Gene profiling of CD11b⁺ and CD11b⁻ B1 cell subsets reveals potential cell sorting artifacts.
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ABSTRACT:
SUBMITTER: Reynaud CA
PROVIDER: S-EPMC3302223 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundCladribine is a highly effective immunotherapy that is applied in two short-term courses over 2 years and reduces relapse rate and disease progression in patients with relapsing multiple sclerosis (MS). Despite the short treatment period, cladribine has a long-lasting effect on disease activity even after recovery of lymphocyte counts, suggesting a yet undefined long-term immune modulating effect.ObjectivesOur aim was to provide a more profound understanding of the detailed effects of cladribine, also with regard to the patients' therapy response.DesignWe performed an open-labeled, explorative, prospective, single-arm study, in which we examined the detailed lymphocyte subset development of MS patients who received cladribine treatment over 2 years.MethodsWe performed in-depth profiling of the effects of cladribine on peripheral blood lymphocytes by flow cytometry, bulk RNA sequencing of sorted CD4+ T cells, CD8+ T cells, and CD19+ B cells as well as single-cell RNA sequencing of peripheral blood mononuclear cells in a total of 23 MS patients before and at different time points up to 24 months after cladribine treatment. Data were correlated with clinical and cranial magnetic resonance imaging (MRI) disease activity.ResultsFlow cytometry revealed a predominant and sustained reduction of memory B cells compared to other B cell subsets after cladribine treatment, whereas T cell subsets were slightly reduced in a more uniform pattern. The overall transcriptional profile of total blood B cells exhibited reduced expression of proinflammatory and T cell activating genes, while single-cell transcriptomics revealed that gene expression within each B cell cluster did not change over time. Stable patients displayed stronger reductions of selected memory B cell clusters as compared to patients with clinical or cerebral MRI disease activity.ConclusionWe describe a pronounced and sustained effect of cladribine on the memory B cell compartment, and the resulting change in B cell subset composition causes a significant alteration of B cell transcriptional profiles resulting in reduced proinflammatory and T cell activating capacities. The extent of reduction in selected memory B cell clusters by cladribine may predict treatment response.
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Project description:BACKGROUND:Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets. METHODOLOGY AND PRINCIPAL FINDINGS:Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p?=?0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC. CONCLUSION:HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.
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Project description:BACKGROUND:The contribution of phenotypic variation of peanut-specific T cells to clinical allergy or tolerance to peanut is not well understood. OBJECTIVES:Our objective was to comprehensively phenotype peanut-specific T cells in the peripheral blood of subjects with and without peanut allergy (PA). METHODS:We obtained samples from patients with PA, including a cohort undergoing baseline peanut challenges for an immunotherapy trial (Consortium of Food Allergy Research [CoFAR] 6). Subjects were confirmed as having PA, or if they passed a 1-g peanut challenge, they were termed high-threshold subjects. Healthy control (HC) subjects were also recruited. Peanut-responsive T cells were identified based on CD154 expression after 6 to 18 hours of stimulation with peanut extract. Cells were analyzed by using flow cytometry and single-cell RNA sequencing. RESULTS:Patients with PA had tissue- and follicle-homing peanut-responsive CD4+ T cells with a heterogeneous pattern of TH2 differentiation, whereas control subjects had undetectable T-cell responses to peanut. The PA group had a delayed and IL-2-dependent upregulation of CD154 on cells expressing regulatory T (Treg) cell markers, which was absent in HC or high-threshold subjects. Depletion of Treg cells enhanced cytokine production in HC subjects and patients with PA in vitro, but cytokines associated with highly differentiated TH2 cells were more resistant to Treg cell suppression in patients with PA. Analysis of gene expression by means of single-cell RNA sequencing identified T cells with highly correlated expression of IL4, IL5, IL9, IL13, and the IL-25 receptor IL17RB. CONCLUSIONS:These results demonstrate the presence of highly differentiated TH2 cells producing TH2-associated cytokines with functions beyond IgE class-switching in patients with PA. A multifunctional TH2 response was more evident than a Treg cell deficit among peanut-responsive T cells.
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