BackgroundThe brain’s initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood–brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.MethodsUsing a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.ResultsWe found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.ConclusionsIn summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0329-1) contains supplementary material, which is available to authorized users.

The peripheral immune system plays a critical role in aging and in the response to brain injury. Emerging data suggest inflammatory responses are exacerbated in older animals following ischemic stroke; however, our understanding of these age-related changes is poor. In this work, we demonstrate marked differences in the composition of circulating and infiltrating leukocytes recruited to the ischemic brain of old male mice after stroke compared to young male mice. Blood neutrophilia and neutrophil invasion into the brain were increased in aged animals. Relative to infiltrating monocyte populations, brain-invading neutrophils had reduced phagocytic potential, and produced higher levels of reactive oxygen species and extracellular matrix-degrading enzymes (i.e., MMP-9), which were further exacerbated with age. Hemorrhagic transformation was more pronounced in aged versus young mice relative to infarct size. High numbers of myeloperoxidase-positive neutrophils were found in postmortem human brain samples of old (> 71 years) acute ischemic stroke subjects compared to non-ischemic controls. Many of these neutrophils were found in the brain parenchyma. A large proportion of these neutrophils expressed MMP-9 and positively correlated with hemorrhage and hyperemia. MMP-9 expression and hemorrhagic transformation after stroke increased with age. These changes in the myeloid response to stroke with age led us to hypothesize that the bone marrow response to stroke is altered with age, which could be important for the development of effective therapies targeting the immune response. We generated heterochronic bone marrow chimeras as a tool to determine the contribution of peripheral immune senescence to age- and stroke-induced inflammation. Old hosts that received young bone marrow (i.e., Young → Old) had attenuation of age-related reductions in bFGF and VEGF and showed improved locomotor activity and gait dynamics compared to isochronic (Old → Old) controls. Microglia in young heterochronic mice (Old → Young) developed a senescent-like phenotype. After stroke, aged animals reconstituted with young marrow had reduced behavioral deficits compared to isochronic controls, and had significantly fewer brain-infiltrating neutrophils. Increased rates of hemorrhagic transformation were seen in young mice reconstituted with aged bone marrow. This work suggests that age alters the immunological response to stroke, and that this can be reversed by manipulation of the peripheral immune cells in the bone marrow.Electronic supplementary materialThe online version of this article (10.1007/s00401-018-1859-2) contains supplementary material, which is available to authorized users.

Human neuronal models of hereditary spastic paraplegias (HSP) that recapitulate disease-specific axonal pathology hold the key to understanding why certain axons degenerate in patients and to developing therapies. SPG4, the most common form of HSP, is caused by autosomal dominant mutations in the SPAST gene, which encodes the microtubule-severing ATPase spastin. Here, we have generated a human neuronal model of SPG4 by establishing induced pluripotent stem cells (iPSCs) from an SPG4 patient and differentiating these cells into telencephalic glutamatergic neurons. The SPG4 neurons displayed a significant increase in axonal swellings, which stained strongly for mitochondria and tau, indicating the accumulation of axonal transport cargoes. In addition, mitochondrial transport was decreased in SPG4 neurons, revealing that these patient iPSC-derived neurons recapitulate disease-specific axonal phenotypes. Interestingly, spastin protein levels were significantly decreased in SPG4 neurons, supporting a haploinsufficiency mechanism. Furthermore, cortical neurons derived from spastin-knockdown human embryonic stem cells (hESCs) exhibited similar axonal swellings, confirming that the axonal defects can be caused by loss of spastin function. These spastin-knockdown hESCs serve as an additional model for studying HSP. Finally, levels of stabilized acetylated-tubulin were significantly increased in SPG4 neurons. Vinblastine, a microtubule-destabilizing drug, rescued this axonal swelling phenotype in neurons derived from both SPG4 iPSCs and spastin-knockdown hESCs. Thus, this study demonstrates the successful establishment of human pluripotent stem cell-based neuronal models of SPG4, which will be valuable for dissecting the pathogenic cellular mechanisms and screening compounds to rescue the axonal degeneration in HSP.

Aging is associated with an increase in basal inflammation in the central nervous system (CNS) and an overall decline in cognitive function and poorer recovery following injury. Growing evidence suggests that leukocyte recruitment to the CNS is also increased with normal aging, but to date, no systematic evaluation of these “age-associated” leukocytes have been performed. In this work the effect of aging on CNS leukocyte recruitment was examined. Aging was associated with an increased number of CD45hi leukocytes, primarily composed of conventional CD8+ T cells. These results were strain-independent and seen in both sexes. Intravascular labeling and immunohistology revealed the presence of parenchymal CD8+ T cells in several regions of the brain including the choroid plexus and meninges. These cells had effector memory (CD44+CD62L−) and tissue-resident phenotypes and expressed markers associated with T cell receptor (TCR) activation. Analysis of TCRvβ repertoire usage suggested that entry into the CNS is likely stochastic rather than antigen-driven. Correlational analyses revealed a positive association between CD8 T cell numbers and decreased pro-inflammatory function of microglia. However, the effects of cerebral ischemia and ex-vivo stimulation of these cells dramatically increased production of tumor necrosis factor (TNF), interferon gamma (IFNγ), and monocyte-chemotactic protein-1 (MCP-1/CCL2). Taken together, we identified a novel population of resident memory, immunosurveillant CD8 T cells that represent a hallmark of CNS aging and appear to modify microglia homeostasis under normal conditions, but are primed to potentiate inflammation and leukocyte recruitment following ischemic injury.

