Multilevel human secondary lymphoid immune system compartmentalization revealed by complementary multiplexing and mass spectrometry imaging approaches
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Abstract
Secondary human lymphoid tissue immune reactions take place in a highly coordinated environment with compartmentalization representing a fundamental feature of this organization. In situ profiling methodologies are indispensable for the understanding of this compartmentalization. Here, we propose a complementary experimental approach aiming to reveal different aspects of this process. The analysis of human tonsils, using a combination of single cell phenotypic analysis based on flow cytometry and multiplex imaging and mass spectrometry-based methodologies, revealed a compartmentalized organization at cellular and molecular level. More specifically, the skewed distribution of highly specialized immune cell subsets and relevant soluble mediators was accompanied by a compartmentalized localization of several lipids across different anatomical areas of the tonsillar tissue. The performance of such combinatorial experimental approaches could lead to the identification of novel in situ interactions and molecular targets for the in vivo manipulation of lymphoid organ, particularly the germinal center, immune reactions.
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further confirming our hypothesis that these cells can be differentiated by their unique lipid signatures serving as molecular ‘barcodes’.
This is such an interesting revelation! It begs atleast three additional questions for future studies. (1) Do these cell types maintain the same lipid composition outside the tonsils, (2) does differential lipid compositioning between the same cell type indicate anything about the state of a cell, and (3) does lipid composition track with the upregulation of specific enzymes involved in the syntheses of these particular lipids (would rummaging through existing transcriptomics or proteomics datasets be helpful?)
This is an archived comment originally written by Peter Thuy-Boun
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Since MSI is performed in situ, the only currently available instruments that can separate isobaric ions prior to the mass analyzer are hybrid ion mobility spectrometer-mass spectrometers.
MALDI imaging on a timstof flex could yield some interesting additional insights in future investigations! Alternatively, a less preferable approach to resolving isobars could involve applying an ozonolysis approach to cleave lipids at sites of unsaturation. This could potentially resolve some PC and SM species differing by acyl-proximal unsaturation points. Setting this up on a MALDI-ready slide may not be trivial but performing this on bulk-extracted samples should be approachable. Bulk extracted samples might also be amenable to structural characterization by UVPD (https://pubs.acs.org/doi/10.1021/acs.analchem.6b03353).
This is an archived …
Since MSI is performed in situ, the only currently available instruments that can separate isobaric ions prior to the mass analyzer are hybrid ion mobility spectrometer-mass spectrometers.
MALDI imaging on a timstof flex could yield some interesting additional insights in future investigations! Alternatively, a less preferable approach to resolving isobars could involve applying an ozonolysis approach to cleave lipids at sites of unsaturation. This could potentially resolve some PC and SM species differing by acyl-proximal unsaturation points. Setting this up on a MALDI-ready slide may not be trivial but performing this on bulk-extracted samples should be approachable. Bulk extracted samples might also be amenable to structural characterization by UVPD (https://pubs.acs.org/doi/10.1021/acs.analchem.6b03353).
This is an archived comment originally written by Peter Thuy-Boun
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Tonsils were anonymized discarded pathologic specimens obtained from Children’s National Medical Center (CNMC) under the auspices of the Basic Science Core of the District of Columbia Developmental Center for AIDS Research.
Were these tonsils extracted from patients undergoing treatment for similar conditions? Would you expect to find more or less concordance in lipid composition between cell types if healthy patients' tonsils were under examination? The answer to these questions may extend beyond the scope of this investigation so please feel free to ignore; these are such cool results that these questions had to be asked.
This is an archived comment originally written by Peter Thuy-Boun
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