Orai, STIM, and PMCA contribute to reduced calcium signal generation in CD8⁺ T cells of elderly mice

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.

INTRODUCTION

Aging of the immune system is a cumulative phenomenon and contributes to morbidity and mortality in man due to the greater incidence of infection, as well as autoimmune phenomena and cancer in elderly individuals [1–3]. Multiple changes in T cell populations are considered to be critical contributors to age-associated immune dysfunction [4]. A rather unexplored field of this dysfunction in aging is the Ca²⁺ homeostasis, which especially in T cells, is tightly regulated by an orchestra of channels, pumps, transporters, and receptors [5, 6]. The increase in cytoplasmic free [Ca²⁺] following T cell receptor activation is an essential element of various activation pathways [7] and necessary for the induction of gene expression in CD8⁺ T cells [8–11]. In addition, Ca²⁺ orchestrates the cytotoxicity of CD8⁺ T cells as the main function of this cell type [12]. For murine and human immune cells, store operated Ca²⁺ entry (SOCE) is the main pathway for Ca²⁺ to enter T cells [6, 13, 14]. Highly Ca²⁺-selective Orai channels and STIM proteins, as Ca²⁺ sensors, are the key players in SOCE and with their distinct characteristic properties [15, 16] they shape the Ca²⁺ pattern in T cells [17]. In contrast to the human SOCE components, four different Orai (Orai1, Orai2S, Orai2L, Orai3) proteins [18] and four STIM (STIM1, STIM1L, STIM2.1 and STIM2.2) proteins [19–21] have been identified in the mouse so far, but their role during aging in T cells is completely unknown.

While STIM/Orai channels are the likely candidates to mediate Ca²⁺ entry in aging T cells, other mechanisms could in principle contribute to store-operated Ca²⁺ signals: K⁺ channels (like K_v1.3 and K_Ca3.1) and TRPM4 may control the membrane potential and thereby the net Ca²⁺ entry through Orai channels [22, 23]. Furthermore, Ca²⁺ ATPases and mitochondria export Ca²⁺ from the cytosol and thereby shape Orai-dependent Ca²⁺ signals [24].

Following T cell activation in mice, several groups have reported a decline in Ca²⁺ signals with age [25, 26]. However, since the molecular mechanisms of these decreased Ca²⁺ signals in the context of aging are still elusive, we performed comprehensive assessments of the molecular repertoire governing Ca²⁺ signals in CD8⁺ T cells and investigated the influence of varying [Ca²⁺]_ext on the main function of these cells, the lysis of target cells.

RESULTS

Elderly mice show an increased proportion of CD8⁺ T cells and a shift in subpopulations

First, we examined the health status of the mice used for experiments. For this, body weight and spleen weight were measured (Table 1). Splenomegaly is a frequently observed sign of non-specific necropsy [27], therefore we excluded those mice from our cohort. A shift in the distribution of T cell subtypes to less naïve and more memory cells is considered as a marker of immunosenescence [26, 28, 29]. To assess the CD8⁺ T cell subtype distribution of the cells we stained them with surface markers CD44 and CD62L to differentiate between naïve (N: CD62L^highCD44^low); effector memory (EM: CD62L^lowCD44^high), and central memory CD8⁺ T cells (CM: CD62L^highCD44^high) (Supplementary Figure 1A, 1D). The untouched cells of adult mice display a pronounced population of CD62L^highCD44^low naϊve cells, a lesser relative amount of CD62L^highCD44^high central memory cells and only a small proportion of CD62L^lowCD44^high effector memory cells (Supplementary Figure 1A, 1C). In contrast, the untouched cells of elderly mice only show a small population of naϊve cells, a prominent population of effector and an even bigger population of central memory cells (Supplementary Figure 1D, 1F). The stimulation with CD3/CD28 beads led to a significant loss of the naïve population of CD8⁺ T cells and a significant increase in the relative amount of effector memory cells for both the adult and the elderly age group (examples in Supplementary Figure 1B, 1E, statistics in Supplementary Figure 1C, 1F). Furthermore, the population of central memory cells showed a significant increase for the CD8⁺ T cells of adult and a significant decrease for the CD8⁺ T cells of elderly mice after stimulation. In summary, the CD8⁺ T cells of adult mice mainly consist of naϊve cells and shifts primarily to central and secondarily to effector memory cells, whereas the CD8⁺ T cell pool of elderly mice mostly consists of memory T cells and shifts to more effector memory cells with T cell activation.