The presence of tumor-infiltrating CD8 þ T cells is associated with tumor regression and better prognosis. Cytomegalovirus (CMV) infection elicits a robust and long-lasting CD8 þ T-cell response, which makes CMV a potentially promising vaccine vector against cancer. In the current study, we used recombinant murine CMV (MCMV) strains as prophylactic and therapeutic vaccines in an aggressive B16 lung metastatic melanoma model. Immunization with MCMV-expressing ovalbumin (OVA) induced a potent OVA-specific CD8 þ T-cell response and was effective in protecting mice from OVAexpressing B16 melanoma in an antigen-dependent manner. We engineered MCMV to express a modified B16 melanoma antigen gp100 (MCMV-gp100KGP). Immunization with MCMV-gp100KGP was highly effective in overcoming immune tolerance to self-antigen and induced a strong, long-lasting gp100-specific CD8 þ T-cell response even in the presence of preexisting anti-CMV immunity. Furthermore, both prophylactic and therapeutic vaccinations of mice with MCMV-gp100KGP effectively protected mice from highly aggressive lung B16-F10 melanoma, and the protection was mediated by gp100-specific CD8 þ T cells. We showed that MCMV is a superior vaccine vector compared with a commonly used vesicular stomatitis virus vector. Collectively, our studies demonstrate that CMV is a promising vaccine vector to prevent and treat tumors.

This study sought to measure the degree of synergy induced by specific small molecule inhibitors of DNA-PK [NU7026 and IC486241 (ICC)], a major component of the non-homologous end-joining (NHEJ) pathway, with SN38 or oxaliplatin. Synergy between the DNA damaging drugs and the DNA-PK inhibitors was assessed using the sulforhodamine-B assay (SRB). Effects of drug combinations on cell cycle and DNA-PK activity were determined using flow cytometry and western blot analysis. DNA damage was assessed via comet assay and quantification of γH2AX. The role of homologous recombination repair (HRR) was determined by nuclear Rad51 protein levels and a GFP reporter recombination assay. Significant reductions in the IC(50) values of SN38 were observed at 5 and 10 μM of DNA-PK inhibitors. Moreover, at 1-2 μM (attainable concentrations with ICC in mice) these DNA-PKcs inhibitors demonstrated synergistic reductions in the IC(50) of SN38. Flow cytometric data indicated that SN38 and SN38 in combination with DNA-PKcs inhibitors showed dramatic G2/M arrest at 24 h. Furthermore, reduced phosphorylation of DNA-PKcs and increased DNA damage were observed at this time point with SN38 in combination with DNA-PKcs inhibitors as compared to cells treated with SN38 alone. SN38 alone and in the presence of ICC increased nuclear Rad51 protein levels. Furthermore, inhibition of DNA-PKcs increased HRR suggesting that NHEJ is a negative regulator of HRR. These data indicate that small molecule inhibitors of DNA-PKcs dramatically enhance the efficacy of SN38 in colon cancer cell lines.

BKM120, a pan class I PI3K inhibitor, was cytotoxic in the majority of primary B-chronic lymphocytic leukemia (CLL) lymphocytes, including samples from patients who have a high-risk for poor response to treatment (patient with del11 and del17) at clinically obtainable concentrations. The PI3Kδ inhibitor Cal-101 is cytotoxic in B-CLL lymphocytes in vitro and is active in the treatment of CLL in vivo. Interestingly, we demonstrated that BKM120 is 3.6 fold more toxic than Cal-101 in malignant B-CLL lymphocytes in vitro. BKM120 cytotoxicity correlated with the basal expression of proteins involved in the PI3K/Akt pathway. A protein signature of PI3K pathway proteins predicts the response to BKM120 treatment. In the primary B-CLL lymphocytes tested in vitro, BKM120 decreased the phosphorylation status of molecular biomarkers used as indicators of PI3K pathway inhibition in vivo. Also, BKM120 induced apoptosis in primary B-CLL cells culture in the presence and absence of stromal cell support. Our findings suggest that BKM120 should be tested clinically in CLL.

With the advent of checkpoint blockade therapies, immunotherapy is now a critical modality for the treatment of some cancers. While some patients respond well to checkpoint blockade, many do not, necessitating the need for other forms of therapy. Vaccination against malignancy has been a long sought goal of science. For cancers holding a microbial etiology, vaccination has been highly effective in reducing the incidence of disease. However, vaccination against established malignancy has been largely disappointing. In this review, we discuss efforts to develop diverse vaccine modalities in the treatment of cancer with a particular focus on melanoma. Recent work has suggested that vaccines targeting patient-specific tumor mutations may be more relevant than those targeting unmutated proteins. Nonetheless, tumor cells utilize many strategies to evade host immunity. It is likely that the full potential of cancer vaccination will only be realized when vaccines are combined with other therapies targeting tumor immunoevasive mechanisms. By modulating inhibitory molecules, regulatory immune cells, and the metabolic resources and demands of T cells, scientists and clinicians can ensure vaccine-stimulated T cells are fully functional within the immunosuppressive tumor microevironment.