Chronic T cell receptor stimulation unmasks NK receptor signaling in peripheral T cell lymphomas via epigenetic reprogramming.

Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.

CD1d-restricted peripheral T cell lymphoma in mice and humans.

Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia, known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans.

Evolution of the Immune Response to Chronic Airway Colonization with Aspergillus fumigatus Hyphae.

Airway colonization by the mold Aspergillus fumigatus is common in patients with underlying lung disease and is associated with chronic airway inflammation. Studies probing the inflammatory response to colonization with A. fumigatus hyphae have been hampered by the lack of a model of chronic colonization in immunocompetent mice. By infecting mice intratracheally with conidia embedded in agar beads (Af beads), we have established an in vivo model to study the natural history of airway colonization with live A. fumigatus hyphae. Histopathological examination and galactomannan assay of lung homogenates demonstrated that hyphae exited beads and persisted in the lungs of mice up to 28 days postinfection without invasive disease. Fungal lesions within the airways were surrounded by a robust neutrophilic inflammatory reaction and peribronchial infiltration of lymphocytes. Whole-lung cytokine analysis from Af bead-infected mice revealed an increase in proinflammatory cytokines and chemokines early in infection. Evidence of a Th2 type response was observed only early in the course of colonization, including increased levels of interleukin-4 (IL-4), elevated IgE levels in serum, and a mild increase in airway responsiveness. Pulmonary T cell subset analysis during infection mirrored these results with an initial transient increase in IL-4-producing CD4(+) T cells, followed by a rise in IL-17 and Foxp3(+) cells by day 14. These results provide the first report of the evolution of the immune response to A. fumigatus hyphal colonization.

Aspergillus galactosaminogalactan mediates adherence to host constituents and conceals hyphal ß-glucan from the immune system.

Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall ß-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.

Role of trehalose biosynthesis in Aspergillus fumigatus development, stress response, and virulence.

Aspergillus fumigatus is a pathogenic mold which causes invasive, often fatal, pulmonary disease in immunocompromised individuals. Recently, proteins involved in the biosynthesis of trehalose have been linked with virulence in other pathogenic fungi. We found that the trehalose content increased during the developmental life cycle of A. fumigatus, throughout which putative trehalose synthase genes tpsA and tpsB were significantly expressed. The trehalose content of A. fumigatus hyphae also increased after heat shock but not in response to other stressors. This increase in trehalose directly correlated with an increase in expression of tpsB but not tpsA. However, deletion of both tpsA and tpsB was required to block trehalose accumulation during development and heat shock. The DeltatpsAB double mutant had delayed germination at 37 degrees C, suggesting a developmental defect. At 50 degrees C, the majority of DeltatpsAB spores were found to be nonviable, and those that were viable had severely delayed germination, growth, and subsequent sporulation. DeltatpsAB spores were also susceptible to oxidative stress. Surprisingly, the DeltatpsAB double mutant was hypervirulent in a murine model of invasive aspergillosis, and this increased virulence was associated with alterations in the cell wall and resistance to macrophage phagocytosis. Thus, while trehalose biosynthesis is required for a number of biological processes that both promote and inhibit virulence, in A. fumigatus the predominant effect is a reduction in pathogenicity. This finding contrasts sharply with those for other fungi, in which trehalose biosynthesis acts to enhance virulence.

Aspergillus fumigatus MedA governs adherence, host cell interactions and virulence.

In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusDeltamedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The DeltamedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro-inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusDeltamedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.

Aspergillus fumigatus induces immunoglobulin E-independent mast cell degranulation.

BACKGROUND: Pulmonary colonization by Aspergillus fumigatus in chronic lung disease is associated with progressive decline in lung function even in the absence of specific allergic response. We hypothesized that A. fumigatus contributes to this decline by inducing pulmonary mast cell degranulation even in the absence of antigen-specific immunoglobulin E (IgE). Therefore, we investigated whether A. fumigatus can induce mast cell degranulation independently of IgE. METHODS: We studied the interactions of Aspergillus species with mast cells in the absence of IgE in vitro with use of scanning electron microscopy. The extent of mast cell degranulation was quantified by measuring the release of beta-hexosaminidase. RESULTS: Mature A. fumigatus hyphae induced mast cell degranulation in the absence of IgE. Hyphae of Aspergillus flavus, Aspergillus niger, and Aspergillus nidulans induced much less mast cell degranulation. Mast cell degranulation required direct contact with mature A. fumigatus hyphae, and was not induced by conidia or immature hyphae. Killed hyphae induced significant degranulation, whereas live hyphae from mutants deficient in the fungal development regulators StuA and MedA induced very little degranulation. CONCLUSIONS: Factors expressed on the surface of mature A. fumigatus hyphae that are controlled by StuA and MedA induce mast cell degranulation in the absence of IgE.