No abstract

T cell-based therapies have induced cancer remissions, though most tumors ultimately progress, reflecting inherent or acquired resistance including antigen escape. Better understanding of how T cells eliminate tumors will help decipher resistance mechanisms. We used a CRISPR/Cas9 screen and identified a necessary role for Fas-FasL in antigen-specific T-cell killing. We also found that Fas-FasL mediated off-target "bystander" killing of antigen-negative tumor cells. This localized bystander cytotoxicity enhanced clearance of antigen-heterogeneous tumors in vivo, a finding that has not been shown previously. Fas-mediated on-target and bystander killing was reproduced in chimeric antigen receptor (CAR-T) and bispecific antibody T-cell models and was augmented by inhibiting regulators of Fas signaling. Tumoral FAS expression alone predicted survival of CAR-T-treated patients in a large clinical trial (NCT02348216). These data suggest strategies to prevent immune escape by targeting both the antigen expression of most tumor cells and the geography of antigen-loss variants.SigNifiCANCe: This study demonstrates the first report of in vivo Fas-dependent bystander killing of antigen-negative tumors by T cells, a phenomenon that may be contributing to the high response rates of antigen-directed immunotherapies despite tumoral heterogeneity. Small molecules that target the Fas pathway may potentiate this mechanism to prevent cancer relapse. intRoductionT cell-based immunotherapies-including adoptive transfer of engineered T cells, bispecific antibodies, and checkpoint blockade-have revolutionized cancer treatment. However, even with the remarkably high response rates of chimeric antigen receptor (CAR)-T-treated patients, most either progress or relapse within one year (1-3). Microenvironmental factors contributing to T-cell priming (4-6) and T cell-intrinsic factors (7, 8) both influence antitumor immunity, but tumor cell-intrinsic factors have the most abundant clinical evidence for contributing to treatment potency and failures.The clearest such mechanism is target antigen (Ag) modulation-expression downregulation, lineage switching, or emergence of splice variants-which is the most common cause of relapse following CAR-T therapy for B-cell acute

Although individual cells vary in behavior during the formation of tissues, the nature of such variations are largely uncharacterized. Here, we tracked the morphologies and motilities of ∼300 human endothelial cells from an initial dispersed state to the formation of capillary-like structures, distilling the dynamics of tissue morphogenesis into an array of ∼36,000 numerical phenotypes. Quantitative analysis of population averages revealed two previously unidentified phases in which the cells spread before forming connections with neighboring cells and where the microvascular plexus stabilized before spatially reorganizing. Analysis at the single-cell level showed that in contrast to the population-averaged behavior, most cells followed distinct temporal patterns that were not reflected in the bulk average. Interestingly, some of these behavioral patterns correlated to the cells' final structural role within the plexus. Knowledge of how individual cells or groups of cells behave enhances our understanding of how native tissues self-organize and could ultimately enable more precise approaches for engineering tissues and synthesizing multicellular communities.vasculogenesis | systems biology | single-cell tracking | multispectral fluorescence microscopy | variability V ariations in the behaviors of individual cells during the morphogenesis of human tissues are likely to be important in shaping the evolution of multicellular structures (1). Knowledge of how individual cells behave and self-organize in native systems are also important in the design of synthetic systems, such as engineered tissues (1) and complex multicellular communities (2, 3). By contrast, population-averaged measurements, although widely used, are the end results of a large number of possible underlying statistical distributions and mask critical cell-to-cell variations (4). For example, the same population-averaged measurement could reflect either all cells behaving close to the average or the sum of many unique cellular behaviors. For the morphogenesis of a human tissue, the extent of cell-to-cell variations has thus far not been systematically studied and is currently largely unknown.Here, we directly tracked the behavior of every individual primary endothelial cell during the early stages of formation of human microvascular structures. Formation of microvessels is central to the etiology of many diseases (5) and critical for vascularizing newly engineered tissues for regenerative medicine (6). Formation of new capillaries, the smallest of the microvessels, can take place via vasculogenesis-the de novo formation of vascular networks from dispersed endothelial cells-both during prenatal development and in adults. For nearly three decades, microvasculogenesis has been studied by using well-established in vitro models (7,8). In particular, beginning with Folkman and up to recent studies (7, 9, 10), soft gels such as Matrigel have remained the most well-established in vitro system for controllably studying the initial steps of microvasculogenesis...

While the cellular origin of lymphoma is often characterized by chromosomal translocations and other genetic aberrations, its growth and development into a malignant neoplasm is highly dependent upon its ability to escape natural host defenses. Neoplastic cells interact with a variety of non-malignant cells in the tumor milieu to create an immunosuppressive microenvironment. The resulting functional impairment and dysregulation of tumor-associated immune cells not only allows for passive growth of the malignancy but may even provide active growth signals upon which the tumor subsequently becomes dependent. In the past decade, the success of immune checkpoint blockade and adoptive cell transfer for relapsed or refractory lymphomas has validated immunotherapy as a possible treatment cornerstone. Here, we review the mechanisms by which lymphomas have been found to evade and even reprogram the immune system, including alterations in surface molecules, recruitment of immunosuppressive subpopulations, and secretion of anti-inflammatory factors. A fundamental understanding of the immune evasion strategies utilized by lymphomas may lead to better prognostic markers and guide the development of targeted interventions that are both safer and more effective than current standards of care.

Immunotherapies directly enhancing anti-tumor CD8+ T cell responses have yielded measurable but limited success, highlighting the need for alternatives. Anti-tumor T cell responses critically depend on antigen presenting dendritic cells (DC), and enhancing mobilization, antigen loading and activation of these cells represent an attractive possibility to potentiate T cell based therapies. Here we show that expansion of DCs by Flt3L administration impacts in situ vaccination with oncolytic Newcastle Disease Virus (NDV). Mechanistically, NDV activates DCs and sensitizes them to dying tumor cells through upregulation of dead-cell receptors and synergizes with Flt3L to promote anti-tumor CD8+ T cell cross-priming. In vivo, Flt3L-NDV in situ vaccination induces parallel amplification of virus- and tumor-specific T cells, including CD8+ T cells reactive to newly-described neoepitopes, promoting long-term tumor control. Cross-presenting conventional Type 1 DCs are indispensable for the anti-tumor, but not anti-viral, T cell response, and type I IFN-dependent CD4+ Th1 effector cells contribute to optimal anti-tumor immunity. These data demonstrate that mobilizing DCs to increase tumor antigen cross-presentation improves oncolytic virotherapy and that neoepitope-specific T cells can be induced without individualized, ex vivo manufactured vaccines.