Project description:PBMCs collected from patients with or without graft dysfunction post heart transplantation were analyzed using the BD Rhapsody CITE-seq and VDJ-seq. The study groups include patients with cardiac allograft dysfunction (CAV), non-specific graft dysfunction (NGD), and normal graft function (Normal HTx).
Project description:To search for new markers of active lesions that might help better understand the molecular basis of MPA and aid in its diagnosis, DNA microarray analysis was performed with peripheral blood mononuclear cells (PBMCs).
Project description:A cryovial that contains frozen PBMCs (10^6 cells/vial) from each of 11 COVID-19 patients was thawed in 37oC water bath. Immediately after, thawed cells were transferred to 15-mL Falcon tube that contained 10 mL of cold culture medium. After centrifugation at 150g for 5 min, the cell pellets were resuspended in complete RPMI + 10% heat-inactivated FCS medium and plated onto 12-well plate at a concentration of 5 x 10^6 cells/3 mL/well. Then a peptide cocktail was added at a concentration of 5 μg/mL for 16hr in 5% CO2 37oC incubator. After 16-hour incubation, cells were washed 3 times and incubated for 10 minutes with Fc receptor block, following each sample was stained with a total of 6 HLA-A2+/peptide fluorescent tetramer (Tet) and pentamer (Pent) for 10 min. After cell washing at twice, each sample was stained with cell hashing antibodies for 20 min, respectively and then washed at three times and finally pooled. The combined cells were stained with CITE-seq and anti-human CD8 FACS antibody for 30 minutes. After cell washing at twice, viable HLA-A2+/peptide Tet/Pent positive CD8+ T cells were sorted by FACS Aria II. The scRNA-Seq libraries including GEX and CITE-seq libraries were prepared using the 10x Chromium single-cell 3’ reagent kits (v3.1 Chemistry), per manufacturer’s instructions. As a result, we have 2 separate groups of the single cell datasets. MKH datasets (MKH_GEX_... and MKH_FB...) contain RNA-seq outcomes of HLA-A2 restricted human CD8+ T cells against SARS-CoV-2 from 5 COVID-19 patients. MKK datasets contain RNA-seq outcomes of HLA-A2 restricted human CD8+ T cells against SARS-CoV-2 from 6 COVID-19 patients. Cell hashing and CITE-seq antibody information is available in the csv files.
Project description:Skin and blood single cell RNA sequencing datasets of IL-22- and IFNγ-secreting CD1a-reactive GAS-responsive T cells were generated from four healthy skins, five healthy PBMCs and three psoriatic PBMCs. Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), we measured T cell specific functional states at both protein and transcriptome levels.
Project description:Single-cell RNA-sequencing (scRNA-Seq) is widely used to characterize immune cell populations. However, mRNA levels correlate poorly with expression of surface proteins, which are well established to define immune cell types. CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) utilizes oligonucleotide-tagged antibodies to simultaneously analyze surface phenotypes and transcriptomes. Considering the high costs of adding surface phenotyping to scRNA-Seq, we aimed to determine which of 188 tested CITE-Seq antibodies can detect their antigens on human peripheral blood mononuclear cells (PBMCs), a commonly interrogated cell population in immunology, and find the optimal concentration for staining. The recommended concentration was optimal for 76 antibodies, whereas staining quality of 7 antibodies improved when the concentration was doubled. 33 and 8 antibodies still worked well when the concentration was reduced to 1/5 or 1/25, respectively. 64 antigens were not detected at any antibody concentration. Optimizing the antibody panel by removing antibodies not able to detect their target antigens and adjusting concentrations of the remaining antibodies could enable a cost reduction of almost 50%. In conclusion, our data are a resource for building an informative and cost-effective panel of CITE-Seq antibodies and use them at their optimal concentrations in future CITE-seq experiments on human PBMCs.
Project description:Mycophenolic acid (MPA), an immunosuppressive drug widely used in kidney transplantation, has been suggested to have anti-fibrotic effects. To analyze at a genomic level these effects, we prospectively studied a group of stable kidney transplant recipients (n=35) on cyclosporine (CyA) and azathioprine treatment. Twenty patients were converted from azathioprine to MPA (MPA group) and 15 patients continued on azathioprine (AZA group). RNA was extracted by peripheral blood mononuclear cells at baseline and 3 months thereafter. Genomic analysis, performed on 5 randomly-selected MPA patients, revealed that 17 genes discriminated the transcriptomic profile after conversion. Neutral endopeptidase (NEP), an enzyme degrading angiotensin-II, was the most significant up-regulated gene. NEP expression level was inversely correlated to proteinuria at baseline and after conversion. Immunohistochemistry on graft biopsy of 33 independent patients demonstrated higher glomerular and tubular NEP protein expression in CyA+MPA (n=13) compared to CyA+AZA (n=12) and CyA alone (n=8). Glomerular NEP levels were inversely correlated to proteinuria and glomerulosclerosis. Tubular NEP expression was inversely correlated to interstitial fibrosis. Incubation of proximal tubular cells with MPA led to a dose- and time-dependent increase of NEP gene expression. The direct influence of MPA on NEP expression may suggest a novel therapeutic effect of this drug.
Project description:ANCA associated vasculitis(AAV) is a systemic vasculitis of small vessels, characterzied by injury of vascular endothelial cells induced by abnormally activated neutrophils. Microscopic polyangiitis (MPA) is a member of AAV with a high risk of kidney involvement. Blood lipid disorder promotes endothelial injury. We aim to investigate the correlation between blood lipid levels and renal prognosis in MPA patients. Firstly, we retrospectively included 110 patients diagnosed with MPA and the primary endpoint was the occurrence of ESRD. The association between blood lipids and renal outcome was evaluated with logistic regression analysis and survival analysis. During a median follow-up period of 23 months, 44 out of 110 patients (40%) developed ESRD. High serum triglycerides (TG) and VLDL at diagnosis were associated with ESRD development after adjusting for several confounding factors. MPA patients with TG >1.45 mmol/L or VLDL > 0.66mmol/L had significantly higher risk of ESRD development than those with TG ≤1.45 mmol/L or VLDL ≤0.66 mmol/L. Secondly, we explored the underlying mechanism of poor renal prognosis in patients with high TG levels using data independent acquisition (DIA) quantitative proteomics. DIA quantitative proteomics analysis suggested that patients in high TG group had up-regulated profibrotic pathway, inflammatory signaling pathways and complement and coagulation cascades compared with those in low TG group. In conclusion, high baseline TG or VLDL is associated with an increased risk of ESRD development in MPA patients. The potential mechanisms may be the upregulation of pro-fibrotic and inflammatory signaling pathways, and the activation of complement and coagulation cascades.