CD11bhigh B Cells Increase after Stroke and Regulate Microglia.

Recent studies have highlighted the deleterious contributions of B cells to post-stroke recovery and cognitive decline. Different B cell subsets have been proposed on the basis of expression levels of transcription factors (e.g., T-bet) as well as specific surface proteins. CD11b (α-chain of integrin) is expressed by several immune cell types and is involved in regulation of cell motility, phagocytosis, and other essential functions of host immunity. Although B cells express CD11b, the CD11bhigh subset of B cells has not been well characterized, especially in immune dysregulation seen with aging and after stroke. Here, we investigate the role of CD11bhigh B cells in immune responses after stroke in young and aged mice. We evaluated the ability of CD11bhigh B cells to influence pro- and anti-inflammatory phenotypes of young and aged microglia (MG). We hypothesized that CD11bhigh B cells accumulate in the brain and contribute to neuroinflammation in aging and after stroke. We found that CD11bhigh B cells are a heterogeneous subpopulation of B cells predominantly present in naive aged mice. Their frequency increases in the brain after stroke in young and aged mice. Importantly, CD11bhigh B cells regulate MG phenotype and increase MG phagocytosis in both ex vivo and in vivo settings, likely by production of regulatory cytokines (e.g., TNF-α). As both APCs and adaptive immune cells with long-term memory function, B cells are uniquely positioned to regulate acute and chronic phases of the post-stroke immune response, and their influence is subset specific.

Peripherally-sourced myeloid antigen presenting cells increase with advanced aging.

Aging is associated with dysfunction of the gut microbiota-immune-brain axis, a major regulatory axis in both brain health and in central nervous system (CNS) diseases. Antigen presenting cells (APCs) play a major role in sensing changes in the gut microbiota and regulation of innate and adaptive immune responses. APCs have also been implicated in various chronic inflammatory conditions, including age-related neurodegenerative diseases. The increase in chronic low-level inflammation seen with aging has also been linked to behavioral decline. Despite their acknowledged importance along the gut microbiota-immune-brain axis, there is limited evidence on how APCs change with aging. In this study, we examined age-related changes in myeloid APCs in the gut, spleen, and brain as well as changes in the gut microbiota and behavioral phenotype in mice ranging in age from 2 months up to 32 months of both sexes. Our data show that the number of peripherally-sourced myeloid APCs significantly increases with advanced aging in the brain. In addition, our data showed that age-related changes in APCs are subset-specific in the gut and sexually dimorphic in the spleen. Our work highlights the importance of studying myeloid APCs in an age-, tissue-, and sex-specific manner.

Microglial IRF5-IRF4 regulatory axis regulates neuroinflammation after cerebral ischemia and impacts stroke outcomes.

Microglial activation plays a central role in poststroke inflammation and causes secondary neuronal damage; however, it also contributes in debris clearance and chronic recovery. Microglial pro- and antiinflammatory responses (or so-called M1-M2 phenotypes) coexist and antagonize each other throughout the disease progress. As a result of this balance, poststroke immune responses alter stroke outcomes. Our previous study found microglial expression of interferon regulatory factor 5 (IRF5) and IRF4 was related to pro- and antiinflammatory responses, respectively. In the present study, we genetically modified the IRF5 and IRF4 signaling to explore their roles in stroke. Both in vitro and in vivo assays were utilized; IRF5 or IRF4 small interfering RNA (siRNA), lentivirus, and conditional knockout (CKO) techniques were employed to modulate IRF5 or IRF4 expression in microglia. We used a transient middle cerebral artery occlusion model to induce stroke and examined both acute and chronic stroke outcomes. Poststroke inflammation was evaluated with flow cytometry, RT-PCR, MultiPlex, and immunofluorescence staining. An oscillating pattern of the IRF5-IRF4 regulatory axis function was revealed. Down-regulation of IRF5 signaling by siRNA or CKO resulted in increased IRF4 expression, enhanced M2 activation, quenched proinflammatory responses, and improved stroke outcomes, whereas down-regulation of IRF4 led to increased IRF5 expression, enhanced M1 activation, exacerbated proinflammatory responses, and worse functional recovery. Up-regulation of IRF4 or IRF5 by lentivirus induced similar results. We conclude that the IRF5-IRF4 regulatory axis is a key determinant in microglial activation. The IRF5-IRF4 regulatory axis is a potential therapeutic target for neuroinflammation and ischemic stroke.

