Preventing trogocytosis by cathepsin B inhibition augments CAR T cell function.

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in cancer treatment. Still, most patients receiving CAR T cells relapse within 5 years of treatment. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism in which cell surface proteins transfer from tumor cells to CAR T cells. CMT results in the emergence of antigen-negative tumor cells, which can evade future CAR detection, and antigen-positive CAR T cells, which is hypothesized to lead to CAR T cell fratricide and dysfunction. Using a system to selectively degrade trogocytosed antigen in CAR T cells, we show that the presence of trogocytosed antigen in CAR T cells directly causes CAR T cell fratricide and exhaustion. By performing a small molecule screening using a custom high throughput CMT-screening assay, we identified the cysteine protease cathepsin B (CTSB) as a key driver of CMT. We show that overexpression of cystatin A (CSTA), an endogenous human inhibitor of CTSB, reduces trogocytosis resulting in prolonged antitumor activity and increased CAR T cell expansion/persistence. Overall, we show that targeting CMT is an effective approach to enhance CAR T cell function, which may improve their clinical efficacy.

Estimating the localization spread function of static single-molecule localization microscopy images.

Single-molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM data set is an essential aspect of the image processing and analysis pipeline. Here, we present a simple method to estimate the localization spread function (LSF) of a static SMLM data set directly from acquired localizations, exploiting the correlated dynamics of individual emitters and properties of the pair autocorrelation function evaluated in both time and space. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labeled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. We show that experimentally obtained images have LSFs that are broader than expected from the localization precision alone, due to additional uncertainty accrued when localizing molecules imaged over time.

A mean shift algorithm for drift correction in localization microscopy.

Single-molecule localization microscopy techniques transcend the diffraction limit of visible light by localizing isolated emitters sampled stochastically. This time-lapse imaging necessitates long acquisition times, over which sample drift can become large relative to the localization precision. Here, we present an efficient and robust method for estimating drift, using a simple peak-finding algorithm based on mean shifts that is effective for single-molecule localization microscopy in two or three dimensions.