Kendall, Benjamin L.

Abstract T-cell immune tolerance is established in part through the activity of the autoimmune regulator (AIRE) transcription factor in medullary thymic epithelial cells (mTEC) of the thymus. AIRE induces expression of peripheral tissue–specific self-antigens for presentation to naïve T cells to promote activation/deletion of autoreactive T cells. This traditional role of AIRE in mTECs is to prevent autoimmunity. Herein, we demonstrate that tumors mimic the role of AIRE in mTECs to evade immune rejection. We found that AIRE induced a profile of “selfness” at the RNA and protein levels which, when presented on major histocompatibility complexes (MHC), shielded the tumor from inherently self-tolerized T cells. Moreover, we describe an in vivo immunotherapy in which engineered changes in AIRE expression in tumor cells altered their profile of selfness, exposing both AIRE-modified and parental unmodified tumor cells to T-cell attack. Therefore, by resetting the immunologic selfness of cancer cells, this AIRE-mediated immunotherapy (i) converted a highly tolerized T-cell compartment into a tumor-reactive T-cell population; (ii) conferred upon nonimmunogenic tumors de novo sensitivity to immune checkpoint blockade; (iii) removed the need to identify potentially immunogenic tumor-associated antigens as targets for generation of T-cell responses; and (iv) lead to potent T cell–mediated rejection of aggressive, immunologically cold, nonimmunogenic tumors. Patient RNA sequencing data showed that the expression of AIRE predicted response to immune therapies with a strong correlation between AIRE expression and markers of T-cell receptor signaling, suggesting that our studies have therapeutic translational value.

Chimeric antigen receptor (CAR) T cell therapy faces many challenges against solid tumors including T cell exhaustion and poor CAR durability. Here, we show that engaging the CAR T cell endogenous T cell receptor (TCR) using an oncolytic virus enhances CAR T cell functionality, durability, and therapy. Upon combination therapy of solid tumors with CAR T cells and vesicular stomatitis virus (VSV), a subpopulation of antiviral, TCR-primed CAR T cells was generated with enhanced effector functions, altered activation states, and differential gene and protein expression when compared to non–TCR-primed CAR T cells. Single-cell RNA sequencing showed clonal expansion of anti-VSV CAR T cells and enhancement of effector-associated genes with VSV-mediated CAR T cell expansion. CD4 T cells played a pivotal role in the development of these TCR-primed CAR T cells. These results provide a strong rationale both for a novel use of systemic oncolytic virotherapy and for directly exploiting the CAR T cell TCR to fine tune the CAR T cell phenotype and function.

Abstract Most oncolytic viruses are delivered by intratumoural injection, and local administration can induce regression of both injected and distant tumours in mice and patients. However, the mechanisms by which local viral infection reprograms systemic immunity remain poorly understood. Here we show that intratumoural RP1, an oncolytic HSV-1 encoding GM-CSF and GALV-GP-R ⁻ , drives regression of injected and uninjected murine melanoma tumours and prolongs survival. RP1 elicits coordinated CD4 ⁺ and CD8 ⁺ T cell infiltration accompanied by local cytokine remodelling, reshaping the immune landscape at both tumour sites. Leveraging the Timer of Cell Kinetics and Activity (Tocky) system and Kaede photoconvertible protein technology, we resolve the temporal dynamics of CD8 ⁺ T cell responses following local virotherapy and identify two systemically induced, virus-driven CD8 ⁺ T cell populations distinguished by TCR engagement kinetics: antigen-engaged Timer-positive ‘viral-induced precursors’ (VIPs) and Timer-negative KLRG1 ⁺ ‘viral-induced terminal effectors’ (VITEs). Single-cell transcriptomic and pseudotime analyses reveal divergent differentiation trajectories; VIPs exhibit sustained antigen engagement within the tumour microenvironment (TME), a transcriptional programme associated with self-renewal, and preferential homing to draining lymph nodes. A VIP-associated gene signature correlates with clinical response to RP1 plus PD-1 blockade in the IGNYTE trial of RP1 and nivolumab in PD-1-refractory melanoma and is independently associated with response to immune checkpoint inhibitor (ICI) therapy in melanoma. These findings establish a mechanistic link between local oncolytic virotherapy, systemic CD8 ⁺ T cell reprogramming, and durable regression of distant lesions in patients.

