Real-Time Analysis of Calcium Signals during the Early Phase of T Cell Activation Using a Genetically Encoded Calcium Biosensor

Abstract

Proper T cell activation is promoted by sustained calcium signaling downstream of the TCR. However, the dynamics of calcium flux following stimulation with an Antigen Presenting Cell in vivo remain to be fully understood. Previous studies focusing on T cell motility suggested that the activation of naïve T cells in the lymph node occurs in distinct phases. In Phase I, T cells make multiple transient contacts with dendritic cells before entering a Phase II, where they exist in stable clusters with DCs. It has been suggested that T cells signal during transient contacts of Phase I, but this has never been shown directly. Since time dependent loss of calcium dyes from cells hamper long-term imaging of cells in vivo following antigenic stimulation, we generated a knock-in mouse expressing the mCameleon FRET reporter for intracellular calcium and examined calcium flux both in vitro and in situ. In vitro we observed transient, oscillatory, and sustained calcium flux following contact with APC but these behaviors were not affected by the type of APC or antigen quantity, but was however moderately dependent on antigen quality. In vivo, we found that during Phase I, T cells exhibit weak calcium fluxes and detectable changes in cell motility. This demonstrates that naïve T cells signal during Phase I and support the hypothesis that accumulated calcium signals are required to signal the beginning of Phase II.

CD45 pre-exclusion from the tips of microvilli establishes a phosphatase-free zone for early TCR triggering

“…The Ca 2+ signals often fluctuated during the microvilli-mediated contacts. Thus, the Ca 2+ flux oscillations previously observed during initial T cell-APC contacts (Le Borgne et al, 2016) were likely due to the brief early contacts made by microvilli, which could not be resolved in the previous study.…”

Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8+ T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei

“…Single-cell calcium imaging of OT-I T cells stimulated with varying doses of OVA peptide on B cell APCs show that reducing the peptide dose increases the proportion of nonresponding cells in the population but, importantly, has no effect on the magnitude of the peak calcium signal in the responding subset ( 16 ). Bone marrow-derived macrophages pulsed with varying doses of OVA peptide also induce calcium responders and nonresponders within a stimulated naive OT-I cell population ( 17 ). These data are consistent with our NFAT1 translocation data and together indicate that the all-or-none calcium response in turn produces an all-or-none response of NFAT1 activation.…”

In vivo multiphoton imaging of immune cell dynamics

“…Intracellular calcium increases [39, 40, 51, 63, 71, 83], nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT) [41, 43, 56], and caspase 3 activation during apoptosis [15] were visualized in T cells or B cells in vivo by using biosensors. There are two types of biosensors available to monitor intracellular calcium increases.…”

“…There are two types of biosensors available to monitor intracellular calcium increases. One is to detect Förster resonance energy transfer (FRET) as changes in either the acceptor-to-donor fluorescence intensity ratio or the donor fluorescence lifetime [39, 40, 51, 64, 71, 80, 82, 83]. The other is to simply detect changes in the fluorescence intensity of biosensors [14, 63, 80].…”