Human fetal dendritic cells promote prenatal T-cell immune suppression through arginase-2.

During gestation the developing human fetus is exposed to a diverse range of potentially immune-stimulatory molecules including semi-allogeneic antigens from maternal cells, substances from ingested amniotic fluid, food antigens, and microbes. Yet the capacity of the fetal immune system, including antigen-presenting cells, to detect and respond to such stimuli remains unclear. In particular, dendritic cells, which are crucial for effective immunity and tolerance, remain poorly characterized in the developing fetus. Here we show that subsets of antigen-presenting cells can be identified in fetal tissues and are related to adult populations of antigen-presenting cells. Similar to adult dendritic cells, fetal dendritic cells migrate to lymph nodes and respond to toll-like receptor ligation; however, they differ markedly in their response to allogeneic antigens, strongly promoting regulatory T-cell induction and inhibiting T-cell tumour-necrosis factor-α production through arginase-2 activity. Our results reveal a previously unappreciated role of dendritic cells within the developing fetus and indicate that they mediate homeostatic immune-suppressive responses during gestation.

Optimization of mass cytometry sample cryopreservation after staining.

The advent of mass cytometry has facilitated highly multi-parametric single-cell analysis allowing for the deep assessment of cellular diversity. While the data and analytical power of this approach are well described, associated technical and experimental hurdles remain. Issues like equipment breakdown and sampling of large-scale batches, which may require multiple days of data acquisition, are minor but critical obstacles that prompt a technical solution, especially when dealing with precious samples. An ability to cryopreserve mass cytometry samples that have already been stained would alleviate numerous technical limitations we face with currently used sample-handling approaches. Here, we evaluated two protocols for freezing of already-stained and fixed cellular samples and compared them with standard sample refrigeration in staining buffer. A comprehensive human T cell staining phenotypic and functional profiling panel was used and the signal intensity and reliability of each marker was assessed over a 4-week period. In general, cellular viability, DNA Ir-Intercalator and barcode staining were minimally affected by freezing compared to refrigeration, and the signal intensities for cell surface markers and receptors were not compromised. Intracellular cytokine staining did show some decreases in signal intensity after freezing, with the decreases more prominent in a methanol-based protocol compared to a protocol involving the use of 10% DMSO in FBS. We conclude that freezing already-stained samples suspended in 10% DMSO in FBS is practical and efficient way to preserve already-stained samples when needed. © 2016 International Society for Advancement of Cytometry.

CD103(+) Dendritic Cells Control Th17 Cell Function in the Lung.

Th17 cells express diverse functional programs while retaining their Th17 identity, in some cases exhibiting a stem-cell-like phenotype. Whereas the importance of Th17 cell regulation in autoimmune and infectious diseases is firmly established, the signaling pathways controlling their plasticity are undefined. Using a mouse model of invasive pulmonary aspergillosis, we found that lung CD103(+) dendritic cells (DCs) would produce IL-2, dependent on NFAT signaling, leading to an optimally protective Th17 response. The absence of IL-2 in DCs caused unrestrained production of IL-23 and fatal hyperinflammation, which was characterized by strong Th17 polarization and the emergence of a Th17 stem-cell-like population. Although several cell types may be affected by deficient IL-2 production in DCs, our findings identify the balance between IL-2 and IL-23 productions by lung DCs as an important regulator of the local inflammatory response to infection.

High-dimensional analysis of the murine myeloid cell system.

Advances in cell-fate mapping have revealed the complexity in phenotype, ontogeny and tissue distribution of the mammalian myeloid system. To capture this phenotypic diversity, we developed a 38-antibody panel for mass cytometry and used dimensionality reduction with machine learning-aided cluster analysis to build a composite of murine (mouse) myeloid cells in the steady state across lymphoid and nonlymphoid tissues. In addition to identifying all previously described myeloid populations, higher-order analysis allowed objective delineation of otherwise ambiguous subsets, including monocyte-macrophage intermediates and an array of granulocyte variants. Using mice that cannot sense granulocyte macrophage-colony stimulating factor GM-CSF (Csf2rb(-/-)), which have discrete alterations in myeloid development, we confirmed differences in barrier tissue dendritic cells, lung macrophages and eosinophils. The methodology further identified variations in the monocyte and innate lymphoid cell compartment that were unexpected, which confirmed that this approach is a powerful tool for unambiguous and unbiased characterization of the myeloid system.

Complement mediated signaling on pulmonary CD103(+) dendritic cells is critical for their migratory function in response to influenza infection.

Trafficking of lung dendritic cells (DCs) to the draining lymph node (dLN) is a crucial step for the initiation of T cell responses upon pathogen challenge. However, little is known about the factors that regulate lung DC migration to the dLN. In this study, using a model of influenza infection, we demonstrate that complement component C3 is critically required for efficient emigration of DCs from the lung to the dLN. C3 deficiency affect lung DC-mediated viral antigen transport to the dLN, resulting in severely compromised priming of virus-specific T cell responses. Consequently, C3-deficient mice lack effector T cell response in the lungs that affected viral clearance and survival. We further show that direct signaling by C3a and C5a through C3aR and C5aR respectively expressed on lung DCs is required for their efficient trafficking. However, among lung DCs, only CD103(+) DCs make a significant contribution to lung C5a levels and exclusively produce high levels of C3 and C5 during influenza infection. Collectively, our findings show that complement has a profound impact on immune regulation by controlling tissue DC trafficking and highlights a potential utility for complement as an adjuvant in novel vaccine strategies.

Mycobacterial antibody levels and immune restoration disease in HIV patients treated in South East Asia.

AIM: Immune restoration disease (IRD) associated with Mycobacterium tuberculosis parallels the reconstitution of a pathogen-specific Th1 response. However, it is not clear whether humoral responses to M. tuberculosis antigens also rise, or whether antibody levels predict IRD. Here, humoral immunity to M. tuberculosis antigens was investigated in four Asian cohorts. METHODS: Plasma samples were obtained from longitudinal prospective studies of HIV patients beginning antiretroviral therapy (ART) in New Delhi (India), Kuala Lumpur (Malaysia), Jakarta (Indonesia) and Phnom Penh (Cambodia). IgG antibodies to purified protein derivative, lipoarabinomannan and 38-kDa antigens of M. tuberculosis were quantitated using in-house ELISAs. IRD was defined as exacerbated symptoms of tuberculosis in patients on anti-tuberculosis therapy or a novel presentation of tuberculosis on ART. RESULTS: Pre-ART IgG levels to purified protein derivative, lipoarabinomannan and 38-kDa antigen were similar in the IRD and control groups from each site. Compared with non-IRD controls, a higher proportion of IRD patients had elevated IgG levels to lipoarabinomannan (defined as a greater than twofold increase) over 12 weeks of ART. However, this trend was not significant for the other antigens and longitudinal analyses did not reveal clear rises in antibody levels at the time of IRD. CONCLUSION: Levels of antibody to mycobacterial antigens do not predict IRD, but levels of antibody reactive with lipoarabinomannan rise during an IRD in some patients.