Rigor and Reproducibility of Cytometry Practices for Immuno-Oncology: A multifaceted challenge.

The rapid advancement of immunotherapy strategies has created a need for technologies that can reliably and reproducibly identify rare populations, detect subtle changes in modulatory signals, and assess antigenic expression patterns that are time-sensitive. Accomplishing these tasks requires careful planning and the employment of tools that provide greater sensitivity and specificity without demanding extensive time. Flow Cytometry has earned its place as a preferred analysis platform. This technology offers a flexible path to the interrogation of protein expression patterns and detection of functional properties in cell populations of interest. Mass Cytometry is a newcomer technology that has generated significant interest in the field. By incorporating mass spectrometry analysis to the traditional principles of flow cytometry, this innovative tool promises to significantly expand the ability to detect multiple proteins on a single cell. The use of these technologies in a manner that is consistent and reproducible through multiple sample sets demands careful attention to experiment design, reagent selection, and instrumentation. Whether applying flow or mass cytometry, reaching successful, reliable results involves many factors. Sample preparation, antibody titrations, and appropriate controls are major biological considerations that impact cytometric analysis. Additionally, instrument voltages, lasers, and run quality assessments are essential for ensuring comparability and reproducibility between analyses. In this article, we aim to discuss the critical aspects that impact flow cytometry, and will touch on important considerations for mass cytometry as well. Focusing on their relevance to immunotherapy studies, we will address the importance of appropriate sample processing and will discuss how selection of suitable panels, controls, and antibodies must follow a carefully designed plan. We will also comment on how educated use of instrumentation plays a significant role in the reliability and reproducibility of results.Through this work, we hope to contribute to the effort toward establishing higher standards for rigor and reproducibility of cytometry practices by researchers, operators, and general cytometry users employing cytometry-based assays in their work. © 2019 International Society for Advancement of Cytometry.

The importance of area scaling with FACS DIVA software.

With the release and use of the Becton Dickenson FACS Diva Software, the use of Area as the default parameter came into play. As such, the use of area as a calculated parameter, methods were needed to be employed to ensure doublet discrimination and proper display on standard FSC/SSC. Improper setting of forward area scaling can alter the display cell populations. This combined with improper area gating strategy can lead to doublet inclusion which in sorting rare events can compromise sort purity. In extreme cases where area scaling with the individual lasers is ignored, differences can exist between Area and Height where compensation will likely not be optimal, particularly if one parameter - usually height is saturated. In addition, area scaling can impact population grouping. As FSC and individual laser area scaling is a function of event size, the most common error is to accept the setting determined by CS&T, which are 3.2 µm particles and proceed with the sample(s) without regard to the sample's actual size. With cellular events smaller or more likely larger than the CS&T beads, this will make the area scaling settings less than optimal. Analysis and sorting rare events with populations larger than the CS&T beads can be compromised if adjustments in FSC area scaling are not addressed. Proper FSC and laser area scaling must be determined empirically for each sample. Examples of the effects of sample size on area scaling will be presented in addition to gating and templates for determining area scaling.

Lineage extrinsic and intrinsic control of immunoregulatory cell numbers by SHIP.

