Abstract
Activation of CD28 on T lymphocytes initiates a cascade of intracellular events, which in concert with activation of the T cell receptor, culminates in production of cytokines and a functional immune response. One of the earliest biochemical changes observed following stimulation of CD28 is tyrosine phosphorylation. We have demonstrated that both the LCK and the EMT/ITK/TSK (EMT) intracellular tyrosine kinases are activated following cross-linking of CD28. Utilizing somatic cell mutants lacking LCK, we demonstrate that functional LCK is required for CD28-induced activation of EMT as evidenced by increased tyrosine phosphorylation and kinase activity. In support of a role for LCK in EMT activation, reconstitution of a LCK-negative Jurkat T cell line by transfection with normal LCK recreates CD28-mediated EMT activation. Furthermore, co-transfection of LCK and EMT into COS-7 cells showed that EMT becomes phosphorylated in the presence of LCK. In addition, increases in EMT association with CD28 were eliminated in a LCK-negative Jurkat cell line, but were restored following transfection of wild type LCK. The data are most compatible with a model in which LCK, either directly or indirectly, initiates EMT activation and association with CD28 following ligation of CD28.
Highlights
Co-stimulation of T lymphocytes requires the cooperation of two signals delivered by antigen presenting cells: one stimulatory signal derived from interaction of the T cell receptor (TCR)1 complex with antigen in the context of the major histocompatibility complex and a second co-stimulatory signal from the ligation of accessory molecules [1]
To test whether LCK expression is required for optimal activation of EMT by CD28, we measured the level of EMT activation, as assessed by tyrosine phosphorylation and kinase activity, after CD28 cross-linking in the JCaM 1.6 Jurkat T cell line that does not express LCK
CD28-induced tyrosine phosphorylation of EMT was readily detectable in parental Jurkat cells (Fig. 1, panels a and d) [21], no CD28-induced increase in EMT phosphorylation was detected in JCaM 1.6 cells (Fig. 1, panels b and d)
Summary
Co-stimulation of T lymphocytes requires the cooperation of two signals delivered by antigen presenting cells: one stimulatory signal derived from interaction of the T cell receptor (TCR) complex with antigen in the context of the major histocompatibility complex and a second co-stimulatory signal from the ligation of accessory molecules [1]. It is clear that cross-linking of CD28 can induce a number of early signals, the role that activation of these biochemical changes plays in the ability of CD28 to synergize with the TCR to induce a functional T cell response remains unclear. The exact function of this domain is currently unknown, but it may play a role in the ability of BTK and EMT to associate with other molecules such as protein kinase C [23, 24]. Since cross-linking mouse Fc⑀RI leads to activation of BTK [28], this kinase may play a role in mast cell activation
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