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Konecny, A. J.

Publications and source records attributed to Konecny, A. J..

4 recordsLinked to original sources

Tissue-specific adaptation of human T cells is preserved during tissue inflammation

T cells play an essential role in protecting tissues against pathogens and regulating tissue homeostasis. Previous studies highlight that T cells display tissue-specific phenotypic and functional properties, suggesting that T cells adapt to their local environment. Whether inflammation disrupts tissue-specific T cell adaptation remains poorly understood. To address this open question, we examined the T cell compartment - including conventional CD4 and CD8 T cells, regulatory T cells, {gamma}{delta} T cells, and MAIT cells - from healthy and inflamed human mucosal tissues. Using high-parameter spectral flow cytometry, we examined phenotype ex vivo and the functional capacity following stimulation, utilizing conventional gating and unsupervised clustering analysis approaches. Overall, we analyzed 65 tissue samples including mild, moderate, and severely inflamed oral gingiva, healthy and inflamed lung, along with healthy and inflamed tissue from the decidual-placental interface. Across these mucosal barrier tissues, we find that tissue location plays a dominant role in shaping the composition, phenotype, and functional capacity of the T cell compartment. Importantly, these tissue-specific adaptations were largely maintained during states of tissue inflammation. This included the ability to exert tissue repair functions, which was preserved across T cell subsets, even in severely inflamed tissues.

immunology↗

CaptureBody - an anti-CD45 x anti-IgG bispecific antibody enables accurate unmixing for spectral flow cytometry

Accurate spectral unmixing is a critical step for flow cytometry data analysis and requires a single stain control for every fluorescent parameter used in an experiment. Currently, compensation particles are often used for making single stain controls when a target protein is of low abundance or a cell type is of low frequency. However, compensation particles introduce incongruencies in emission spectra compared to cells resulting in spectral unmixing or compensation errors. To enable the use of cells regardless of the abundance of target proteins or immune cell type, we generated a bispecific antibody that links a human anti-CD45 and mouse anti-IgG variable region. We refer to this new bispecific tool as CaptureBody (CB) and highlight the benefits of its final nanobody-based design. We provide all sequences and methods necessary for the in-house expression of a CaptureBody to disseminate their use for spectral flow cytometry experiments.

immunology↗

Measurement and prediction of unmixing-dependent spreading in spectral flow cytometry panels

Advances in spectral cytometry instrumentation and fluorescent reagents have led to the possibility of ultra-high-parameter panels exceeding 50 colors. However, panel size is limited in practice by unmixing-dependent spreading (UDS), a mathematical phenomenon which leads to a progressive deterioration of unmixed signal-to-noise ratios in panels that contain fluorochrome combinations with significant spectral overlap. Choosing spectrally compatible sets of fluorochromes that avoid UDS is a complex and labor-intensive task involving substantial trial-and-error experimentation. Here, we provide a detailed explanation of UDS and practical strategies for handling UDS in large spectral panels. We describe the empirical hallmarks of UDS, demonstrate how to quantify its impact, and dissect its underlying mathematical cause in terms of spectral collinearity. We present novel computational metrics that can be used to select optimal combinations of fluorochromes in a platform-agnostic fashion based on publicly available reference data, providing a general tool for spectral panel design.

bioinformatics↗

50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in tissue biopsies and other human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.

immunology↗