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Oncolytic viruses have the potential to induce immunogenic cell death (ICD) that may provoke potent and long-lasting anti-cancer immunity. Here we aimed to characterize the ICD-inducing ability of wild-type Adenovirus (Ad), Semliki Forest virus (SFV) and Vaccinia virus (VV). We did so by investigating the cell death and immune-activating properties of virus-killed tumor cells. Ad-infection of tumor cells primarily activates autophagy, but also activate events of necroptotic and pyroptotic cell death. SFV infection on the other hand primarily activates immunogenic apoptosis while VV activates necroptosis. All viruses mediated lysis of tumor cells leading to the release of danger-associated molecular patterns, triggering of phagocytosis and maturation of dendritic cells (DCs). However, only SFV-infected tumor cells triggered significant T helper type 1 (Th1)-cytokine release by DCs and induced antigen-specific T-cell activation. Our results elucidate cell death processes activated upon Ad, SFV, and VV infection and their potential to induce T cell-mediated anti-tumor immune responses. This knowledge provides important insight for the choice and design of therapeutically successful virus-based immunotherapies.

Background Many lines of evidence suggest that accumulation of aggregated alpha-synuclein (αSYN) in the Parkinson’s disease (PD) brain causes infiltration of T cells. However, in which ways the stationary brain cells interact with the T cells remain elusive. Here, we identify astrocytes as potential antigen-presenting cells capable of activating T cells in the PD brain. Astrocytes are a major component of the nervous system, and accumulating data indicate that astrocytes can play a central role during PD progression. Methods To investigate the role of astrocytes in antigen presentation and T-cell activation in the PD brain, we analyzed post mortem brain tissue from PD patients and controls. Moreover, we studied the capacity of cultured human astrocytes and adult human microglia to act as professional antigen-presenting cells following exposure to preformed αSYN fibrils. Results Our analysis of post mortem brain tissue demonstrated that PD patients express high levels of MHC-II, which correlated with the load of pathological, phosphorylated αSYN. Interestingly, a very high proportion of the MHC-II co-localized with astrocytic markers. Importantly, we found both perivascular and infiltrated CD4 + T cells to be surrounded by MHC-II expressing astrocytes, confirming an astrocyte T cell cross-talk in the PD brain. Moreover, we showed that αSYN accumulation in cultured human astrocytes triggered surface expression of co-stimulatory molecules critical for T-cell activation, while cultured human microglia displayed very poor antigen presentation capacity. Notably, intercellular transfer of αSYN/MHC-II deposits occurred between astrocytes via tunneling nanotubes, indicating spreading of inflammation in addition to toxic protein aggregates. Conclusions In conclusion, our data from histology and cell culture studies suggest an important role for astrocytes in antigen presentation and T-cell activation in the PD brain, highlighting astrocytes as a promising therapeutic target in the context of chronic inflammation.

Tumor angiogenesis occurs through regulation of genes that orchestrate endothelial sprouting and vessel maturation, including deposition of a vessel-associated extracellular matrix. CD93 is a transmembrane receptor that is upregulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates β1 integrin signaling and organization of fibronectin fibrillogenesis during tumor vascularization. In endothelial cells and mouse retina, CD93 was found to be expressed in endothelial filopodia and to promote filopodia formation. The CD93 localization to endothelial filopodia was stabilized by interaction with multimerin-2 (MMRN2), which inhibited its proteolytic cleavage. The CD93-MMRN2 complex was required for activation of β1 integrin, phosphorylation of focal adhesion kinase (FAK), and fibronectin fibrillogenesis in endothelial cells. Consequently, tumor vessels in gliomas implanted orthotopically in CD93-deficient mice showed diminished activation of β1 integrin and lacked organization of fibronectin into fibrillar structures. These findings demonstrate a key role of CD93 in vascular maturation and organization of the extracellular matrix in tumors, identifying it as a potential target for therapy.

Gliomas are brain tumors characterized by an immunosuppressive microenvironment. Immunostimulatory agonistic CD40 antibodies (αCD40) are in clinical development for solid tumors, but are yet to be evaluated for glioma. Here, we demonstrate that systemic delivery of αCD40 in preclinical glioma models induces the formation of tertiary lymphoid structures (TLS) in proximity of meningeal tissue. In treatment-naïve glioma patients, the presence of TLS correlates with increased T cell infiltration. However, systemic delivery of αCD40 induces hypofunctional T cells and impairs the response to immune checkpoint inhibitors in pre-clinical glioma models. This is associated with a systemic induction of suppressive CD11b + B cells post-αCD40 treatment, which accumulate in the tumor microenvironment. Our work unveils the pleiotropic effects of αCD40 therapy in glioma and reveals that immunotherapies can modulate TLS formation in the brain, opening up for future opportunities to regulate the immune response. Agonistic CD40 antibodies (αCD40) have broad immunostimulatory properties, however their efficacy in glioma remains unclear. Here the authors show that αCD40 promotes the formation of tertiary lymphoid structures but does not improve survival and impairs the response to immune checkpoint blockade in murine glioma models.

