Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires

Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota

QFMatch: multidimensional flow and mass cytometry samples alignment

Part of the flow/mass cytometry data analysis process is aligning (matching) cell subsets between relevant samples. Current methods address this cluster-matching problem in ways that are either computationally expensive, affected by the curse of dimensionality, or fail when population patterns significantly vary between samples. Here, we introduce a quadratic form (QF)-based cluster matching algorithm (QFMatch) that is computationally efficient and accommodates cases where population locations differ significantly (or even disappear or appear) from sample to sample. We demonstrate the effectiveness of QFMatch by evaluating sample datasets from immunology studies. The algorithm is based on a novel multivariate extension of the quadratic form distance for the comparison of flow cytometry data sets. We show that this QF distance has attractive computational and statistical properties that make it well suited for analysis tasks that involve the comparison of flow/mass cytometry samples

Earth Mover’s Distance (EMD): A True Metric for Comparing Biomarker Expression Levels in Cell Populations

Changes in the frequencies of cell subsets that (co)express characteristic biomarkers, or levels of the biomarkers on the subsets, are widely used as indices of drug response, disease prognosis, stem cell reconstitution, etc. However, although the currently available computational “gating” tools accurately reveal subset frequencies and marker expression levels, they fail to enable statistically reliable judgements as to whether these frequencies and expression levels differ significantly between/among subject groups. Here we introduce flow cytometry data analysis pipeline which includes the Earth Mover’s Distance (EMD) metric as solution to this problem. Well known as an informative quantitative measure of differences between distributions, we present three exemplary studies showing that EMD 1) reveals clinically-relevant shifts in two markers on blood basophils responding to an offending allergen; 2) shows that ablative tumor radiation induces significant changes in the murine colon cancer tumor microenvironment; and, 3) ranks immunological differences in mouse peritoneal cavity cells harvested from three genetically distinct mouse strains

QFMatch: multidimensional flow and mass cytometry samples alignment

Part of the flow/mass cytometry data analysis process is aligning (matching) cell subsets between relevant samples. Current methods address this cluster-matching problem in ways that are either computationally expensive, affected by the curse of dimensionality, or fail when population patterns significantly vary between samples. Here, we introduce a quadratic form (QF)-based cluster matching algorithm (QFMatch) that is computationally efficient and accommodates cases where population locations differ significantly (or even disappear or appear) from sample to sample. We demonstrate the effectiveness of QFMatch by evaluating sample datasets from immunology studies. The algorithm is based on a novel multivariate extension of the quadratic form distance for the comparison of flow cytometry data sets. We show that this QF distance has attractive computational and statistical properties that make it well suited for analysis tasks that involve the comparison of flow/mass cytometry samples

Development of B Cells and Erythrocytes Is Specifically Impaired by the Drug Celastrol in Mice

Background: Celastrol, an active compound extracted from the root of the Chinese medicine ‘‘Thunder of God Vine’’ (Tripterygium wilfordii), exhibits anticancer, antioxidant and anti-inflammatory activities, and interest in the therapeutic potential of celastrol is increasing. However, described side effects following treatment are significant and require investigation prior to initiating clinical trials. Here, we investigated the effects of celastrol on the adult murine hematopoietic system. Methodology/Principal Findings: Animals were treated daily with celastrol over a four-day period and peripheral blood, bone marrow, spleen, and peritoneal cavity were harvested for cell phenotyping. Treated mice showed specific impairment of the development of B cells and erythrocytes in all tested organs. In bone marrow, these alterations were accompanied by decreases in populations of common lymphoid progenitors (CLP), common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP). Conclusions/Significance: These results indicate that celastrol acts through regulators of adult hematopoiesis and could be used as a modulator of the hematopoietic system. These observations provide valuable information for further assessmen

Celastrol treatment results in multiple defects in BM progenitors.

<p>Representative FACS analysis of Megakaryocyte-Erythrocyte Progenitors (MEP), Common Myeloid Progenitors (CMP), Granulocyte-Monocyte Progenitors (GMP), LSK CD34⁻ (Lin⁻ Sca-1⁺ c-Kit⁺ CD34⁻) cells and Common Lymphoid Progenitors (CLP) from bone marrow of control (left) and celastrol treated-mice (right) (n = 10). Cells were harvested from animals treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone-0035733-g002" target="_blank">Figure 2</a> and percentages shown correspond to raw data numbers. FACS-analysis of the different sub-populations of multipotent progenitors was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Kusy1" target="_blank">[23]</a>.</p

Changes in peripheral blood parameters and cellularity in celastrol treated-BALB/c mice.

<p>(A) Peripheral blood parameters in celastrol treated-mice. Mice received four consecutive daily IP injections of DMSO 5% or various concentrations of celastrol (0.01, 0.1, 1 or 5 mg/kg/day). Peripheral blood was harvested the day following the last injection. Values are percent of control. RBC: red blood cells; HGB: Hemoglobin; HCT: hematocrit; WBC: white blood cells. Mean ± SEM, n = 10. (B) Cellularity in control and celastrol treated-mice. Mice received four consecutive daily IP injections of DMSO 5% or celastrol (5 mg/kg). Cells from bone marrow (1 femur+1 tibia), spleen and peritoneal cavity were harvested the day following the last injection. Mean ± SEM, n = 10.</p

Celastrol treatment results in multiple defects in mature lineages.

<p>(A) Representative FACS analysis of mature lineages from peripheral blood of control (left) and celastrol treated-mice (right). Mice received four consecutive daily intraperitoneal injections and peripheral blood cells were harvested the day following the last injection, processed and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#s2" target="_blank">Materials and Methods</a>. The percentages shown correspond to raw data numbers. Data shown are representative of 10 mice. (B) Representative FACS analysis of total B cells (gated as LIVE/DEAD⁺, CD5⁻, CD19⁺, and analyzed for IgD and IgM expression) from bone marrow, spleen and peritoneal cavity of control (left) and celastrol treated-mice (right) (n = 10). FACS-analysis of the different sub-populations of B lymphoid progenitors was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Ghosn1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Yang2" target="_blank">[20]</a>. MZ B cells: Marginal Zone B cells. (C) Representative FACS analysis of total red cells (gated as LIVE/DEAD⁺, CD5⁻, CD19⁻, CD11b⁻, Gr-1⁻, SSC-A^low and analyzed for CD71 and Ter119 expressions) from bone marrow, spleen and peritoneal cavity of control (left) and celastrol treated-mice (right) (n = 10). FACS-analysis of the different sub-populations of red cell progenitors was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Cao1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Liu1" target="_blank">[22]</a>.</p

Monocyte depletion enhances neutrophil influx and proneural to mesenchymal transition in glioblastoma

Myeloid cells are the predominant cell type in the tumor microenvironment of human and murine glioblastoma (GBM). By generating a mouse model deficient for all monocyte chemoattractant proteins, here the authors show that blocking monocyte recruitment promotes a compensatory neutrophil influx and that concomitant neutrophil inhibition is required to improve survival in GBM preclinical models