T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. While all the CD3 subunits possess at least one ITAM, CD3 ε subunit also contains a proline-rich sequence (PRS) and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. Herein, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P2, and PtdIns(3,4,5)P3 with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to stably accumulate at the immunological synapse during T cell-antigen presenting cell interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures following T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.

SUMMARY Most solid tumors are aneuploid, and p53 has been implicated as the guardian of the euploid genome. Previous experiments using human cell lines showed that aneuploidy induction leads to p53 accumulation and p21-mediated G1 cell cycle arrest. We find that adherent 2-dimensional (2D) cultures of human immortalized or cancer cell lines activate p53 upon aneuploidy induction, whereas suspension cultures of a human lymphoid cell line undergo a p53-independent cell cycle arrest. Surprisingly, 3D human and mouse organotypic cultures from neural, intestinal, or mammary epithelial tissues do not activate p53 or arrest in G1 following aneuploidy induction. p53-deficient colon organoids have increased aneuploidy and frequent lagging chromosomes and multipolar spindles during mitosis. These data suggest that p53 may not act as a universal surveillance factor restricting the proliferation of aneuploid cells but instead helps directly or indirectly ensure faithful chromosome transmission likely by preventing polyploidization and influencing spindle mechanics.

Tissue-engineered small intestinal implants are being widely investigated as a potential treatment for children with short bowel syndrome, yet are currently limited by their growth potential and relatively low surface area. To address this gap in the field, several investigators have utilized whole organ decellularization of the small intestine as a platform for subsequent growth of intestinal tissue. However, such scaffold-cell constructs require sterilization as a prerequisite for implantation, and the effects of the different pathogen-clearance techniques used on the tissue architecture remains unknown. The effects of four different published protocols for pathogen clearance of decellularized intestine, namely 0.1% peracetic acid (PAA), 0.18% PAA +4.8% ethanol (EtOH), 0.08% PAA +1% hydrogen peroxide (H 2 O 2 ), and ultraviolet (UV) sterilization were compared using qualitative and quantitative techniques to assess changes to the extracellular matrix, cytocompatibility, and biocompatibility. All methods of sterilization of decellularized intestine were found to be equally effective and each method had similar histologic and scanning electron microscopy appearance of the sterilized tissue. In addition, collagen and glycosaminoglycan quantities, and the ability to support cell growth were similar among all methods. This study provides insights into the change in crypt villous architecture of the extracellular matrix with all sterilization techniques studied. Our findings demonstrate that sterilization affects the microarchitecture significantly, which has not been well accounted for in studies to date, and we were unable to identify a single best agent to achieve tissue sterilization while preserving the microarchitectural features of the tissue.

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