Chimeric antigen receptor (CAR)-T cell therapy is rapidly advancing as cancer treatment, however, designing an optimal CAR remains challenging. A single-chain variable fragment (scFv) is generally used as CAR targeting moiety, wherein the complementarity-determining regions (CDRs) define its specificity. We report here that the CDR loops can cause CAR clustering, leading to antigen-independent tonic signalling and subsequent CAR-T cell dysfunction. We show via CARs incorporating scFvs with identical framework and varying CDR sequences that CARs may cluster on the T cell surface, which leads to antigen-independent CAR-T cell activation, characterized by increased cell size and interferon (IFN)-γ secretion. This results in CAR-T cell exhaustion, activation-induced cell death and reduced responsiveness to target-antigen-expressing tumour cells. CDR mutagenesis confirms that the CAR-clustering is mediated by CDR-loops. In summary, antigen-independent tonic signalling can be induced by CDR-mediated CAR clustering, which could not be predicted from the scFv sequences, but could be tested for by evaluating the activity of unstimulated CAR-T cells. The challenge of designing chimeric antigen receptor (CAR)-T cells for cancer therapy is not limited to finding targetable cellular proteins, but also in optimising the effector properties. Here authors show that single-chain variable fragment targeting moieties could unpredictably prompt spontaneous CAR-T cell activation via CAR clustering, which argues for empirical screening for tonic signalling.

Tertiary lymphoid structures (TLS) are lymph node-like aggregates that can form in association with chronic inflammation or cancer. Mature TLS are organized into B and T cell zones, and are not encapsulated but include all cell types necessary for eliciting an adaptive immune response. TLS have been observed in various cancer types and are generally associated with a positive prognosis as well as increased sensitivity to cancer immunotherapy. However, a comprehensive understanding of the roles of TLS in eliciting anti-tumor immunity as well as the mechanisms involved in their formation and function is still lacking. Further studies in orthotopic, immunocompetent cancer models are necessary to evaluate the influence of TLS on cancer therapies, and to develop new treatments that promote their formation in cancer. Here, we review key insights obtained from functional murine studies, discuss appropriate models that can be used to study cancer-associated TLS, and suggest guidelines on how to identify TLS and distinguish them from other antigen-presenting niches.

Semliki Forest virus (SFV) is a neuropathogenic alphavirus which is of interest both as a model neurotropic alphavirus and as an oncolytic virus with proven potency in preclinical cancer models. In laboratory mice, peripherally administered SFV infiltrates the central nervous system (CNS) and causes encephalitis of varying severity. The route of SFV CNS entrance is poorly understood but has been considered to occur through the blood-brain barrier. Here we show that neuroinvasion of intravenously administered SFV is strictly dependent on very-low-density-lipoprotein receptor (VLDLR) which acts as an entry receptor for SFV. Moreover, SFV primarily enters the CNS through the blood-cerebrospinal fluid (B-CSF) barrier via infecting choroid plexus epithelial cells which show distinctly high expression of VLDLR. This is the first indication of neurotropic alphavirus utilizing choroid plexus for CNS entry, and VLDLR playing a specific and crucial role for mediating SFV entry through this pathway. The authors present that encephalitic Semliki Forest virus uses choroid plexus epithelial cells as neuroinvasion pathway in mice and show that entry is specifically dependent on expression of very low-density-lipoprotein receptor in these cells.

Background Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model. Methods CD4 + T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery. Results The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs. Conclusion CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

Glioblastoma is the most common and aggressive brain tumor, which is uniformly lethal due to its extreme invasiveness and the absence of curative therapies. Immune checkpoint inhibitors have not yet proven efficacious for glioblastoma patients, due in part to the low prevalence of tumor-reactive T cells within the tumor microenvironment. The priming of tumor antigen-directed T cells in the cervical lymph nodes is complicated by the shortage of dendritic cells and lack of appropriate lymphatic vessels within the brain parenchyma. However, recent data suggest that naive T cells may also be primed within brain tumor-associated tertiary lymphoid structures. Here, we review the current understanding of the formation of these structures within the central nervous system, and hypothesize that promotion of tertiary lymphoid structures could enhance priming of tumor antigen-targeted T cells and sensitize glioblastomas to cancer immunotherapy.

Chimeric antigen receptor (CAR) T‐cell therapy is a new successful treatment for refractory B‐cell leukemia. Successful therapeutic outcome depends on long‐term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long‐term cell engineering method using non‐integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV‐S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV‐S/MAR‐engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19 + target cell recognition as LV‐engineered T cells and are as effective in controlling tumor growth in vivo . We propose that NILV‐S/MAR vectors are superior to current options as they enable long‐term transgene expression without the risk of insertional mutagenesis and genotoxicity. Synopsis CAR T cells for cancer immunotherapy are commonly engineered ex vivo via a viral vector with integration of the transgene and possible tumorigenic risk. A new vector, NILV‐S/MAR, allows long‐term episomal engineering of T cells to reduce such risk. The NILV‐S/MAR vector is maintained as self‐replicating DNA circles in engineered T cells, which are passed on to daughter cells. The NILV‐S/MAR vector is suitable for both long‐term transgene expression and delivery of shRNA for gene silencing. CAR T cells engineered with the NILV‐S/MAR vector exhibit phenotype and function similar to CAR T cells engineered by a conventional lentiviral vector. The NILV‐S/MAR vector can be used for the safe engineering of stem cells as well as induced pluripotent stem cells. Graphical Abstract CAR T cells for cancer immunotherapy are commonly engineered ex vivo via a viral vector with integration of the transgene and possible tumorigenic risk. A new vector, NILV‐S/MAR, allows long‐term episomal engineering of T cells to reduce such risk.