No abstract

T cell function can be compromised during chronic infections or through continuous exposure to tumor antigens by the action of immune checkpoint receptors, such as programmed cell death protein 1 (PD-1). Systemic administration of blocking antibodies against the PD-1 pathway can restore T cell function, and has been approved for the treatment of several malignancies, although there is a risk of adverse immune-related side-effects. We have developed a method for generating gene knockouts in human antigen (Ag)-specific cytotoxic T-Lymphocyte (CTLs) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. Using this method, we generated several transduced CD4+ or CD8+ antigen-specific polyclonal CTL lines and clones, and validated gene modifications of the PD-1 gene. We compared these T-cell lines and clones with control groups in the presence of programmed death-ligand 1 (PD-L1) and observed improved effector functions in the PD1-disrupted cell group. Overall, we have developed a versatile tool for functional genomics in human antigen-specific CTL studies. Furthermore, we provide an alternative strategy for current cell-based immunotherapy that will minimize the side effects caused by antibody blockade therapy.

The maintenance of tight endothelial junctions requires the establishment of proper cell polarity, which includes not only the apicobasal and front-rear polarity but also the left-right (L-R) polarity. The cell possesses an intrinsic mechanism of orienting the L-R axis with respect to the other axes, following a left-hand or right-hand rule, termed cell chirality. We have previously reported that endothelial cells exhibit a clockwise or rightward bias on ring-shaped micropatterns. Now we further characterize the chirality of individual endothelial cells on micropatterns by analyzing the L-R positioning of the cell centroid relative to the nucleus-centrosome axis. Our results show that the centroids of endothelial cells preferably polarized towards the right side of the nucleus-centrosome axis. This bias is consistent with cell chirality characterized by other methods. These results suggest that the positioning of cell organelles is intrinsically L-R biased inside individual cells. This L-R bias provides an opportunity for determining cell chirality in situ, even in vivo, without the limitations of using isolated cells in in vitro engineered platforms.

The cardiovascular system demonstrates left-right (LR) asymmetry: most notably, the LR asymmetric looping of the bilaterally symmetric linear heart tube. Similarly, the orientation of the aortic arch is asymmetric as well. Perturbations to the asymmetry have been associated with several congenital heart malformations and vascular disorders. The source of the asymmetry, however, is not clear. Cell chirality, a recently discovered and intrinsic LR asymmetric cellular morphological property, has been implicated in the heart looping and vascular barrier function. In this paper, we summarize recent advances in the field of cell chirality and describe various approaches developed for studying cell chirality at multi- and single-cell levels. We also examine research progress in asymmetric cardiovascular development and associated malformations. Finally, we review evidence connecting cell chirality to cardiac looping and vascular permeability and provide thoughts on future research directions for cell chirality in the context of cardiovascular development and disease.