ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
T cell activation is initiated upon ligand engagement of the T cell receptor (TCR) and costimulatory receptors. The CD28 molecule acts as a major costimulatory receptor in promoting full activation of naive T cells. However, despite extensive studies, why naive T cell activation requires concurrent stimulation of both the TCR and costimulatory receptors remains poorly understood. Here, we explore this issue by analyzing calcium response as a key early signaling event to elicit T cell activation. Experiments using mouse naive CD4+ T cells showed that engagement of the TCR or CD28 with the respective cognate ligand was able to trigger a rise in fluctuating calcium mobilization levels, as shown by the frequency and average response magnitude of the reacting cells compared with basal levels occurred in unstimulated cells. The engagement of both TCR and CD28 enabled a further increase of these two metrics. However, such increases did not sufficiently explain the importance of the CD28 pathways to the functionally relevant calcium responses in T cell activation. Through the autocorrelation analysis of calcium time series data, we found that combined but not separate TCR and CD28 stimulation significantly prolonged the average decay time (τ) of the calcium signal amplitudes determined with the autocorrelation function, compared with its value in unstimulated cells. This increasement of decay time (τ) uniquely characterizes the fluctuating calcium response triggered by concurrent stimulation of TCR and CD28, as it could not be achieved with either stronger TCR stimuli or by co-engaging both TCR and LFA-1, and likely represents an important feature of competent early signaling to provoke efficient T cell activation. Our work has thus provided new insights into the interplay between the TCR and CD28 early signaling pathways critical to trigger naive T cell activation.
Subject(s)
CD28 Antigens/metabolism , CD4-Positive T-Lymphocytes/immunology , Calcium Signaling/immunology , Lymphocyte Activation , Receptors, Antigen, T-Cell/metabolism , Animals , Antigen-Presenting Cells , CD28 Antigens/immunology , CD4-Positive T-Lymphocytes/metabolism , COS Cells , Cells, Cultured , Chlorocebus aethiops , Coculture Techniques , Lymphocyte Function-Associated Antigen-1/immunology , Lymphocyte Function-Associated Antigen-1/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred CBA , Mice, Transgenic , Primary Cell Culture , Receptors, Antigen, T-Cell/immunologyABSTRACT
Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.