Modifying T cell differentiation via protein based delivery of signalling attenuators

Abstract

In recent years, evidence has accumulated that hierarchical thresholds for proliferation and differentiation may determine the generation of various effector T cell modes. It is as yet poorly understood, however, how changes in TCR-mediated signalling and its downstream signalling cascades drive the differentiation of the naive T cell into different response modes. This project aims to study whether manipulation of the signalling pathways directly downstream of the TCR/CD28 receptor can result in distinct functional differentiation of monoclonal CD4+ T cell populations with known antigenic specificity. To this end, two potential protein transfer mechanisms were tested for their suitability to act as inert delivery vehicles to deliver proteins into ex vivo naive T cells. The B subunit pentamer of enterotoxin B induced high levels apoptosis in naive T cells, which precluded its use as an inert delivery vehicle. The protein transduction domain of the HIV-1 protein Tat, however, did not influence T cell physiology and was subsequently successfully tested for its ability to transduce ex vivo T cells genetically fused to large proteins. Two fusion partners, the kinase truncated forms of ZAP-70 and Lck, were tested for their ability to interfere with TCR-mediated signalling. The kinase- truncated form of ZAP-70 showed a concentration dependent effect on calcium mobilisation, which was reliant on the strength of CD3 crosslinking. The kinase- truncated form of Lck also altered calcium mobilisation in a concentration dependent manner, but independent of TCR strength. In addition, it was shown to influence T cell activation, to be capable of altering the signal transduction cascade after T cell priming, and to dramatically change the cytokine profile