Table 1. Spleen to body weight ratio of adult (n = 91) and elderly (n = 71) mice ± SEM.

	body weight (g)	spleen weight (mg)	spleen to body weight ratio (%)
adult	24.65 (± 0.24)	93.70 (± 1.59)	0.380 (± 0.005)
elderly	33.10 (± 0.58)	139.41 (± 3.53)	0.425 (± 0.011)

Untouched CD8⁺ T cells from elderly mice show reduced SOCE

Ca²⁺ influx is an essential step in T cell activation and regulation of diverse cellular functions and the main pathway of Ca²⁺ in immune cells is through STIM-gated Orai channels [5]. We first investigated store-operated Ca²⁺ responses of elderly and adult untouched CD8⁺ T cells activated by thapsigargin (TG), an irreversible inhibitor of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pumps. We measured Ca²⁺ mobilization with two different approaches. The first approach combined the measurement of Ca²⁺ release and entry (combined protocol, Figure 1A) in the presence of 0.5 mM [Ca²⁺]_ext, whereas the second one separates them (Figure 1D) by carrying out the classical Ca²⁺ re-addition protocol. In the latter protocol, cells are initially treated with TG in Ca²⁺-free Ringer’s solution to deplete the endoplasmic reticulum (ER) Ca²⁺ stores, and Ca²⁺ influx is assured by subsequent addition of Ca²⁺. CD8⁺ T cells from elderly and adult mice differ in their ability to increase the internal calcium concentration ([Ca²⁺]_int) after TG-triggered stimulation in both approaches (Figure 1A, 1D). Resting [Ca²⁺]_int in CD8⁺ T cells from both mice was not significantly changed in combined and re-addition protocol (Figure 1A, 1D, Supplementary Tables 1, 2). Treatment of cells with TG in the absence of [Ca²⁺]_ext resulted in a transient rise in [Ca²⁺]_int revealing no significant change in the size of TG-releasable peak (Figure 1D, Supplementary Table 2). Upon re-addition of [Ca²⁺]_ext but also with the combined Ca²⁺ protocol, [Ca²⁺]_int mobilization was reduced in elderly CD8⁺ T cells by about 25 % (Figure 1A, 1B, 1D, 1E, Supplementary Tables 1, 2). The rate of Ca²⁺ entering CD8⁺ T cells is an indirect read-out for Orai channel function. For the re-addition protocol, the Ca²⁺ entry rate was significantly slower in cells from elderly compared to adult mice (Figure 1F); a similar tendency was observed in the combined protocol (Figure 1C). Similar results were obtained in case [Ca²⁺]_ext was increased to 2 mM (Supplementary Figure 2, Supplementary Tables 3, 4).

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.

Reduced I_CRAC in untouched CD8⁺ T cells from elderly mice

Since mainly Ca²⁺ release-activated Ca²⁺ currents (I_CRAC) through Orai channels are responsible for Ca²⁺ influx in T cells, we assessed I_CRAC in untouched CD8⁺ T cells from adult and elderly mice to determine age-related changes (Figure 2). We performed whole-cell patch-clamp experiments by perfusing cells with a high concentration of the Ca²⁺ chelator BAPTA, and IP₃ to deplete the Ca²⁺ stores. Both adult and elderly CD8⁺ T cells immediately developed a small, inwardly rectifying current (I_CRAC) with a reversal potential above +40 mV. Elderly CD8⁺ T cells showed reduced currents by 46 % (-1.89 ± 0.37 compared with -3.51 ± 0.36 (pA/pF) at 120 sec, p < 0.0001) (Figure 2A) with the typical inwardly rectifying current-voltage relationship (IV, Figure 2B).

