Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. While T cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell–inflamed tumor microenvironment are not fully understood. We used an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment (TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified ephrin-A receptor 2 (EPHA2) as a candidate tumor-intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that prostaglandin endoperoxide synthase 2 (PTGS2), the gene encoding cyclooxygenase-2, lies downstream of EPHA2 signaling through TGF-β and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2/PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2/TGF-β/PTGS2 pathway inhibitors with antitumor immunotherapy and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.

The origin and nature of age-associated B cells (ABCs) in mice are poorly understood. In this article, we show that their emergence required MHC class II and CD40/CD40L interactions. Young donor B cells were adoptively transferred into congenic recipients and allowed to remain for 1 mo in the absence of external Ag. B cells expressing the T-bet transcription factor, a marker for ABCs, were generated after multiple cell divisions from C57BL/6 donors but not from MHC class II– or CD40-deficient donors. Furthermore, old CD154 (CD40L)-deficient mice did not accrue ABCs, confirming that they arise primarily through T-dependent interactions. To determine what Igs ABCs express, we sequenced V_H and Vκ rearranged genes from unimmunized 22-mo-old C57BL/6 mice and showed that they had a heterogeneous repertoire, which was comparable to that seen in old follicular and marginal zone B cell subsets. However, in contrast to the follicular and marginal zone cells, ABCs displayed significant somatic hypermutation. The mutation frequency was lower than found in germinal center cells after deliberate immunization, suggesting that ABCs have undergone mild stimulation from endogenous Ags over time. These observations show that quiescent ABCs are Ag-experienced cells that accumulate during T cell–dependent responses to diverse Ags during the life of an individual.

Aberrations in the esophageal proliferation-differentiation gradient are histologic hallmarks in eosinophilic esophagitis (EoE) and gastroesophageal reflux disease. A reliable protocol to grow 3-dimensional (3D) esophageal organoids is needed to study esophageal epithelial homeostasis under physiological and pathologic conditions. We modified keratinocyte-serum free medium to grow 3D organoids from endoscopic esophageal biopsies, immortalized human esophageal epithelial cells, and murine esophagi. Morphologic and functional characterization of 3D organoids was performed following genetic and pharmacologic modifications or exposure to EoE-relevant cytokines. The Notch pathway was evaluated by transfection assays and by gene expression analyses in vitro and in biopsies. Both murine and human esophageal 3D organoids displayed an explicit proliferation-differentiation gradient. Notch inhibition accumulated undifferentiated basal keratinocytes with deregulated squamous cell differentiation in organoids. EoE patient-derived 3D organoids displayed normal epithelial structure ex vivo in the absence of the EoE inflammatory milieu. Stimulation of esophageal 3D organoids with EoE-relevant cytokines resulted in a phenocopy of Notch inhibition in organoid 3D structures with recapitulation of reactive epithelial changes in EoE biopsies, where Notch3 expression was significantly decreased in EoE compared with control subjects. Esophageal 3D organoids serve as a novel platform to investigate regulatory mechanisms in squamous epithelial homeostasis in the context of EoE and other diseases. Notch-mediated squamous cell differentiation is suppressed by cytokines known to be involved in EoE, suggesting that this may contribute to epithelial phenotypes associated with disease. Genetic and pharmacologic manipulations establish proof of concept for the utility of organoids for future studies and personalized medicine in EoE and other esophageal diseases.