CD19-CAR T cells undergo exhaustion DNA methylation programming in patients with acute lymphoblastic leukemia.

CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acute lymphoblastic leukemia (ALL); however, limited persistence of the CAR T cells enables tumor relapse for many patients. To gain a deeper understanding of the molecular characteristics associated with CAR T cell differentiation, we performed longitudinal genome-wide DNA methylation profiling of CD8 CD19-CAR T cells post-infusion in ALL patients. We report that CAR T cells undergo a rapid and broad erasure of repressive DNA methylation reprograms at effector-associated genes.

Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity.

Chimeric antigen receptor (CAR) T cell therapy is revolutionizing cancer immunotherapy for patients with B cell malignancies and is now being developed for solid tumors and chronic viral infections. Although clinical trials have demonstrated the curative potential of CAR T cell therapy, a substantial and well-established limitation is the heightened contraction and transient persistence of CAR T cells during prolonged antigen exposure. The underlying mechanism(s) for this dysfunctional state, often termed CAR T cell exhaustion, remains poorly defined.

Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 T cell effector differentiation.

To gain insight into the signaling determinants of effector-associated DNA methylation programming among CD8 T cells, we explore the role of interleukin (IL)-12 in the imprinting of IFNg expression during CD8 T cell priming. We observe that anti-CD3/CD28-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promoter. However, anti-CD3/CD28 stimulation in the presence of the inflammatory cytokine, IL-12, results in stable demethylation of the IFNg locus that is commensurate with IFNg expression.

In vivo electroporation of E1 chick embryos.

In ovo electroporation of chick embryos at ages ≥ E2 is simple to conduct and widely used to manipulate gene function. However, in ovo electroporation at early E1 stages has so far been unsuccessful because of unacceptable levels of tissue damage and embryonic lethality. Early E1 manipulations in the chick have therefore relied on in vitro electroporation, posing problems for morphogenetic studies in which the long-term preservation (>24 h) of three-dimensional tissue organization is critical.

A novel role for FOXA2 and SHH in organizing midbrain signaling centers.

The floor plate (FP) is a midline signaling center, known to direct ventral cell fates and axon guidance in the neural tube. The recent identification of midbrain FP as a source of dopaminergic neurons has renewed interest in its specification and organization, which remain poorly understood. In this study, we have examined the chick midbrain and spinal FP and show that both can be partitioned into medial (MFP) and lateral (LFP) subdivisions.

A simple technique for early in vivo electroporation of E1 chick embryos.

Background: The amenability of the chick embryo to a variety of manipulations has made it an ideal experimental model organism for over 100 years. The ability to manipulate gene function via in ovo electroporations has further revolutionized its value as an experimental model in the last 15 years. Although in ovo electroporations are simple to conduct in embryos ≥ E2, in ovo electroporations at early E1 stages have proven to be technically challenging due to the tissue damage and embryonic lethality such electroporations produce.