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. 2020 Feb 12;12(4):3266-3286.
doi: 10.18632/aging.102809. Epub 2020 Feb 12.

Orai, STIM, and PMCA Contribute to Reduced Calcium Signal Generation in CD8 + T Cells of Elderly Mice

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Free PMC article

Orai, STIM, and PMCA Contribute to Reduced Calcium Signal Generation in CD8 + T Cells of Elderly Mice

Adrian Angenendt et al. Aging (Albany NY). .
Free PMC article

Abstract

Ca2+ is a crucial second messenger for proper T cell function. Considering the relevance of Ca2+ signals for T cell functionality it is surprising that no mechanistic insights into T cell Ca2+ signals from elderly individuals are reported. The main Ca2+ entry mechanism in T cells are STIM-activated Orai channels. Their role during lymphocyte aging is completely unknown. Here, we report not only reduced Ca2+ signals in untouched and stimulated, but also in central and effector memory CD8+ T cells from elderly (18-24 months) compared to adult (3-6 months) mice. Two mechanisms contribute to the overall reduction in Ca2+ signals of CD8+ T cells of elderly mice: 1) Reduced Ca2+ currents through Orai channels due to decreased expressions of STIMs and Orais. 2) A faster extrusion of Ca2+ owing to an increased expression of PMCA4. The reduced Ca2+ signals correlated with a resistance of the cytotoxic efficiency of CD8+ T cells to varying free [Ca2+]ext with age. In summary, reduced STIM/Orai expression and increased Ca2+ clearing rates following enhanced PMCA4 expression contribute to reduced Ca2+ signals in CD8+ T cells of elderly mice. These changes are apparently relevant to immune function as they reduce the Ca2+ dependency of CTL cytotoxicity.

Keywords: CD8 T cells; Orai; PMCA; STIM; calcium.

Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Untouched CD8+ T cells from elderly mice show reduced thapsigargin (TG)-induced Ca2+ signals. (A) Fura2-AM based Ca2+ Imaging with 1 μM TG as stimulus applied in the presence of 0.5 mM [Ca2+]ext of CD8+ T cells (combined Ca2+ protocol) from adult (black, n = 5) and elderly (red, n = 7) mice. The scatter dot plot in (B) displays the corresponding statistics of Ca2+ influx peak and Ca2+ plateau and in (C) the corresponding influx rates. (D) Ca2+ Imaging with 1 μM TG applied in the absence of [Ca2+]ext before re-addition of 0.5 mM Ca2+ (re-addition protocol) of CD8+ T cells from adult (black, n = 5) and elderly (red, n = 7) mice. The scatter dot plot in (E) displays the corresponding statistics of Ca2+ influx peak and Ca2+ plateau and (F) the corresponding influx rates. Ca2+ signalling curves are presented as mean ± SEM. Scatter dot plots are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 2
Figure 2
Untouched CD8+ T cells from elderly mice show reduced lP3-induced CRAC currents. (A) Average IP3-induced CRAC current amplitudes at –80 mV normalized to cell size from CD8+ T cells of adult (black, n = 8) and elderly (red, n = 7) mice. (B) Average current-voltage relationship of CRAC currents from cells presented in (A) after CRAC had fully developed. Data obtained are presented as mean ± SEM.
Figure 3
Figure 3
Stimulated CD8+ T cells from elderly mice show reduced thapsigargin (TG)-induced Ca2+ signals. (A) Fura2-AM based Ca2+ Imaging with 1 μM TG as stimulus applied in the presence of 0.5 mM [Ca2+]ext of CD8+ T cells (combined Ca2+ protocol) from adult (black, n = 4) and elderly (red, n = 4) mice. The scatter dot plot in (B) displays the corresponding statistics of Ca2+ influx peak and Ca2+ plateau and in (C) the corresponding influx rates. (D) Ca2+ Imaging with 1 μM TG applied in the absence of [Ca2+]ext before re-addition of 0.5 mM Ca2+ (re-addition protocol) of CD8+ T cells from adult (black, n = 4) and elderly (red, n = 4) mice. The scatter dot plot in (E) displays the corresponding statistics of Ca2+ influx peak and Ca2+ plateau and (F) the corresponding influx rates. Ca2+ data are presented as mean ± SEM. Scatter dot plots are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
CD8+ T cells from elderly mice show reduced Ca2+ signals after T cell receptor stimulation and are less affected in their cytotoxic function by varying free external Ca2+ concentrations. Fura2-AM based Ca2+ Imaging with 2 μg/ml anti-CD3 antibody as stimulus applied in the presence of 0.5 mM [Ca2+]ext of (A) untouched (black: adult, n = 664 cells; red: elderly, n = 327 cells) and (B) stimulated (black: adult, n = 155 cells; red: elderly, n = 116 cells) CD8+ T cells from adult and elderly mice. (C) The cytotoxic function of CD8+ T cells from elderly mice is less affected by varying free [Ca2+]ext. Changes in end-point lysis with the addition of Ca2+ or the Ca2+ chelating agent EGTA to the medium of a cytotoxicity assay for CD8+ T cells of adult (grey, n = 3 - 5) and elderly (n = 2 - 3) mice. Data obtained are presented as mean ± SEM.
Figure 5
Figure 5
mRNA and protein levels of distinct STIM and Orai isoforms are reduced in CD8+ T cells of elderly mice. (A) Normalised relative mRNA expressions of Orai1, 2 and 3 and STIM1 and 2 of untouched CD8+ T cells from adult (grey, n = 12) and elderly (red, n = 11) mice. (C) Normalised relative mRNA expressions of SOCE components of stimulated CD8+ T cells from adult (grey, n = 12) and elderly (red, n = 11) mice. Protein quantification after normalization to GAPDH of SOCE components from (B) untouched (n = 7 - 9) and (D) stimulated (n = 5 - 7) CD8+ T cells lysates isolated from adult (black) and elderly (red) mice. Data obtained are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 6
Figure 6
CD8+ T cells from elderly mice show a faster efflux of Ca2+. Exemplary combined protocol measurement of untouched (A) and stimulated (D) CD8+ T cells with a highlight on the Ca2+ plateau and efflux. Rate constants of untouched (B) and stimulated (E) CD8+ T cells from adult and elderly mice plotted against their respective Ca2+ plateaus. (C) Relative mRNA expressions of PMCA1 and 4 of untouched CD8+ T cells from adult (grey, n = 7) and elderly (red, n = 7) mice. (F) Relative mRNA expressions of PMCA1 and 4 of stimulated CD8+ T cells from adult (grey, n = 7) and elderly (red, n = 8) mice. Data obtained are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 7
Figure 7
The most abundant subtypes of activated CD8+ T cells exhibit age-related reductions of TG-induced Ca2+ signals. Combined (A) and re-addition (B) protocol of CD8+ central memory T cells (CM) from adult (black, n = 6) and elderly (red, n = 6) mice. The bar graphs in (C) and (E) display the corresponding statistics of Ca2+ influx peak and Ca2+ plateau and in (D) and (F) the corresponding influx rates of combined and re-addition protocol, respectively. Combined (G) and re-addition (H) protocol of CD8+ effector memory T cells (EM) from adult (black, n = 6) and elderly (red, n = 6) mice. The scatter dot plots in (I) and (K) display the corresponding statistics of Ca2+ influx peak and Ca2+ plateau and in (J) and (L) the corresponding influx rates of combined and re-addition protocol, respectively. Ca2+ signalling curves show one exemplary out of six measurements with equal tendencies of central and effector memory cells of adult and elderly mice as mean ± SEM. Bar graphs show values of CD8+ T cells from elderly mice normalized to the values of CD8+ T cells from adult mice as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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