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mitochondrial DNA (mtDNA) and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro . Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci as an example of near-complete induction of mtDNA heteroplasmy in vivo . Gene editing specificity as precise as a single nucleotide was observed for a protein-coding gene. Nondestructive genotyping enables single-animal mtDNA analyses for downstream biological functional genomic applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

Abstract The paradigm in the field of oncolytic virotherapy proposes that tumor cell killing by an oncolytic virus (OV) culminates in the priming of antitumor CD8 T cells. However, this ignores the impact a highly immunodominant antiviral response against the OV has on the antitumor response which has been weakened by mechanisms of central tolerance. Here, we show that inflammatory Vesicular Stomatitis Virus (VSV) failed to prime an adoptively transferred, or pre-existing, population of tumor-reactive T cells.Combination with αPD1 immune checkpoint blockade therapy improved survival only when VSV expressed tumor associated antigens (TAA). These data show that, in this model, the highly inflammatory OV VSV alone actively outcompetes antitumor immunity. However, we also show that viral expression of a mutant near-self TAA can break central tolerance expanding heteroclitic self-reactive and near-self-reactive T cells, thus overcoming viral immunodominance by promoting tumor-specific T cell proliferation in parallel with expanding antiviral T cells.

Abstract T cell immune tolerance is established in part through the activity of the Auto-immune Regulator (AIRE) transcription factor in the medullary Thymic Epithelial Cells (mTEC) of the thymus. AIRE induces expression of SELF peripheral tissue-specific antigens for presentation to naïve T cells to promote activation/deletion of potentially autoreactive T cells. We show, for the first time to our knowledge, that tumors mimic the role of AIRE in mTEC to evade immune rejection. Thus, by expressing a broad range of SELF epitopes against which minimal functional T cell reactivities exist because of thymic deletion, AIRE acts as a master controller of SELFNESS, effectively cloaking the tumor from T cell attack. Moreover, we describe a completely novel immunotherapy in which engineered changes in AIRE expression in tumor cells alters their profile of SELFNESS, exposing both AIRE-modified, and parental unmodified, tumor cells to T cell attack. Consistent with our studies, patient RNAseq shows expression of AIRE predicts response to immune therapies with a strong correlation between AIRE expression and markers of TCR signaling. Therefore, by re-setting the immunological SELFNESS of cancer cells, this novel AIRE-mediated immunotherapy 1). converts a highly tolerized T cell compartment into a heteroclitic tumor-reactive T cell population; 2) confers de novo sensitivity to immune checkpoint blockade upon non-immunogenic tumors; 3). completely removes the need to identify potentially immunogenic tumor-associated antigens as targets for generation of de novo CD8+ and helper CD4+ T cell responses; and 4) leads to potent T cell-mediated rejection of aggressive, immunologically cold, non-immunogenic tumors.

Repurposing FDA-approved drugs that treat respiratory infections caused by coronaviruses, such as SARS-CoV-2 and MERS-CoV, could quickly provide much needed antiviral therapies. In the current study, the potency and cellular toxicity of four fluoroquinolones (enoxacin, ciprofloxacin, levofloxacin, and moxifloxacin) were assessed in Vero cells and A549 cells engineered to overexpress ACE2, the SARS-CoV-2 entry receptor. All four fluoroquinolones suppressed SARS-CoV-2 replication at high micromolar concentrations in both cell types, with enoxacin demonstrating the lowest effective concentration 50 value (EC50) of 126.4 μM in Vero cells. Enoxacin also suppressed the replication of MERS-CoV-2 in Vero cells at high micromolar concentrations. Cellular toxicity of levofloxacin was not found in either cell type. In Vero cells, minimal toxicity was observed following treatment with ≥37.5 μM enoxacin and 600 μM ciprofloxacin. Toxicity in both cell types was detected after moxifloxacin treatment of ≥300 μM. In summary, these results suggest that the ability of fluoroquinolones to suppress SARS-CoV-2 and MERS-CoV replication in cultured cells is limited.