Calcium Dynamics of <i>Ex Vivo</i> Long-Term Cultured CD8⁺ T Cells Are Regulated by Changes in Redox Metabolism

<div><p>T cells reach a state of replicative senescence characterized by a decreased ability to proliferate and respond to foreign antigens. Calcium release associated with TCR engagement is widely used as a surrogate measure of T cell response. Using an ex vivo culture model that partially replicates features of organismal aging, we observe that while the amplitude of Ca²⁺ signaling does not change with time in culture, older T cells exhibit faster Ca²⁺ rise and a faster decay. Gene expression analysis of Ca²⁺ channels and pumps expressed in T cells by RT-qPCR identified overexpression of the plasma membrane CRAC channel subunit ORAI1 and PMCA in older T cells. To test whether overexpression of the plasma membrane Ca²⁺ channel is sufficient to explain the kinetic information, we adapted a previously published computational model by Maurya and Subramaniam to include additional details on the store-operated calcium entry (SOCE) process to recapitulate Ca²⁺ dynamics after T cell receptor stimulation. Simulations demonstrated that upregulation of ORAI1 and PMCA channels is not sufficient to explain the observed alterations in Ca²⁺ signaling. Instead, modeling analysis identified kinetic parameters associated with the IP₃R and STIM1 channels as potential causes for alterations in Ca²⁺ dynamics associated with the long term ex vivo culturing protocol. Due to these proteins having known cysteine residues susceptible to oxidation, we subsequently investigated and observed transcriptional remodeling of metabolic enzymes, a shift to more oxidized redox couples, and post-translational thiol oxidation of STIM1. The model-directed findings from this study highlight changes in the cellular redox environment that may ultimately lead to altered T cell calcium dynamics during immunosenescence or organismal aging.</p></div

Age related Ca²⁺ changes in CD8⁺ T cells.

<p>a) Cytoplasmic Ca²⁺ levels in resting T cells. Mean and SEM from 4 different donors. AU = arbitrary units b) Representative trace of Ca²⁺ dynamics following TCR stimulation with the related parameters studied, peak time, peak amplitude, baseline and decay. The integral corresponds to the colored area under the curve. c) Time to peak in seconds. d) Fast decay time constant in minutes. For c-d), the data represents the mean of each calculated parameters for each donor and its standard deviation. The red diamonds correspond to the parameter calculated if the Ca²⁺ time courses are averaged for all donors for a specific day in culture.</p

An oxidative shift in redox potential with ex vivo T cell expansion.

<p>Total glutathione levels (a) and corresponding GSH/GSSG redox potential (b) in CD8⁺ T cells with time in culture (n = 3). Statistical analysis: one-way ANOVA (p = 0.04) followed by Scheffe’s post-hoc test * p<0.05 between day 8 and day 16. Trx1 levels in CD8⁺ T cells with time in culture. c) Total, reduced and oxidized Trx1 levels for a representative donor. DTT-treated lysate is included as a reduced control and diamide-treated lysate is included as an oxidized control. d) Trx1 redox potential (n = 4). Statistical analysis: one-way Anova (p = 0.007) followed by Scheffe’s post-hoc test * p<0.05 between day 4 and days 16–24. ** p<0.05 between days 8–12 and days 20–24.</p

Difference in fit parameter values between the Young and Old CD8⁺ T Cell Models.

<p>Difference in fit parameter values between the Young and Old CD8⁺ T Cell Models.</p

Changes in mRNA levels of Ca²⁺ channels and pumps with age (n = 6).

<p>Points above the red line represent targets that show significant statistical difference (at p < 0.05) between young and old samples. The blue lines represent fold changes that are above 1.5 fold up or down.</p

Model parameters bounds for optimization.

<p>Model parameters bounds for optimization.</p

Oxidation of STIM1 thiols occurs during ex vivo aging of primary T cells.

<p>a) Validation of assay for detection of oxidation (streptavidin signal) of STIM1 in Jurkat lysates. Reduced thiols were labelled with PEO-biotin-iodoacetamide (BIAM) as described in the Methods. b) Oxidation of STIM1 in young and old primary human CD8⁺ T cells.</p

Young CD8⁺ T Cell Model predictions of IP₃ and Ca²⁺ in the various cellular compartments in response to TCR signaling in CD8⁺ T cells.

<p>Cytosolic Ca²⁺ dynamics in the absence (a) or presence of inhibitors (b,c) at the same concentration as for Jurkat cells. d-f) model predictions for other state variables in the no inhibitor simulation. The average trace across all optimized parameter sets is illustrated by the blue solid line and the shaded region represents the standard deviation of the dynamic behavior.</p

Schematic of the Ca²⁺ signaling model.

<p>Schematic of the Ca²⁺ signaling model.</p