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

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

Fetal Hematopoietic Stem Cell Transplantation Fails to Fully Regenerate the B-Lymphocyte Compartment

B cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system

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

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

Abstract 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

Bmi1 maintains the self-renewal property of innate-like B lymphocytes

The self-renewal ability is a unique property of fetal-derived innate-like B-1a lymphocytes, which survive and function without being replenished by bone marrow (BM) progenitors. However, the mechanism by which IgM-secreting mature B-1a lymphocytes self-renew is poorly understood. In this study, we showed that Bmi1 was critically involved in this process. Although Bmi1 is considered essential for lymphopoiesis, the number of mature conventional B cells was not altered when Bmi1 was deleted in the B cell lineage. In contrast, the number of peritoneal B-1a cells was significantly reduced. Peritoneal cell transfer assays revealed diminished self-renewal ability of Bmi1-deleted B-1a cells, which was restored by additional deletion of Ink4-Arf, the well-known target of Bmi1 Fetal liver cells with B cell-specific Bmi1 deletion failed to repopulate peritoneal B-1a cells, but not other B-2 lymphocytes after transplantation assays, suggesting that Bmi1 may be involved in the developmental process of B-1 progenitors to mature B-1a cells. Although Bmi1 deletion has also been shown to alter the microenvironment for hematopoietic stem cells, fat-associated lymphoid clusters, the reported niche for B-1a cells, were not impaired in Bmi1 -/- mice. RNA expression profiling suggested lysine demethylase 5B (Kdm5b) as another possible target of Bmi1, which was elevated in Bmi1-/- B-1a cells in a stress setting and might repress B-1a cell proliferation. Our work has indicated that Bmi1 plays pivotal roles in self-renewal and maintenance of fetal-derived B-1a cells

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

B-1 lymphocyte participation in experimental cryptococcosis

A criptococose é uma infecção fúngica causada pela levedura encapsulada Cryptococcus neoformans que acomete freqüentemente o meningoencéfalo de pacientes imunocomprometidos. No Brasil, cerca de 6% dos pacientes com AIDS desenvolvem a criptococose. A construção da resposta imune na criptococose envolve fagócitos e linfócitos T e B presentes no granuloma. Atualmente, são descritos três subtipos de linfócitos B: linfócitos B-1 a, B-1 b e B-2, o último conhecido como linfócito B \"convencional\". Os linfócitos B-1 (B-1a e B-1b) expressam concomitantemente imunofenótipo de macrófagos, células B e células T. Estes linfócitos receberam o nome de fagócitos mononucleares derivados de linfócitos B-1 e possuem a capacidade de migrar do peritônio até um foco inflamatório não específico, fagocitarem, expressarem antígeno e secretarem anticorpos e citocinas. Portanto, avaliamos a participação destes linfócitos B-1 na criptococose através de ensaios in vitro e in vivo com duas cepas diferentes de Cryptococcus neoformans variedade neoformans sorotipo A. Os linfócitos B-1 fagocitaram e destruiram as leveduras de Cryptococcus neoformans, principalmente na presença de complemento. Altas produções de NO encontradas no sobrenadante de cultura do ensaio de fagocitose associada à alta atividade peroxidásica e capacidade destes linfócitos em gerar EROs, poderiam justificar a alta atividade de \"killing\" encontrada nestes linfócitos B-1. Ainda, quando estimuladas com o fungo, os linfócitos B-1 secretaram principalmente IL-12 e IFN-γ, enquanto a IL-10 diminuiu significativamente. Na infecção experimental os camundongos BALB/xid, desprovidos de linfócitos B-1, apresentaram maior susceptibilidade à criptococose, com ambas as cepas, quando comparados aos camundongos BALB controle (BALB/c). Estes animais BALB/xid apresentaram maior carga fúngica nos órgãos baço, fígado e pulmão e morreram precocemente. A concentração de IL-10 foi maior nos órgãos dos camundongos BALB/xid, enquanto a IL-4 aumentou nos camundongos BALB/c. A concentração das citocinas de padrão Th1 (IL-12, IFN-γ e IL-2). consideradas protetoras na criptococose foi maior nos camundongos BALB/c. Além· da produção de citocinas protetoras, os órgãos dos camundongos BALB/c infectados com a cepa de menor cápsula apresentaram um maior população de linfócitos B-1. Estes linfócitos provavelmente estavam participando do processo inflamatório e contribuindo para a cura da infecção. Embora outras investigações sejam necessárias para melhor compreendermos a participação de linfócitos B-1 na criptococose, podemos considerar que estas células interagem com leveduras de Cryptococcus neoformans e podem ser decisivas para uma resposta protetora na criptococose experimental.Abstract not available