We previously showed that germline or induced SHIP deficiency expands immuno-regulatory cell numbers in T lymphoid and myeloid lineages. We postulated these increases could be interrelated. Here, we show that myeloid-specific ablation of SHIP leads to the expansion of both myeloid-derived suppressor cell (MDSC) and regulatory T (Treg) cell numbers, indicating SHIP-dependent control of Treg-cell numbers by a myeloid cell type. Conversely, T-lineage specific ablation of SHIP leads to expansion of Treg-cell numbers, but not expansion of the MDSC compartment, indicating SHIP also has a lineage intrinsic role in limiting Treg-cell numbers. However, the SHIP-deficient myeloid cell that promotes MDSC and Treg-cell expansion is not an MDSC as they lack SHIP protein expression. Thus, regulation of MDSC numbers in vivo must be controlled in a cell-extrinsic fashion by another myeloid cell type. We had previously shown that G-CSF levels are profoundly increased in SHIP(-/-) mice, suggesting this myelopoietic growth factor could promote MDSC expansion in a cell-extrinsic fashion. Consistent with this hypothesis, we find that G-CSF is required for expansion of the MDSC splenic compartment in mice rendered SHIP-deficient as adults. Thus, SHIP controls MDSC numbers, in part, by limiting production of the myelopoietic growth factor G-CSF.

Major remodelling of the murine stem cell kinome following differentiation in the hematopoietic compartment.

The changes in signal transduction associated with the acquisition of specific cell fates remain poorly understood. We performed massive parallel assessment of kinase signatures of the radiations of the hematopoietic system, including long-term repopulating hematopoietic stem cells (LT-HSC), short-term repopulating HSC (ST-HSC), immature natural killer (iNK) cells, NK cells, B cells, T cells, and myeloid cells. The LT-HSC kinome is characterized by noncanonical Wnt, Ca(2+) and classical protein kinase C (PKC)-driven signaling, which is lost upon the transition to ST-HSC, whose kinome signature prominently features receptor tyrosine kinase (RTK) activation of the Ras/MAPK signaling cassette. Further differentiation to iNK maintains signaling through this cassette but simultaneously leads to activation of a PI3K/PKB/Rac signaling, which becomes the dominant trait in the kinase signature following full differentiation toward NK cells. Differentiation along the myeloid and B cell lineages is accompanied by hyperactivation of both the Ras/MAPK and PI3K/PKB/Rac signaling cassette. T cells, however, deactivate signaling and only display residual G protein-coupled pathways. Thus, differentiation along the hematopoietic lineage is associated with major remodelling of cellular kinase signature.

Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity.

Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2(-/-) p53(515C/515C) mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS and cell cycle arrest, senescence, and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized because of ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.

SHIP is required for a functional hematopoietic stem cell niche.

SH2-domain-containing inositol 5'-phosphatase-1 (SHIP) deficiency significantly increases the number of hematopoietic stem cells (HSCs) present in the bone marrow (BM). However, the reconstitution capacity of these HSCs is severely impaired, suggesting that SHIP expression might be an intrinsic requirement for HSC function. To further examine this question, we developed a model in which SHIP expression is ablated in HSCs while they are resident in a SHIP-competent milieu. In this setting, we find that long-term repopulation by SHIP-deficient HSCs is not compromised. Moreover, SHIP-deficient HSCs from this model repopulate at levels comparable with wild-type HSCs upon serial transfer. However, when HSCs from mice with systemic ablation of SHIP are transplanted, they are functionally compromised for repopulation. These findings demonstrate that SHIP is not an intrinsic requirement for HSC function, but rather that SHIP is required for the BM milieu to support functionally competent HSCs. Consistent with these findings, cells that comprise the BM niche express SHIP and SHIP deficiency profoundly alters their function.

SHIP deficiency enhances HSC proliferation and survival but compromises homing and repopulation.

The SH2 domain-containing inositol 5'-phosphatase-1 (SHIP) has the potential to modulate multiple signaling pathways downstream of receptors that impact hematopoietic stem cell (HSC) biology. Therefore, we postulated that SHIP might play an important role in HSC homeostasis and function. Consistent with this hypothesis, HSC proliferation and numbers are increased in SHIP(-/-) mice. Despite expansion of the compartment, SHIP(-/-) HSCs exhibit reduced capacity for long-term repopulation. Interestingly, we observe that SHIP(-/-) stem/progenitor cells home inefficiently to bone marrow (BM), and consistent with this finding, have reduced surface levels of both CXCR4 and vascular cell adhesion marker-1 (VCAM-1). These studies demonstrate that SHIP is critical for normal HSC function, homeostasis, and homing.