Dataset Information

In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization.

ABSTRACT: CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper CD4 T cells, particularly those residing in germinal centers known as follicular helper T cells (Tfh), provide critical help to B-cells to promote their survival, isotype switching and selection of high affinity memory B-cells. On the other hand, the important role of Tfh cells for the maintenance of HIV reservoir is well documented. Thus, interrogating and better understanding the tissue specific micro-environment and immune subsets that contribute to optimal Tfh cell differentiation and function is important for designing successful prevention and cure strategies. Here, we describe the development and optimization of eight multispectral confocal microscopy immunofluorescence panels designed for in depth characterization and immune-profiling of relevant immune cells in formalin-fixed paraffin-embedded human lymphoid tissue samples. We provide a comprehensive library of antibodies to use for the characterization of CD4+ T-cells -including Tfh and regulatory T-cells- as well as CD8 T-cells, B-cells, macrophages and dendritic cells and discuss how the resulting multispectral confocal datasets can be quantitatively dissected using the HistoCytometry pipeline to collect information about relative frequencies and immune cell spatial distributions. Cells harboring actively transcribed virus are analyzed using an in-situ hybridization assay for the characterization of HIV mRNA positive cells in combination with additional protein markers (multispectral RNAscope). The application of this methodology to lymphoid tissues offers a means to interrogate multiple relevant immune cell targets simultaneously at increased resolution in a reproducible manner to guide CD4 T-cell studies in infection and vaccination.

SUBMITTER: Moysi E

PROVIDER: S-EPMC8221112 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundThe implementation of successful strategies to achieve an HIV cure has become a priority in HIV research. However, the current location and size of HIV reservoirs is still unknown since there are limited tools to evaluate HIV latency in viral sanctuaries such as gut-associated lymphoid tissue (GALT). As reported in the so called "Boston Patients", despite undetectable levels of proviral HIV-1 DNA in blood and GALT, viral rebound happens in just few months after ART interruption. This fact might imply that current methods are not sensitive enough to detect residual reservoirs. Showing that, it is imperative to improve the detection and quantification of HIV-1 reservoir in tissue samples. Herein, we propose a novel non-enzymatic protocol for purification of Lamina Propria Leukocytes (LPL) from gut biopsies combined to viral HIV DNA (vDNA) quantification by droplet digital PCR (ddPCR) to improve the sensitivity and accuracy of viral reservoir measurements (LPL-vDNA assay).MethodsEndoscopic ileum biopsies were sampled from 12 HIV-1-infected cART-suppressed subjects. We performed a DTT/EDTA-based treatment for epithelial layer removal followed by non-enzymatic disruption of the tissue to obtain lamina propria cell suspension (LP). CD45+ cells were subsequently purified by flow sorting and vDNA was determined by ddPCR.ResultsvDNA quantification levels were significantly higher in purified LPLs (CD45+) than in bulk LPs (p<0.01). The levels of vDNA were higher in ileum samples than in concurrent PBMC from the same individuals (p = 0.002). As a result of the increased sensitivity of this purification method, the Poisson 95% confidence intervals of the vDNA quantification data from LPLs were narrower than that from bulk LPs. Of note, vDNA was unambiguously quantified above the detection limit in 100% of LPL samples, while only in 58% of bulk LPs.ConclusionWe propose an innovative combined protocol for a more sensitive detection of the HIV reservoir in gut-associated viral sanctuaries, which might be used to evaluate any proposed eradication strategy.

Project description:Optimal surgical therapy for brain tumors is the combination of complete resection with minimal invasion and damage to the adjacent normal tissue. To achieve this goal, we need advanced imaging techniques on a scale from macro- to microscopic resolution. In the last decade, the development of fluorescence-guided surgery has been the most influential breakthrough, marginally improving outcomes in brain tumor surgery. Multispectral fluorescence microscopy (MFL) is a novel imaging technique that allows the overlapping of a fluorescent image and a white light image in real-time, with delivery of the merged image to the surgeon through the eyepieces of a surgical microscope. MFL permits the detection and characterization of brain tumors using fluorescent molecular markers such as 5-aminolevulinic acid (5-ALA) or indocyanine green (ICG), while simultaneously obtaining high definition white light images to create a pseudo-colored composite image in real-time. Limitations associated with the use of MFL include decreased light imaging intensity and decreased levels of magnification that may compromise maximal tumor resection on a cellular scale. Confocal laser endomicroscopy (CLE) is another novel advanced imaging technique that is based on miniaturization of the microscope imaging head in order to provide the possibility of in vivo microscopy at the cellular level. Clear visualization of the cellular cytoarchitecture can be achieved with 400-fold-1,000-fold magnification. CLE allows on the one hand the intra-operative detection and differentiation of single tumor cells (without the need for intra-operative histologic analysis of biopsy specimens) as well as the definition of borders between tumor and normal tissue at a cellular level, dramatically improving the accuracy of surgical resection. The application and implementation of CLE-assisted surgery in surgical oncology increases not only the number of options for real-time diagnostic imaging, but also the therapeutic options by extending the resection borders of cancer at a cellular level and, more importantly, by protecting the functionality of normal tissue in the adjacent areas of the human brain. In this article, we describe our experience using these new techniques of confocal-assisted fluorescent surgery including analysis on the technology, usability, indications, limitations, and further developments.

Dataset Information

In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets