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1.
Cell Rep Med ; 4(7): 101125, 2023 07 18.
Article in English | MEDLINE | ID: mdl-37467715

ABSTRACT

Acute graft-versus-host disease (aGvHD) is a significant complication after allogeneic hematopoietic stem cell transplantation (aHSCT), but major factors determining disease severity are not well defined yet. By combining multiplexed tissue imaging and single-cell RNA sequencing on gastrointestinal biopsies from aHSCT-treated individuals with fecal microbiome analysis, we link high microbiome diversity and the abundance of short-chain fatty acid-producing bacteria to the sustenance of suppressive regulatory T cells (Tregs). Furthermore, aGvHD severity strongly associates with the clonal expansion of mainly CD8 T cells, which we find distributed over anatomically distant regions of the gut, persistent over time, and inversely correlated with the presence of suppressive Tregs. Overall, our study highlights the pathophysiological importance of expanded CD8 T cell clones in the progression of aGvHD toward more severe clinical manifestations and strongly supports the further development of microbiome interventions as GvHD treatment via repopulation of the gut Treg niche to suppress inflammation.


Subject(s)
Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Microbiota , Humans , Graft vs Host Disease/pathology , Microbiota/genetics , Hematopoietic Stem Cell Transplantation/adverse effects , Gastrointestinal Tract/pathology , CD8-Positive T-Lymphocytes/pathology
2.
STAR Protoc ; 3(2): 101374, 2022 06 17.
Article in English | MEDLINE | ID: mdl-35586313

ABSTRACT

In this protocol, we describe the use of ChipCytometry to combine RNA in situ hybridization and antibody staining for multiplexed tissue imaging of human formalin-fixed and paraffin-embedded tissue samples. The advantages of ChipCytometry are long-term storage for re-interrogation and advanced image quality by high dynamic range imaging of staining and background. A titrated pretreatment of tissue samples bypasses challenges because of the retrieval of antigens on coverslips and achieves an optimal staining quality at the minimal expense of tissue integrity. For complete details on the use and execution of this protocol, please refer to Jarosch et al. (2021).


Subject(s)
Formaldehyde , RNA , Humans , In Situ Hybridization , RNA/genetics , RNA, Messenger/genetics
3.
Cell Rep Methods ; 1(7): 100104, 2021 11 22.
Article in English | MEDLINE | ID: mdl-35475000

ABSTRACT

Deciphering the spatial composition of cells in tissues is essential for detailed understanding of biological processes in health and disease. Recent technological advances enabled the assessment of the enormous complexity of tissue-derived parameters by highly multiplexed tissue imaging (HMTI), but elaborate machinery and data analyses are required. This severely limits broad applicability of HMTI. Here we demonstrate for the first time the application of ChipCytometry technology, which has unique features for widespread use, on formalin-fixed paraffin-embedded samples, the most commonly used storage technique of clinically relevant patient specimens worldwide. The excellent staining quality permits workflows for automated quantification of signal intensities, which we further optimized to compensate signal spillover from neighboring cells. In combination with the high number of validated markers, the reported platform can be used from unbiased analyses of tissue composition to detection of phenotypically complex rare cells, and can be easily implemented in both routine research and clinical pathology.


Subject(s)
Formaldehyde , Humans , Tissue Fixation/methods , Paraffin Embedding/methods
4.
J Biol Chem ; 291(47): 24735-24746, 2016 Nov 18.
Article in English | MEDLINE | ID: mdl-27729449

ABSTRACT

Single-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Co-immunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.


Subject(s)
Ceramides/metabolism , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Photobleaching , Ceramides/chemistry , Endoplasmic Reticulum/chemistry , HeLa Cells , Humans , Membrane Proteins/chemistry , Microscopy, Fluorescence
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