Biology subjects

Bolognesi, M. M.

Publications and source records attributed to Bolognesi, M. M..

6 recordsLinked to original sources

bioRxiv2025

The normal human lymph node cell classification and landscape defined by high-dimensional spatial proteomic.

Lymph nodes (LN) are key secondary lymphoid organs (SLO) for a coordinated immune response. They have been extensively characterized by numerous investigative techniques chiefly as single cell suspensions because they are composed of vagile yet crowded hematolymphoid elements, unfriendly to spatial tissue organization-saving techniques. We comprehensively classify in situ all cells of 19 human LN free of pathology with a 78-marker antibody panel, an hyperplexed cyclic staining method, MILAN, and an analytical bioinformatic pipeline, BRAQUE. A total of 77 cell types were classified, encompassing T, B, innate immune and stromal cells. CD4 and CD8 T-cells were classified into 27 unique subsets by leveraging the expression profiles of TCF7, the presence of co-inhibitory receptors and the spatial distribution. CD5 and TCF7 expression defined novel B-cell types. CD27+ mature B-cells occupied previously unrecognized nodal spaces non-overlapping with the cortex and the plasma-cell rich medullary cords. Type 2 conventional dendritic cells were located in nodular paracortical aggregates. Statistically controlled pairwise neighborhood analysis showed sparse cell-cell interactions, known and new neighbors, established and novel LN landscape niches. A high-dimensional proteomic interrogation of the normal human LN provides spatial allocation of known cell types, novel interactions and the landscape organization.

immunology↗

bioRxiv2024

Seeing or believing in hyperplexed spatial proteomics via antibodies. New and old biases for an image-based technology.

Hyperplexed in-situ targeted proteomics via antibody immunodetection (i.e. > 15 markers) is changing how we classify cells and tissues. Differently from other high-dimensional single-cell assays (flow cytometry, single cell RNA sequencing), the human eye is a necessary component in multiple procedural steps: image segmentation, signal thresholding, antibody validation and iconographic rendering. Established methods complement the human image evaluation, but may carry undisclosed biases in such a new context, therefore we re-evaluate all the steps in hyperplexed proteomics. We found that the human eye can discriminate less than 64 out of 256 gray levels and has limitations in discriminating luminance levels in conventional histology images. Furthermore, only images containing visible signals are selected and eye-guided digital thresholding separates signal from noise. BRAQUE, a hyperplexed proteomic tool, can extract, in a marker-agnostic fashion, granular information from markers which have a very low signal-to-noise ratio and therefore are not visualized by traditional visual rendering. By analyzing a public human lymph node dataset, we also found unpredicted staining results by validated antibodies, which highlight the need to upgrade the definition of antibody specificity in hyperplexed immunostaining. Spatially hyperplexed methods upgrade and supplant traditional image-based analysis of tissue immunostaining, beyond the human eye contribution. IMPACT STATEMENTStaining with multiple biomarkers a single tissue section, multiplex staining, is changing how we examine in-situ normalcy, pathology and the interrelationship of good and bad biological actors. Bioinformatic pipelines developed to deal with high-dimensional datasets such as single cell RNA sequencing or multiparameter flow-cytometry have been adapted to analogous types of data derived from tissue, and co-exist with conventional image analysis tools and human eye-guided image evaluation. We wanted to evaluate if multiplex staining with more than 15 markers (hyperplexed) has comparable sensitivity to conventional image analysis and if these latter analysis tools carry undisclosed biases and should not be used together with hyperplexed staining. We found that the human eye has a reduced discriminative power for grayscale and luminance levels compared to the 8-bit available spectrum, affecting positive signal recognition above the noise. We also found that the granular analytical power of recent bioinformatic pipelines can extract information from images which defy human eye perception and deliver information unattainable with existing image analysis tools for single-stain images.

systems biology↗

bioRxiv2020

NOT ALL ANTIBODIES HAVE BEEN CREATED EQUAL: Antibodies validated for routinely processed tissue seldom apply to frozen tissue sections.

ABSTRACTA customized context-dependent validation of antibodies for the prospective use, rather than a general stamp of validity, is required for reproducibility and data validity, besides the need of standardized reagents. In-situ antibody staining is a technique broadly used in experimental settings and human diagnostic practice. The first typically, but not exclusively uses lightly fixed material (cell smears, frozen sections), the second, routinely processed formalin-fixed, paraffin-embedded (FFPE) tissue. Differently from techniques based on tissue extraction, there is little awareness of the context-dependent constraints inherent with either type of in situ staining except that antigen masking associated with routine tissue processing limits the range of useful antibodies. We applied a panel of 126 antibodies validated for FFPE to lightly fixed (acetone, formalin) frozen sections and found that less than 30% performed conservatively with all fixations, 35% preferred one fixation over another, 13% gave non-specific staining, 23% did not stain at all. Individual antibody variegation of the paratope fitness for the differentially fixed antigen may be the cause. Re-validation of established antibody panels is required when applied to sections whose fixation and processing is different from the tissue where they have been initially validated.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pathology↗

bioRxiv2018

High-dimensional single cell-analysis of lymphocytic activation in melanoma

In melanoma, the lymphocytic infiltrate is a prognostic parameter classified morphologically into "brisk", "non-brisk" and "absent" entailing a functional association that has never been proved. Recently, it has been shown that lymphocytic populations can be very heterogeneous, and that anti-PD-1 immunotherapy supports activated T cells. Here, we characterize the immune landscape in primary melanoma by high-dimensional single cell multiplex analysis in tissue sections (MILAN technique) followed by image analysis, RT-PCR and shotgun proteomics. We observed that the brisk and non-brisk patterns are heterogeneous functional categories that can be further sub-classified into active, transitional or exhausted. The classification of primary melanomas based on the functional paradigm also shows correlation with spontaneous regression, and an improved prognostic value than that of the brisk classification. Finally, the main inflammatory cell subpopulations that are present in the microenvironment associated with activation and exhaustion and their spatial relationships are described using neighbourhood analysis.

cancer biology↗

bioRxiv2018

The landscape of S100B+ and HLA-DR+ dendritic cell subsets in tonsils at the single cell level via high-parameter mapping

Dendritic cells (DC) (classic, plasmacytoid, inflammatory) are an intense focus of interest because of their role in inflammation, autoimmunity, vaccination and cancer. We present a tissue-based classification of human DC subsets in tonsils with a high-parameter (>40 markers) immunofluorescent approach, cell type-specific image segmentation and the use of bioinformatics platforms. Through this deep phenotypic and spatial examination, classic cDC1, cDC2, pDC subsets have been further refined and a novel subset of DC co-expressing IRF4 and IRF8 identified. Based on unique tissue locations within the tonsil, and close interactions with T cells (cDC1) or B cells (cDC2), DC subsets can be further subdivided by correlative phenotypic changes associated with these interactions. In addition, monocytes and macrophages expressing HLA-DR or S100AB are identified and localized in the tissue. This study thus provides a whole tissue in situ catalog of human DC subsets and their cellular interactions within spatially defined niches.

immunology↗

bioRxiv2017

Multiplex staining by sequential immunostaining and antibody removal on routine tissue sections

Multiplexing (mplx), labeling for multiple immunostains the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labelled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities.\n\nWe have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than thirty different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Mplx on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory and normal cells.

pathology↗