Activation of TNFR2 with a novel agonist expands T reg cells in vivo and protects allo-HCT recipients from acute GvHD while sparing antilymphoma and antiinfectious properties of transplanted donor T cells.

Understanding the spatiotemporal changes of cellular and molecular events within an organism is crucial to elucidate the complex immune processes involved in infections, autoimmune disorders, transplantation, and neoplastic transformation and metastasis. Here we introduce a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and T cell responses in Peyer's patches following stimulation of the immune system. In addition, we employed LSFM to map individual T cell subsets after hematopoietic cell transplantation and detected rare cellular events. Thus, we present a versatile imaging technology that should be highly beneficial in biomedical research.

Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations. IMPORTANCE The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus. There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.

Introduction: Invasive pulmonary Aspergillus fumigatus infections cause high morbidity and mortality in neutropenic patients. Granulocyte transfusions have been tested as an alternative therapy for the management of high-risk neutropenic patients with invasive A. fumigatus infections. To increase the granulocyte yield for transfusion, donors are treated with corticosteroids. Yet, the efficacy of granulocyte transfusion and functional defense mechanisms of granulocytes collected from corticosteroid treated donors remain largely elusive. Therefore, we investigated the efficacy of granulocyte transfusion and functional defense mechanisms of corticosteroid treated granulocytes using mouse models. Methods: To determine the effects of corticosteroids on granulocytes to control A. fumigatus infections, we performed granulocyte adoptive cell transfers using in vivo mouse models, in vitro human and mouse granulocyte and A. fumigatus functional co-culture experiments in combination with flow cytometry, cytokine analysis, fluorescence and electron microscopy. Results: Transfusion of granulocytes from corticosteroid treated mice did not protect cyclophosphamide immunosuppressed mice against lethal A. fumigatus infection in contrast to granulocytes from untreated mice. Upon infection increased levels of inflammatory cytokines helped to recruit granulocytes to the lungs without any recruitment defects in corticosteroid treated and infected mice or in cyclophosphamide immunosuppressed and infected mice that had received the granulocytes from corticosteroid treated mice. However, corticosteroid treated human or mouse neutrophils failed to form neutrophil extracellular traps (NETs) under in vitro and in vivo conditions. Furthermore, corticosteroid treated granulocytes exhibited impaired ROS production against A. fumigatus. Notably, corticosteroids impaired the β-glucan receptor Dectin-1 (CLEC7A) on mouse and human granulocytes to efficiently recognize and phagocytize A. fumigatus, which markedly impaired fungal killing. Conclusions: We conclude that corticosteroid treatment of granulocyte donors for increasing neutrophil yields or patients with ongoing corticosteroid treatment could result in deleterious effects on granulocyte antifungal functions, thereby limiting the benefit of granulocyte transfusion therapies against invasive fungal infections. Disclosures Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau.

Adoptive transfer of donor-derived cytolytic T-lymphocytes (CTL) has evolved as a promising strategy to improve graft-versusleukemia (GvL) effects in allogeneic hematopoietic stem-cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)-reactive CD8