Here we report the potential of modified LAT to improve the effector functions of primary postthymic human T cells. In one study Balagopalan and colleagues (2011
) showed that expression of ubiquitylation-resistant LAT 2KR increased signaling in human T leukemia cells by two mechanisms: more LAT was expressed and LAT 2KR was more potent in a molecule-to-molecule comparison. We find here that electroporation of mRNA results in efficient expression of LAT 2KR and that this results in increased signaling and function of CD4+
human T cells. We found that in primary T cells, the phosphorylation of wild-type LAT was rapid in onset and transient, whereas phosphorylation of 2KR LAT was augmented and sustained. Because phosphorylation and ubiquitylation reactions occur within seconds (; and Pierce et al.
), it is likely that once phosphorylated, wild-type LAT becomes rapidly ubiquitylated. Ubiquitylation likely leads to a change in LAT localization, dephosphorylation, and finally degradation. In contrast, phosphorylated 2KR LAT persists because it is resistant to ubiquitylation.
Stimulation of CD4+
T cells that express LAT 2KR induced a pattern of cytokine secretion consistent with Th1 differentiation, whereas cytokine secretion in LAT knockdown cells was consistent with a Th2 pattern of differentiation. Signaling through the TCR can regulate Th1 and Th2 differentiation (Constant et al.
; Nakayama and Yamashita, 2010
). Previous studies have shown that LAT has a role in T cell development in the balance of Th1 and Th2 differentiation (Malissen et al.
We also found that expression of LAT 2KR increased the activation of CD8+ T cells after tumor antigen presentation. It was notable that cytotoxicity was enhanced in LAT 2KR cells, and that this augmentation was observed not only after antigen presentation to MHC class I-restricted TCRs but also after antigen stimulation of T cells expressing chimeric antigen receptors. The enhanced cytotoxicity was specific for T cells expressing the ubiquitin-resistant form of LAT.
An unexpected finding in our studies was that LAT knockdown T cells had an elevated basal level of intracellular calcium concentration. This suggests that one function of LAT is to negatively regulate calcium homeostasis in quiescent cells. Others have previously reported that LAT can have negative regulatory function in mouse T cells (Mingueneau et al.
). In lymph nodes, there is evidence of continuous “tonic” signaling through the TCR, and that this is required to maintain antigen responsiveness (Hochweller et al.
). Consistent with a role for LAT in basal T cell signaling, imaging studies have shown that membrane domains of both LAT and TCRζ significantly overlap under both activating and nonactivating conditions (Sherman et al.
). Our studies suggest that LAT may play a role in regulating the tonic resting state of the lymphocyte (Hochweller et al.
) as well as the activated state during antigen stimulation (Lillemeier et al.
In these initial studies we have used transient expression of modified LAT to evaluate the potential of LAT 2KR for potential clinical applications. This would provide safety but may limit the potency in the clinic. Further studies will be required to evaluate the safety and efficacy of engineered T cells that are permanently modified to express LAT 2KR. Thus a limitation of the present studies is that the safety of this approach with constitutive expression of ubiquitin-resistant LAT remains unknown. Another limitation of this approach is that it is possible that augmented LAT signaling could result in the induction of activation-induced cell death (AICD). Our results show that the enhanced signaling mediated by LAT does not decrease proliferation during the first 3 days of stimulation; however, it is possible that during longer term stimulation this could result in the induction of AICD and impaired proliferation of LAT-engineered T cells in vivo.
Ubiquitylation-resistant LAT is a novel approach to enhance T cell signaling and has the potential to augment effector T cell function for adoptive cell therapy. We found that the function of T cells expressing an MHC class I-restricted TCR was enhanced by ubiquitin-resistant LAT. It is possible that this approach might improve the effector functions of T cells expressing low-affinity TCRs that are typical of many tumor-associated antigens (Kessels et al.
; Zehn et al.
). Last, the combination of an antigen-specific CAR with ubiquitylation-resistant LAT could be considered an example of a “next-generation CAR” that has promise to further enhance the effector functions of engineered T cells for cancer and chronic viral infections.