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.

Reduced store-operated Ca²⁺ entry in in vitro stimulated CD8⁺ T cells from elderly mice

Since the negatively isolated and untouched cells do not have a long life span (24 hours) and, just like virus-infected or cancer cells, stimulation leads to activated CD8⁺ T cells, we decided to validate Ca²⁺ signals in CD8⁺ T cells after in vitro stimulation. We therefore stimulated the CD8⁺ T cells with anti-CD3/CD28 stimulation beads and examined SOCE on day 3 after stimulation. The overall Ca²⁺ signals analyzed in combined and re-addition protocols were reduced in stimulated CD8⁺ T cells between 60 to 64 % compared to untouched cells (Figures 1A, 1D, 3A, 3D, Supplementary Table 1, 2). This suggested that the molecular composition of the CRAC channel and STIM sensors may change during T cell stimulation. Still, TG-induced SOCE, measured as a peak of the Ca²⁺ response was significantly reduced in stimulated elderly CD8⁺ T cells compared to adult as control (Figure 3B, 3E). Besides the peak, also the Ca²⁺ plateau, as an important determinant of Ca²⁺ dependent cellular responses, was reduced in elderly CD8⁺ T cells (Figure 3B, 3E). For the re-addition protocol, the Ca²⁺ entry rate was significantly slower in cells from elderly compared to adult mice (Figure 3F); a similar tendency was observed in the combined protocol (Figure 3C). In contrast to untouched CD8⁺ T cells, the application of 2 mM [Ca²⁺]_ext was able to rescue the impaired Ca²⁺ signal in the elderly CD8⁺ T cells at least to some extend (Supplementary Figure 3). Measurements of I_CRAC in CD3/CD28 bead-stimulated CD8⁺ T cells were not successful due to their already overall small whole-cell currents that were presumably even more reduced in the T cells from elderly mice.

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.

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

To test for a functional relevance of reduced [Ca²⁺]_int we investigated SOCE in response to a more physiological stimulus. Antibody binding to the CD3/T-cell receptor complex activates T cells and evokes Ca²⁺ signals [30]. To explore the differences in TCR-induced [Ca²⁺]_int mobilization between adult and elderly CD8⁺ T cells we activated the TCR by application of a soluble anti-CD3ε antibody. Figure 4 shows that TCR activation leads to increased Ca²⁺ influx in untouched (Figure 4A) and stimulated (Figure 4B) CD8⁺ T cells but could not reach the levels seen in TG-experiments (Figure 1A, 3A). Mean [Ca²⁺]_int mobilization of the untouched cells was faster and reached overall a higher plateau compared to the stimulated counterparts. As in TG-induced SOCE, CD8⁺ T cells isolated from elderly mice show less efficient TCR-induced [Ca²⁺]_int mobilization compared to adult mice.

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.

The characteristic function of CD8⁺ T cells is to eliminate virus-infected and degenerated targets cells [31]. In our previous paper, we reported how changes in [Ca²⁺]_ext influence human CD8⁺ T cell cytotoxicity [12]. Therefore, we investigated, whether different concentrations of [Ca²⁺]_ext have an impact on the killing efficiency of CD8⁺ T cells in the context of aging. Either EGTA or CaCl₂ were added to AIM V, a medium optimized for the cultivation of lymphocytes, to decrease or increase free [Ca²⁺]_ext. Quantification of cancer cell killing revealed that free [Ca²⁺]_ext higher or lower than the value of 798 μm of AIM V reduced the cytotoxicity of CD8⁺ T cells of adult mice (Figure 4C) similarly as for human CD8⁺ T cells [12]. In contrast, CD8⁺ T cells of elderly mice where largely unaffected by fluctuations in free [Ca²⁺]_ext (Figure 4C). Averaging all experiments showed that elderly CD8⁺ T cells showed similar cytotoxic efficiency against target cells at a relatively wide range of free [Ca²⁺]_ext between 74 up to 1715 μM compared to their adult counterpart. Thus, Ca²⁺ dependent regulation of cytotoxicity is reduced in CD8⁺ cells from elderly mice.

