Single-cell profiling of human dura and meningioma reveals cellular meningeal landscape and insights into meningioma immune response

BACKGROUND: Recent investigations of the meninges have highlighted the importance of the dura layer in central nervous system immune surveillance beyond a purely structural role. However, our understanding of the meninges largely stems from the use of pre-clinical models rather than human samples. METHODS: Single-cell RNA sequencing of seven non-tumor-associated human dura samples and six primary meningioma tumor samples (4 matched and 2 non-matched) was performed. Cell type identities, gene expression profiles, and T cell receptor expression were analyzed. Copy number variant (CNV) analysis was performed to identify putative tumor cells and analyze intratumoral CNV heterogeneity. Immunohistochemistry and imaging mass cytometry was performed on selected samples to validate protein expression and reveal spatial localization of select protein markers. RESULTS: In this study, we use single-cell RNA sequencing to perform the first characterization of both non-tumor-associated human dura and primary meningioma samples. First, we reveal a complex immune microenvironment in human dura that is transcriptionally distinct from that of meningioma. In addition, we characterize a functionally diverse and heterogenous landscape of non-immune cells including endothelial cells and fibroblasts. Through imaging mass cytometry, we highlight the spatial relationship among immune cell types and vasculature in non-tumor-associated dura. Utilizing T cell receptor sequencing, we show significant TCR overlap between matched dura and meningioma samples. Finally, we report copy number variant heterogeneity within our meningioma samples. CONCLUSIONS: Our comprehensive investigation of both the immune and non-immune cellular landscapes of human dura and meningioma at single-cell resolution builds upon previously published data in murine models and provides new insight into previously uncharacterized roles of human dura

Biomarker-defined clusters by level of Type 2 inflammatory involvement in severe asthma

Peer reviewedPostprin

Exploring definitions and predictors of severe asthma clinical remission post-biologic in adults

Peer reviewe

Immunologic targeting of FOXP3 in inflammatory breast cancer cells.

The forkhead transcription factor FOXP3 is necessary for induction of regulatory T lymphocytes (Tregs) and their immunosuppressive function. We have previously demonstrated that targeting Tregs by vaccination of mice with murine FOXP3 mRNA-transfected dendritic cells (DCs) elicits FOXP3-specific T cell responses and enhances tumor immunity. It is clear that FOXP3 expression is not restricted to T-cell lineage and herein, using RT-PCR, flow cytometry, and western immunoblot we demonstrate for the first time that FOXP3 is expressed in inflammatory breast cancer (IBC) cells, SUM149 (triple negative, ErbB1-activated) and SUM190 (ErbB2-overexpressing). Importantly, FOXP3-specific T cells generated in vitro using human FOXP3 RNA-transfected DCs as stimulators efficiently lyse SUM149 cells. Interestingly, an isogenic model (rSUM149) derived from SUM149 with an enhanced anti-apoptotic phenotype was resistant to FOXP3-specific T cell mediated lysis. The MHC class I cellular processing mechanism was intact in both cell lines at the protein and transcription levels suggesting that the resistance to cytolysis by rSUM149 cells was not related to MHC class I expression or to the MHC class I antigen processing machinery in these cells. Our data suggest that FOXP3 may be an effective tumor target in IBC cells however increased anti-apoptotic signaling can lead to immune evasion

Sex-Dependent Differences in Physical Exercise-Mediated Cognitive Recovery Following Middle Cerebral Artery Occlusion in Aged Rats

Stroke remains a leading cause of death and disability in the United States. No current treatments exist to promote cognitive recovery in survivors of stroke. A previous study from our laboratory determined that an acute bout of forced treadmill exercise was able to promote cognitive recovery in 3 month old male rats after middle cerebral artery occlusion (MCAo). In this study, we tested the hypothesis that 6 days of intense acute bout of forced treadmill exercise (physical exercise – PE) promotes cognitive recovery in 11–14 month old male rats. We determined that PE was able to ameliorate cognitive deficits as determined by contextual fear conditioning. Additionally, we also tested the hypothesis that PE promotes cognitive recovery in 11–13 month old reproductive senescent female rats. In contrast to males, the same intensity of exercise that decrease cognitive deficits in males was not able to promote cognitive recovery in female rats. Additionally, we determined that exercise did not lessen infarct volume in both male and female rats. There are many factors that contribute to higher stroke mortality and morbidities in women and thus, future studies will investigate the effects of PE in aged female rats to identify sex differences

FOXP3 expression in IBC cells. A.

