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.

Enhanced T-cell activation and differentiation in lymphocytes from transgenic mice expressing ubiquitination-resistant 2KR LAT molecules

“…We had previously shown that exogenous expression of 2KR LAT enhances proximal TCR signaling to a greater extent than expression of WT LAT. 7,8 The levels of TCR signaling are intrinsically linked to intrathymic T-cell development, and alterations in signaling can shift the balance between positive and negative selection, thereby altering the pool of mature T cells. 9 To avoid any differences in development that expression of a 2KR LAT transgene might cause, transgenic mice were generated using the distal Lck promoter, which promotes transgene expression after positive and negative selection are completed.…”

“…7,8 To assess the overall activation of signaling components downstream of the TCR in LAT-expressing transgenic murine T cells, western blot analysis was performed on lysates of purified CD4 + and CD8 + T cells from LN cells. Using phospho-specific antibodies, we found that whereas activation of the upstream kinase ZAP-70 was similar in WT and 2KR LAT cells, there was a prominent increase in the levels of phosphorylated LAT in 2KR LAT-expressing CD4 + and CD8 + T cells (Figures 1a and b).…”

“…Human T cells expressing 2KR LAT and a chimeric antigen receptor or an major histocompatibility complex class I-restricted TCR showed augmented function as assessed by enhanced cytokine secretion and cytotoxicity. 8 These results indicate that disruption of LAT ubiquitination could be a promising strategy to augment T-cell function for adoptive antitumor immunotherapy.…”

Enabling T Cell Recruitment to Tumours as a Strategy for Improving Adoptive T Cell Therapy

“…Enabling T Cell Recruitment to Tumours to Improve Adoptive T Cell Therapy [67][68][69][70][71][72][73] However, the clinical value of these modifications is unclear, as comparative clinical studies have yet to be pursued. As T cells used in ACT are, in general, as sensitive to suppression as endogenous cells, a growing body of evidence points towards the combination of ACT with immune-activating strategies.…”

Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells

“…Compared to T cells that express CAR alone, this modification also results in enhanced antitumour cytolytic activity, accompanied by increased cytokine production. [54]. …”