STIM and Orai molecules are differently expressed in untouched and stimulated CD8⁺ T cells from elderly mice

The Ca²⁺ influx profile in lymphocytes is strictly determined by the composition of Orai channels and STIM Ca²⁺ sensors [5, 32, 33]. The ratio of Orai to STIM determines the characteristics and properties of I_CRAC [34, 35]. Therefore, we performed a detailed analysis of the expression levels of Orais (Orai1, 2, and 3) and STIMs (STIM1 and 2) in untouched and stimulated CD8⁺ T cells from adult and elderly mice by quantitative real-time PCR and western blot analysis. As expected, mRNA of both STIMs and all three Orais are abundantly expressed in untouched (Figure 5A) and stimulated (Figure 5C) CD8⁺ T cells from both age groups. To facilitate comparison, expression levels of Orai and STIM genes from elderly mice were normalized to reference genes and shown as relative fold change to the adult group. The untouched CD8⁺ T cells from elderly mice showed a significant reduction in mRNA transcript levels for Orai2 and STIM1 (Figure 5A), which could also be confirmed at protein levels after densitometry analysis (Figure 5B, Supplementary Figure 4C). Surprisingly, as shown in Figure 5A, mRNA levels of STIM2 were not changed in untouched CD8⁺ T cells of elderly mice but the statistical analysis of the western blots revealed a significant reduction on protein levels (Figure 5B). The mRNA reduction of Orai1 (Figure 5A) could not be confirmed at the protein level (Figure 5B). In addition, a significant downregulation for STIM1 and STIM2 was seen in stimulated CD8⁺ T cells from elderly mice at mRNA transcript and protein levels (Figure 5C, 5D). The level of Orai3 mRNA remained unchanged in untouched and stimulated cells (Figure 5A, 5C). Protein analysis of Orai2 or Orai3 was hampered by limited sensitivity of commercially available antibodies. Since the patch-clamp attempts with CD3/CD28 bead-stimulated CD8⁺ T cells from adult and elderly mice were unsuccessful, we were wondering if this is due to the reduced levels of Orai and STIM after stimulation. Indeed, the stimulated CD8⁺ T cells showed a significant downregulation of all STIM and Orai mRNA transcript levels compared to the untouched CD8⁺ T cells for both age groups (Supplementary Figure 4A, 4B). In conclusion, the overall expression levels of STIM and Orai correlate well with the reduction of Ca²⁺ entry in CD8⁺ cells from elderly compared to adult cells and also with the reduction of Ca²⁺ signals in stimulated compared to untouched cells.

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.