<p>Flow cytometry-based detection of FOXP3 expression in various cell lines. Monocytes (high Side Scatter (SSC), high Forward Scatter (FSC) cells within human PBMCs) were used as a negative control for FOXP3 expression. Each histogram represents the cell line FOXP3 peak compared to that of isotype peak (white underlaid plot). The mean fluorescence intensity (MFI) ratio (FOXP3/isotype) is shown in each histogram. <b>B.</b> Expression of FOXP3 mRNA by RT-PCR in HME1, breast cancer cells (MCF-7, BT474, SKBR3), IBC cell lines (SUM149, SUM190, rSUM149), Jurkat, human PBMCs, CD4+ T cells isolated from human PBMCs. Actin mRNA is used to verify the integrity of the cDNA preparations.</p

Lysis of IBC cell line, SUM149 by FOXP3-specific T-cells stimulated with FOXP3-encoding RNA-transfected DCs. A.

<p>Immunoblot analysis of 48 kDa FOXP3 and 37 kDa control GAPDH protein expression in 293T, SUM149 cell lysates and tonsil tissue lysates. <b>B.</b> Non-adherent PBMCs and DCs were generated from cells obtained from HLA-A2+ healthy donors. DCs were transfected with FOXP3 RNA, survivin RNA and actin RNA and used to stimulate autologous T cells as described in Methods. Post-stimulation the T cells were assayed for lytic activity using a europium-release assay. T cell lytic activity was measured against IBC cells SUM149 and SUM190 and 293T cells as controls. <b>C.</b> DCs expressing FOXP3 and actin were used as targets to demonstrate specificity using effector T cells described in 2A. <b>D.</b> FOXP3-specific T cell cytolytic activity on SUM149 and lapatinib-resistant, rSUM149 cells. Non-adherent PBMCs and DCs were generated from cells obtained from HLA-A2+ healthy donors. DCs were transfected with FOXP3 RNA and actin RNA and used to stimulate autologous T cells as described in Methods. Post-stimulation the T cells were assayed for lytic activity using a europium-release assay. T cell lytic activity was measured against IBC cells SUM149 and rSUM149 cells.</p

Evaluating MHC class I processing and presentation in SUM149 and rSUM149 cells. A.

<p>Cells used as targets in the CTL assay described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053150#pone-0053150-g002" target="_blank">Figure 2</a> were analyzed for the cell surface expression of HLA-A2 class I molecule as indicated in the panel. The bold black histogram in each panel represents staining with the HLA-A2 specific antibody (clone BB7-2) versus the isotype control. <b>B.</b> Basal expression in the SUM149 and rSUM149 cells. The data shown is representative of replicated experiments analyzed using the ΔΔC(t) method and represented as relative expression (2^−ΔΔC(t)). GAPDH was used as the normalization control. <b>C.</b> Expression after treatment with IFN-γ<b>.</b> The data shown is the average of replicated experiments analyzed using the ΔΔC(t) method and represented as relative expression (2^−ΔΔC(t)). GAPDH was used as the normalization control with the untreated sample set to 1 and compared to the IFN-γ treated group. <b>D.</b> FOXP3-specific T cells were generated as described in Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053150#pone-0053150-g002" target="_blank">Figure 2</a> above. T cells were allowed to rest overnight in RPMI medium with 10% FCS in the absence of cytokines. The following day, T cells were harvested and cocultured with target cells (SUM149, rSUM149 and 293T cells) at a 1∶1 ratio. T cells and target cells were maintained at 10⁶ cells/ml and a 100 µl of each cell suspension was added to a 96-well V-bottom tissue culture plate in triplicates and incubated at 37°C for 16 hours. Supernatant was harvested and IFN-γ secretion was measured using a human IFN-γ ELISA kit from eBioscience. Controls used are indicated in the figure.</p

Expression of p27, XIAP and SKP2 in SUM149 and rSUM149 cells.

<p>Comparative expression of p27, an anti-apoptotic protein, XIAP, an inhibitor of apoptosis, and SKP2, an oncogene, in the GW58830-treated SUM149 and rSUM149 cells. GAPDH is shown as loading control in the immunoblots. <b>A.</b> Immunoblot analysis of expression of p27 <b>B.</b> Immunoblot analysis of expression of SKP2 and XIAP.</p