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Abstract While technologies for multiplexed imaging have provided an unprecedented understanding of tissue composition in health and disease, interpreting this data remains a significant computational challenge. To understand the spatial organization of tissue and how it relates to disease processes, imaging studies typically focus on cell-level phenotypes. However, images can capture biologically important objects that are outside of cells, such as the extracellular matrix. Here, we describe a pipeline, Pixie, that achieves robust and quantitative annotation of pixel-level features using unsupervised clustering and show its application across a variety of biological contexts and multiplexed imaging platforms. Furthermore, current cell phenotyping strategies that rely on unsupervised clustering can be labor intensive and require large amounts of manual cluster adjustments. We demonstrate how pixel clusters that lie within cells can be used to improve cell annotations. We comprehensively evaluate pre-processing steps and parameter choices to optimize clustering performance and quantify the reproducibility of our method. Importantly, Pixie is open source and easily customizable through a user-friendly interface.

Abstract Neurodegenerative disorders are characterized by phenotypic changes and hallmark proteopathies. Quantifying these in archival human brain tissues remains indispensable for validating animal models and understanding disease mechanisms. We present a framework for nanometer-scale, spatial proteomics with multiplex ion beam imaging (MIBI) for capturing neuropathological features. MIBI facilitated simultaneous, quantitative imaging of 36 proteins on archival human hippocampus from individuals spanning cognitively normal to dementia. Customized analysis strategies identified cell types and proteopathies in the hippocampus across stages of Alzheimer’s disease (AD) neuropathologic change. We show microglia-pathologic tau interactions in hippocampal CA1 subfield in AD dementia. Data driven, sample independent creation of spatial proteomic regions identified persistent neurons in pathologic tau neighborhoods expressing mitochondrial protein MFN2, regardless of cognitive status, suggesting a survival advantage. Our study revealed unique insights from multiplexed imaging and data-driven approaches for neuropathologic analysis and serves broadly as a methodology for spatial proteomic analysis of archival human neuropathology. Teaser Multiplex Ion beam Imaging enables deep spatial phenotyping of human neuropathology-associated cellular and disease features.

Mass Based Imaging (MBI) technologies such as Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and Imaging Mass Cytometry (IMC) allow for the simultaneous measurement of the expression levels of 40 or more proteins in biological tissue, providing insight into cellular phenotypes and organization in situ. Imaging artifacts, resulting from the sample, assay or instrumentation complicate downstream analyses and require correction by domain experts. Here, we present MBI Analysis User Interface (MAUI), a series of graphical user interfaces that facilitate this data pre-processing, including the removal of channel crosstalk, noise and antibody aggregates. Our software streamlines these steps and accelerates processing by enabling real-time and interactive parameter tuning across multiple images.

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) plays a pivotal role in immune homeostasis and has been associated with human autoimmune and chronic inflammatory diseases. Though PTPN2 is well-characterized in lymphocytes, little is known about its function in innate immune cells. Our findings demonstrate that dendritic cell (DC)-intrinsic PTPN2 might be the key to explain the central role for PTPN2 in the immune system to maintain immune tolerance. Partial genetic PTPN2 ablation in DCs resulted in spontaneous inflammation, particularly in skin, liver, lung and kidney 22 weeks post-birth. DC-specific PTPN2 controls steady-state immune cell composition and even incomplete PTPN2 deficiency in DCs resulted in enhanced organ infiltration of conventional type 2 DCs, accompanied by expansion of IFNγ-producing effector T-cells. Consequently, the phenotypic effects of DC-specific PTPN2 deficiency were abolished in T-cell deficient Rag knock-out mice. Our data add substantial knowledge about the molecular mechanisms to prevent inflammation and maintain tissue tolerance.

BackgroundIt is unclear whether antibodies can prevent Mycobacterium tuberculosis (Mtb) infection. In this study, we examined the relationship between total plasma IgG levels, IgG elicited by childhood vaccines and soil-transmitted helminths, and Mtb infection prevalence, defined by positive QuantiFERON (QFT) test.MethodsWe studied 100 Mtb uninfected infants, aged 4–6 months. Ten infants (10%) converted to positive QFT test (QFT+) within 2 years of follow-up for Mtb infection. Antibody responses in plasma samples acquired at baseline and tuberculosis investigation were analyzed by enzyme-linked immunosorbent assay and ImmunoCAP® assay.ResultsQFT− infants displayed a significant increase in total IgG titers when re-tested, compared to IgG titers at baseline, which was not observed in QFT+ infants. Bacille Calmette-Guérin (BCG) vaccine-specific IgG2 and live-attenuated measles vaccine-specific IgG were raised in QFT− infants, and infants who acquired an Mtb infection did not appear to launch a BCG-specific IgG2 response. IgG titers against the endemic helminth Ascaris lumbricoides increased from baseline to QFT re-testing in all infants.ConclusionThese data show raised IgG associates with a QFT-status. Importantly, this effect was also associated with a trend showing raised IgG titers to BCG and measles vaccine. Our data suggest a possible protective association between raised antibody titers and acquisition of Mtb infection, potentially mediated by exposure to antigens both related and unrelated to Mtb.

BACKGROUND:The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined. METHODOLOGY/PRINCIPAL FINDINGS:Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection. CONCLUSION:These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization.