title: Enhanced function of redirected human T cells expressing linker for activation of T cells that is resistant to ubiquitylation.
abstract: It is likely that the enhancement of signaling after antigenic stimulation, particularly in the tumor microenvironment, would improve the function of adoptively transferred T cells. Linker for activation of T cells (LAT) plays a central role in T cell activation. We hypothesized that the ubiquitylation-resistant form of LAT in cells would enhance T cell signaling and thus augment antitumor activity. To test this, human CD4(+) or CD8(+) T cells were electroporated with small interfering RNA (siRNA) to repress endogenous LAT and ubiquitylation-resistant LAT 2KR or wild-type LAT mRNA was introduced for reexpression. Significantly enhanced phosphorylation of LAT and phospholipase C-γ (PLCγ) was observed, and augmented calcium signaling after T cell receptor (TCR) triggering was observed in LAT 2KR-expressing T cells. TCR-induced calcium signaling was abrogated in LAT knockdown cells, but the baseline was higher than that of control siRNA-electroporated cells, suggesting a fundamental requirement of LAT to maintain calcium homeostasis. Redirected LAT 2KR T cells expressing a chimeric antigen receptor or an MHC class I-restricted TCR showed augmented function as assessed by enhanced cytokine secretion and cytotoxicity. These results indicate that interruption of LAT ubiquitylation is a promising strategy to augment effector T cell function for adoptive cell therapy.
authors.affiliation: Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-5156, USA.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kunii
authors.forename: Yangbing
authors.initials: Y
authors.lastname: Zhao
authors.forename: Shuguang
authors.initials: S
authors.lastname: Jiang
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Liu
authors.forename: John
authors.initials: J
authors.lastname: Scholler
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Balagopalan
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Samelson
authors.forename: Michael C
authors.initials: MC
authors.lastname: Milone
authors.forename: Carl H
authors.initials: CH
authors.lastname: June
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D019264:Adoptive Transfer / Q000379:methods*; D020013:Calcium Signaling / Q000276:immunology*; D018274:Electroporation; D055785:Gene Knockdown Techniques; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009369:Neoplasms / Q000276:immunology*; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C110810:LAT protein, human; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D011948:Receptors, Antigen, T-Cell; D051966:Phospholipase C gamma