Project description:We performed scRNA-seq of non-hematopoietic cells from non-treated control (WT), BMT and aGVHD mice. Unsupervised clustering, sub-clustering based on gene expression and trajectory analysis converged on the conclusion that aGVHD likely disrupts MSC differentiation potential, especially osteogenesis.
Project description:Acute graft-versus-host disease (aGVHD) is a severe complication of allogeneic hematopoietic stem cell transplantation. Hematopoietic dysfunction accompanied by severe aGVHD, which may be caused by niche impairment, is a long-standing clinical problem. However, how the bone marrow (BM) niche is damaged in aGVHD hosts is poorly defined. To comprehensively address this question, we employed a haplo-MHC-matched transplantation aGVHD murine model and performed single-cell RNA sequencing of non-hematopoietic BM cells. Transcriptional analysis showed that BM mesenchymal stromal cells (BMSCs) were severely affected with a reduction in cell ratio, abnormal metabolism, compromised differentiation potential and defective hematopoietic supportive function, which were validated by functional assays.
Project description:Donor OTUD1 deficiency significanltly ameliorates the severity of aGVHD mice. We used single cell RNA sequencing (scRNA-seq) to analyze the immune cells in aGVHD mice transferred wild type or OTUD1-deficient cells.
Project description:BM cells were isolated from C57BL/6 (CD45.2) Tet2–/– mice and Tet2+/+ littermates, as well as from congenic C57BL/6.SJL (CD45.1) mice (all genotypes purchased from JAX). lethally irradiated (9.5 Gy) CD45.1 mice received 10% Tet2–/– CD45.2 BM cells and 90% WT CD45.1 BM cells (2x105 Tet2–/– CD45.2 BM cells and 18x105 WT CD45.1 BM cells; total of 2x106 BM cells/recipient mouse). This group was designated ‘10% Tet2–/– BMT’ (experimental). For the control group that was designated ‘10% Tet2+/+ BMT’, lethally irradiated (9.5 Gy) CD45.1 mice received 10% Tet2+/+ CD45.2 BM cells and 90% WT CD45.1 cells (2x105 Tet2+/+ CD45.2 BM cells & 18x105 WT CD45.1 BM cells, thus exclusively TET2-sufficient cells; total of 2x106 BM cells/recipient mouse). At 12 weeks post-BMT (when there is complete reconstitution of hematopoiesis from transplanted long-term HSCs), ‘10% Tet2–/– BMT’ and ‘10% Tet2+/+ BMT’ mice either received no further treatment (and examined for natural periodontal bone loss) or were subjected to LIP for 5 days.
Project description:The goal of this study are to identify critical biomarkers distinguishing alloHSCT recipients with aGvHD from alloHSCT recipients without aGvHD in two separate cohorts. Total RNA was isolated from the peripheral blood mononucleated cells and then quantified on a Bioanalyzer. Whole transcriptome sequencing libraries (3 pairs of aGvHD and controls, 6 libraries in total) were prepared following the manufacturer’s instructions for the Whole Transcriptome Sample Prep Kit (Illumina) by GENEWIZ Company. 1148 presented significant alterations in cases with aGvHD (t test, FDR corrected P < 0.05, |fold change|>1.5). Transcriptomic pathway analysis showed that the GPL metabolism pathway was one of the most significant pathways with Bonferroni correction P value (Q value) less than 0.001, indicating that it was a critical pathway related to the development of aGvHD.
Project description:Allogeneic peripheral blood stem cell transplantation (PBSCT) is indicated for the treatment of high-risk hematological malignancies, and in some cases is a cancer cure. A major complication associated with PBSCT (affecting approximately half of recipients) is acute graft-versus-host disease (aGVHD). In aGVHD, alloreactive donor T cells becoming activated by host dendritic cells (DC) in response to pro-inflammatory cytokines released during the conditioning regimen. The result is donor-derived CD4+ T helper-type 1 (Th1) cells maturing and subsequently imposing cytolytic effector responses on host tissues, resulting in multi-organ damage. Despite this well-defined pathophysiological mechanism, there are no definitive markers for predicting aGVHD development or progression to clinically advanced stages. In the current study, we enrolled 8 PBSCT recipients in an IRB-approved study and collected peripheral blood at the time to engraftment. Of these, four patients developed aGVHD within 5-10 days post transplant. The remaining four patients were aGVHD-free. Using total RNA collected from whole blood leukocytes, we analyzed each recipientâs gene expression profile on Affymetrix GeneChip Human Genome U133 Plus 2.0 microarrays. Experiment Overall Design: Samples 1-4 are control patients; 5-8 are aGVHD patients. Peripheral blood was collected at engraftment and used for microarray analysis. In analysing the data, the type of transplant (MUD, related) was used as a covariate to assess if there were significant changes in gene expression between the control (1-4) and aGVHD (5-8) groups, based on a 10% FDR with a pooled error estimate.