It has been demonstrated in this preclinical large animal survival study that ex vivo treatment with AdhIL-10 is safe and improves post-transplant lung function over EVLP alone. Improved function and an immunological advantage in both the innate and adaptive immune responses have been demonstrated.

BACKGROUND: Improved understanding of lung transplant disease states is essential because failure rates are high, often due to chronic lung allograft dysfunction. However, histologic assessment of lung transplant transbronchial biopsies (TBBs) is difficult and often uninterpretable even with 10 pieces. METHODS: We prospectively studied whether microarray assessment of single TBB pieces could identify disease states and reduce the amount of tissue required for diagnosis. By following strategies successful for heart transplants, we used expression of rejection-associated transcripts (annotated in kidney transplant biopsies) in unsupervised machine learning to identify disease states. RESULTS: All 242 single-piece TBBs produced reliable transcript measurements. Paired TBB pieces available from 12 patients showed significant similarity but also showed some sampling variance. Alveolar content, as estimated by surfactant transcript expression, was a source of sampling variance. To offset sampling variation, for analysis, we selected 152 single-piece TBBs with high surfactant transcripts. Unsupervised archetypal analysis identified 4 idealized phenotypes (archetypes) and scored biopsies for their similarity to each: normal; T-cell-mediated rejection (TCMR; T-cell transcripts); antibody-mediated rejection (ABMR)-like (endothelial transcripts); and injury (macrophage transcripts). Molecular TCMR correlated with histologic TCMR. The relationship of molecular scores to histologic ABMR could not be assessed because of the paucity of ABMR in this population. CONCLUSIONS: Molecular assessment of single-piece TBBs can be used to classify lung transplant biopsies and correlated with rejection histology. Two or 3 pieces for each TBB will probably be needed to offset sampling variance.

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The present article is the second in a series on rare lung diseases. It focuses on pulmonary alveolar proteinosis (PAP), a disorder in which lipoproteinaceous material accumulates in the alveolar space. PAP was first described in 1958, and for many years the nature of the material accumulating in the lungs was unknown. Major insights into PAP have been made in the past decade, and these have led to the notion that PAP is an autoimmume disorder in which autoantibodies interfere with signalling through the granulocyte-macrophage colony-stimulating factor receptor, leading to macrophage and neutrophil dysfunction. This has spurred new therapeutic approaches to this disorder. The discussion of PAP will begin with a case report, then will highlight the classification of PAP and review recent insights into the pathogenesis of PAP. The approach to therapy and the prognosis of PAP will also be discussed.