KO of A20 drove a proinflammatory state resistant to suppressive repolarization and enhanced cytotoxicity in HER2-CAR macrophages. With TNFAIP3 surfacing as a brake on antitumor function now in both T cells and macrophages, it may be an attractive target for cell therapies.

Our screens converged on the gene TNFAIP3, which codes for the protein A20, as a master regulator of macrophage inflammatory polarization.

To make pooled screening possible we developed SLICeVLP, sgRNA delivery by lentivirus with Cas9 delivery by eVLPs. We used it for pooled loss-of-function screens and Perturb-seq in primary human macrophages, identifying regulators of inflammatory signaling.

This platform supports knockout, base editing, and epigenetic silencing, and paired with AAV donor delivery it enables site-specific integration of large sequences by homology-directed repair, even in these post-mitotic cells.

@HyuncheolJung and I adapted these VLP-based methods to deliver CRISPR ribonucleoproteins to primary human monocytes, macrophages, and dendritic cells at high efficiency while preserving both viability and innate immune responsiveness.

Big 'Thank you' to @SavvidesLab @DanielleMClancy for writing this thoughtful spotlight @ImmunityCP on our recent work on the 'The pros and con(formation)s of IL-18 activation' doi.org/10.1016/j.immuni.202… cell.com/immunity/abstract/S… nature.com/articles/s41586-0… nature.com/articles/s41586-0…

17/ When comparing human caspase-4 to mouse caspase-11, we found that critical residues in the interfaces (e.g. K356 and K293) are not conserved between the two homologs. Introduction of human-specific mutations at these sites greatly enhanced pro-IL-18 cleavage by caspase-11.

16/ We found that caspase-bound pro-IL-18 adopts a conformation that is distinct from mature IL-18: the pro-domain inserts itself into the mature domain to form active- and exosite. Cleavage may then trigger a structural change to mediate release of mature IL-18 from caspase-4

14/ We confirmed by site-directed mutagenesis that both interfaces need to be intact for caspase-4 to bind and cleave pro-IL-18 efficiently (both in vitro and in cells).

13/ The active site interface on the other hand is dominated by electrostatic interactions, which perfectly orient the the pro-domain of pro-IL-18 and the tetrapeptide motif in in the catalytic center of caspase-4 for cleavage.