Jeremy M. Grenier

Loss of Spastin Function Results in Disease-Specific Axonal Defects in Human Pluripotent Stem Cell-Based Models of Hereditary Spastic Paraplegia

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. Stem Cells 2014;32:414–423

Age-Associated Resident Memory CD8 T Cells in the Central Nervous System Are Primed To Potentiate Inflammation after Ischemic Brain Injury

Aging is associated with an increase in basal inflammation in the 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 has been performed. In this work, the effect of aging on CNS leukocyte recruitment was examined. Aging was associated with more CD45high 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 TCR activation. Analysis of TCRvβ repertoire usage suggested that entry into the CNS is most likely stochastic rather than Ag driven. Correlational analyses revealed a positive association between CD8 T cell numbers and decreased proinflammatory function of microglia. However, the effects of cerebral ischemia and ex vivo stimulation of these cells dramatically increased production of TNF, IFN-γ, and 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.

Cytomegalovirus-Based Vaccine Expressing a Modified Tumor Antigen Induces Potent Tumor-Specific CD8+ T-cell Response and Protects Mice from Melanoma

Qiu and colleagues used cytomegalovirus (CMV)-based prophylactic and therapeutic vaccines expressing foreign or modified self-tumor antigens in a B16 lung metastatic melanoma model and show that these vaccines induced protective antitumor CD8+ T-cell responses even in the presence of preexisting anti-CMV immunity. The presence of tumor-infiltrating CD8+ T cells is associated with tumor regression and better prognosis. Cytomegalovirus (CMV) infection elicits a robust and long-lasting CD8+ 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+ T-cell response and was effective in protecting mice from OVA-expressing 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+ 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+ 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. Cancer Immunol Res; 3(5); 536–46. ©2015 AACR.

Combining Adoptive Cell Therapy with Cytomegalovirus-Based Vaccine Is Protective against Solid Skin Tumors

Despite many years of research, cancer vaccines have largely been ineffective in the treatment of established cancers. Many barriers to immune-mediated destruction of malignant cells exist, and these likely limit the efficacy of cancer vaccines. In this study, we sought to enhance the efficacy of a cytomegalovirus (CMV)-based vaccine targeting melanoma by combining vaccination with other forms of immunotherapy. Adoptive cell therapy in humans and in animal models has been shown to be effective for tumor regression. Thus, in this study, we assessed whether CMV-based vaccines in combination with adoptively transferred antitumor T cells could provide greater antitumor protection than either therapy alone. Our results show that adoptive cell therapy greatly enhanced the antitumor effects of CMV-based vaccines targeting the foreign model antigen, OVA, or the melanoma differentiation antigen, gp100. Combination adoptive cell therapy and vaccination induced the upregulation of the inhibitory ligands, PD-L1, and Qa-1b, on B16 tumor cells. This expression paralleled the infiltration of tumors by vaccine-stimulated T cells which also expressed high levels of the receptors PD-1 and NKG2A/C/E, suggesting a potential mechanism of tumor immune evasion. Surprisingly, therapeutic blockade of the PD-1/PD-L1 and NKG2A/Qa-1b axes did not delay tumor growth following vaccination, suggesting that the presence of inhibitory ligands within malignant tissue may not be an effective biomarker for successful combination therapy with CMV-based vaccines. Overall, our studies show that therapeutic CMV-based vaccines in combination with adoptive T cell transfer alone are effective for tumor rejection.

Reviving virus based cancer vaccines by using cytomegalovirus vectors expressing modified tumor antigens.

Cancer vaccines that have utilized various immunization strategies to induce antitumor immunity have largely failed in clinical settings. We have recently developed a cancer vaccine using a cytomegalovirus (CMV) based vector that expressed a modified melanoma antigen that elicited a robust antitumor CD8+ T cell response and tumor rejection.

Combination Immunotherapy: Taking Cancer Vaccines to the Next Level

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.

Tilting the balance of M1/M2 microglial phenotypes: Inhibition of Ezh2 leads to decreased M1 and enhanced M2 phenotype

Infection of Theilers murine encephalomyelitis virus (TMEV) in the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Alpha 4 integrins function as adhesive structures, providing mechanical support for cell adhesion andmigration and, in addition, as bona fide signaling receptors. HCA3551 is a newly synthesized, orally active small molecule alpha 4 integrin antagonist. In this study, we examined the therapeutic effect of HCA3551 in the development of TMEV-induced demyelinating disease (TMEV-IDD). HCA3551 is provided by Ajinomoto Pharmaceuticals Co., Ltd. Micewere orally administrated twice daily with vehicle or HCA3551 (100 mg/kg) every 12 hours. HCA3551 treatment significantly suppressed the disease development of TMEV-IDD both clinically and histologically. The number of infiltrating mononuclear cells (MNCs) in the CNS was significantly decreased in mice treated with HCA3551 (p b 0.05) compared to control mice orally administrated with vehicle. The significant increase in peripheral lymphocyte countwas observed on day 20 (p b 0.01), day 30 (p b 0.05) and day 40 (p b 0.01) post infection compared to vehicle treated control mice. Flow cytometric analysis of cytokine staining revealed that absolute cell numbers of TNF-producing CD4 and IFN-gamma-producing CD8 T cells were significantly decreased in the CNS ofmice treatedwith HCA3551 compared to vehicle treated control mice (p b 0.01). Taken together these data suggest that HCA3551 treatment may ameliorate TMEV-IDD by inhibiting alpha 4 integrin accompanied with the decreasing number of MNCs and proinflammatory cytokine producing cells in the CNS. Therefore, HCA3551 could be used as a novel therapeutic treatment of MS. doi:10.1016/j.jneuroim.2014.08.422