Subpopulations of bovine WC1+ γδ T cells rather than CD4+CD25highFoxp3+ T cells act as immune regulatory cells ex vivo

Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis. Especially CD4+CD25high T cells are considered to be important regulators of immune reactivity. In humans and rodents these natural Treg are characterized by their anergic nature, defined as a non-proliferative state, suppressive function and expression of Foxp3. In this study the potential functional role of flowcytometry-sorted bovine white blood cell populations, including CD4+CD25high T cells and γδ T cell subpopulations, as distinct ex vivo regulatory cells was assessed in co-culture suppression assays. Our findings revealed that despite the existence of a distinct bovine CD4+CD25high T cell population, which showed Foxp3 transcription/expression, natural regulatory activity did not reside in this cell population. In bovine co-culture suppression assays these cells were neither anergic nor suppressive. Subsequently, the following cell populations were tested functionally for regulatory activity: CD4+CD25low T cells, WC1+, WC1.1+ and WC1.2+ γδ T cells, NK cells, CD8+ T cells and CD14+ monocytes. Only the WC1.1+ and WC1.2+ γδ T cells and CD14+ monocytes proved to act as regulatory cells in cattle, which was supported by the fact that these regulatory cells showed IL-10 transcription/expression. In conclusion, our data provide first evidence that cattle CD4+CD25highFoxp3+ and CD4+CD25low T cells do not function as Treg ex vivo. The bovine Treg function appears to reside in the γδ T cell population, more precisely in the WC1.1+ and the WC1.2+ subpopulation, major populations present in blood of cattle in contrast to non-ruminant species

In vitro Chicken Bone Marrow-Derived Dendritic Cells Comprise Subsets at Different States of Maturation

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types

In vitro Chicken Bone Marrow-Derived Dendritic Cells Comprise Subsets at Different States of Maturation

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types

In vitro Chicken Bone Marrow-Derived Dendritic Cells Comprise Subsets at Different States of Maturation

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types

Considerations for MESF-bead based assignment of absolute fluorescence values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry is a promising technique to characterize nanoparticles (NPs) and extracellular vesicles (EVs). However, the majority of reported experiments, use arbitrary units to indicate fluorescence intensity. This hampers comparison of results from different laboratories and different platforms. We investigated the advised use of calibrated molecules of equivalent soluble fluorophores (MESF)-beads for assignment of absolute fluorescence to NPs and EVs. Firstly, we evaluated the use of two different FITC MESF bead sets as calibrators on three different flow cytometry platforms (BD Influx, CytoFLEX LX and SORP BD FACSCelesta). Secondly, NPs and biological 4T1 mammary carcinoma-EVs were analyzed using the BD Influx and their fluorescence signals calibrated by using different sets of FITC and PE MESF beads. Although fluorescence calibration, using bright calibrators designed for cellular flow cytometry, makes inter-platform comparison possible for fluorescently labeled cells and brightly labeled particles, but the uncertainty of the currently available calibrators, which are far out of the fluorescence range of the sub-micron particles, hampers a reliable assignment of absolute MESF numbers based on extrapolation into the dim fluorescence range. Our results illustrate the need for calibration materials specifically designed for NPs and EVs to enable a reliable assignment of absolute fluorescence values in the lower fluorescent ranges

Considerations for MESF-bead based assignment of absolute fluorescence values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry is a promising technique to characterize nanoparticles (NPs) and extracellular vesicles (EVs). However, the majority of reported experiments, use arbitrary units to indicate fluorescence intensity. This hampers comparison of results from different laboratories and different platforms. We investigated the advised use of calibrated molecules of equivalent soluble fluorophores (MESF)-beads for assignment of absolute fluorescence to NPs and EVs. Firstly, we evaluated the use of two different FITC MESF bead sets as calibrators on three different flow cytometry platforms (BD Influx, CytoFLEX LX and SORP BD FACSCelesta). Secondly, NPs and biological 4T1 mammary carcinoma-EVs were analyzed using the BD Influx and their fluorescence signals calibrated by using different sets of FITC and PE MESF beads. Although fluorescence calibration, using bright calibrators designed for cellular flow cytometry, makes inter-platform comparison possible for fluorescently labeled cells and brightly labeled particles, but the uncertainty of the currently available calibrators, which are far out of the fluorescence range of the sub-micron particles, hampers a reliable assignment of absolute MESF numbers based on extrapolation into the dim fluorescence range. Our results illustrate the need for calibration materials specifically designed for NPs and EVs to enable a reliable assignment of absolute fluorescence values in the lower fluorescent ranges

Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles

Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications. From the Clinical Editor: The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology

Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles

Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications. From the Clinical Editor: The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology