Most clinically applied cancer immunotherapies rely on the ability of CD8⁺ cytolytic T cells to directly recognize and kill tumour cells^1–3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment^4–6. The ability of CD4⁺ effector cells to contribute to antitumour immunity independently of CD8⁺ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified^7–10. Here, we describe a mechanism whereby a small number of CD4⁺ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8⁺ T cell targeting. The CD4⁺ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II⁺CD11c⁺ antigen-presenting cells. We show that T helper type 1 cell-directed CD4⁺ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4⁺ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4⁺ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8⁺ T cells and natural killer cells and advance cancer immunotherapies.

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.

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.

Chimeric antigen receptor T cells (CAR T cells) are effective against haematologic malignancies. However, in solid tumours, their potency is hampered by local immunosuppression and by the heterogeneous expression of the antigen that the CAR targets. Here we show that CAR T cells expressing a pluripotent pro-inflammatory neutrophil-activating protein (NAP) from Helicobacter pylori trigger endogenous bystander T-cell responses against solid cancers. In mice with subcutaneous murine pancreatic ductal adenocarcinomas, neuroblastomas or colon carcinomas, CAR(NAP) T cells led to slower tumour growth and higher survival rates than conventional mouse CAR T cells, regardless of target antigen, tumour type and host haplotype. In tumours with heterogeneous antigen expression, NAP secretion induced the formation of an immunologically ‘hot’ microenvironment that supported dendritic cell maturation and bystander responses, as indicated by epitope spreading and infiltration of cytotoxic CD8⁺ T cells targeting tumour-associated antigens other than the CAR-targeted antigen. CAR T cells armed with NAP neither increased off-tumour toxicity nor hampered the efficacy of CAR T cells, and hence may have advantageous translational potential.

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.

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.