A single infusion of engineered long-lived and multifunctional T cells confers durable remission of asthma in mice [scRNA-Seq]

The majority of common chronic human diseases remain incurable, impacting a significant portion of the population and necessitating lifelong treatments that impose a substantial burden on health, the economy, and society. Asthma, the most prevalent respiratory disease, exemplifies this challenge, affecting over 300 million people and causing more than 250,000 deaths annually. Here, we demonstrate the achievement of long-term remission of type 2-high asthma through a single infusion of engineered CAR T cells. By utilizing IL-5 as the targeting domain and depleting BCOR and ZC3H12A, we engineer long-lived CAR T cells designed to eradicate IL-5R+ eosinophils, termed Immortal-like and Functional IL-5 CAR T (5TIF) cells. Furthermore, we enhance 5TIF cells by engineering them to secrete an IL-4 mutein that blocks the signaling of both IL-4 and IL-13, two inflammatory cytokines driving asthma pathology, resulting in the creation of 5TIF4 cells. In multiple asthma models, a single infusion of 5TIF4 cells in fully immunocompetent mice, without any conditioning regimen, leads to long-term depletion of pathological eosinophils and blockade of IL-4/IL-13 actions. This results in sustained repression of type 2 inflammation and alleviation of asthmatic symptoms. Additionally, 5TIF4 cells can be induced in human T cells in NSG mice. These findings demonstrate that asthma, a prevalent and incurable disease, can undergo durable remission through a single infusion of engineered CAR T cells. This breakthrough paves the way for the potential functional cure of chronic noncancerous diseases using long-lived CAR T cells. A single-cell RNA sequencing (scRNA-seq) assay was conducted to comprehensively investigate the phenotype of human 5TIF4 cells. One month after transfer, GFP+ human 5TIF4 cells from recipient mice were sorted by flow cytometry. And the single cell suspensions were directly loaded on a microfluidic chip (Singleron GEXSCOPETM Single Cell RNA-seq Kit, Singleron Biotechnologies) and processed for scRNA-seq library preparation according to manufacturer’s protocol. The ultimate constructed and purified library with the target recovery of ~ 13,000 single cells was sequenced on Illumina novaseq 6000.