Enhanced Ca²⁺ clearance rate in CD8⁺ T cells from elderly mice

In addition to the influx mechanisms, [Ca²⁺]_int of course depends on Ca²⁺ efflux that could contribute to the difference in Ca²⁺ signals of CD8⁺ T cells from adult and elderly mice. We therefore compared the clearance rates of both untouched (Figure 6A) and stimulated (Figure 6D) cells from elderly and adult mice. Because the rate of Ca²⁺ extrusion in T cells depends on [Ca²⁺]_int [36, 37], steady-state [Ca²⁺]_int was measured just before Ca²⁺ removal. The clearance rates were directly calculated as exponential decays after removal of [Ca²⁺]_ext. To isolate the effect of [Ca²⁺]_int we performed iso-cell analysis [37, 38] to compare the rate constant at approximately the same levels of [Ca²⁺]_int. The rate constants from untouched (Figure 6B) and stimulated (Figure 6E) CD8⁺ T cells isolated from adult and elderly mice were plotted against the respective Ca²⁺ plateaus. Both the untouched and the stimulated CD8⁺ T cells from the elderly mice show significantly faster Ca²⁺ extrusion rates compared to the iso-cells from the adult mice (Figures 6B, 6E, Supplementary Table 5). PMCA1 and PMCA4 are the two, out of four, known PMCA isoforms that are ubiquitously distributed in all tissues and they are also the major Ca²⁺ extrusion pathway in T cells [37]. PMCA4b is highly expressed in T cells and important to shape [Ca²⁺]_int in T cells [37]. It may therefore contribute to the higher Ca²⁺ clearance rate seen in elderly CD8⁺ T cells. We tested the mRNA expression levels of PMCA1 and 4 in untouched and stimulated CD8⁺ T cells from adult and elderly mice (Figure 6C, 6F). In agreement with the Ca²⁺ clearance rate analysis, PMCA4b expression was increased in CD8⁺ cells from elderly compared to adult mice; the values for the PMCA1 isoform remained constant (Figure 6C, 6F). In conclusion, PMCA4b upregulation in CD8⁺ T cells from elderly mice might contribute to the observed phenotype of reduced [Ca²⁺]_int.

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.

Altered SOCE in central and effector memory CD8⁺ T cells from elderly mice

While changes in the (relative) frequency of distinct T cell subsets have been described, nothing is known about Ca²⁺ signal differences between subsets and if these explain the differences in the total population of CD8⁺ T cells from elderly and adult mice. In order to characterize the [Ca²⁺]_int mobilization of the most abundant subtypes of stimulated CD8⁺ T cells, we performed FACS sorting to separate the subtypes of adult and elderly mice and measured their Ca²⁺ signals. The normalized data of the adult CD8⁺ T cells subpopulations is summarized in Figure 7. Overall the [Ca²⁺]_int reduction of CD8⁺ T cells from elderly compared to adult mice is more prominent in the central memory (CM) than the effector memory (EM) population (Figure 7). The CM from elderly mice showed a more drastic Ca²⁺ influx peak reduction (20 - 25 %) (Figure 7A–7C, 7E) than the EM (~ 10 %) (Figure 7G–7I, 7K). The same applies for the Ca²⁺ plateaus of CM (~ 25 %) (Figure 7A–7C, 7E) and EM (~ 10 %) (Figure 7G–7I, 7K). The influx rates of both CM and EM showed a significant reduction for the more physiological combined protocol (CM: ~ 35 %; EM: ~ 23 %) (Figure 7D, 7F, 7H, 7J). In conclusion, from the two predominant subtypes of stimulated CD8⁺ T cells, the CM from elderly mice revealed a more distinctly reduced overall Ca²⁺ response compared to those of adult animals (Figure 7A, 7B). For the EM this age-related reduction was less pronounced (Figure 7G, 7H). Considering the percentage distribution of the subpopulations in both age cohorts (Supplementary Figure 1), the CM/EM ratios reflect the Ca²⁺ signal differences seen in the total population of CD8⁺ T cells (Figure 3A, 3D).

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.

DISCUSSION

Proper Ca²⁺ homeostasis is essential for the immune system to act fast and specific to eliminate tumor cells at early stages. Altered Ca²⁺ fluctuations have already been associated with numerous age-related diseases, such as neurodegenerative [39–41], autoimmune and inflammatory disorders [42]. Ca²⁺ responses are regulated negatively and positively by several mechanisms involving channels, pumps and sensors [43]. During aging, a continuous and steady decrease of all immune function takes places [2, 44–46] and especially for T cells alterations in Ca²⁺ homeostasis have been reported [25, 26].

Although Ca²⁺ is an important second messenger the underlying cause or mechanism of Ca²⁺ defects at molecular level during aging are not well understood and remain elusive. In this study, we perform for the first time a detailed analysis of Ca²⁺ signals in CD8⁺ T cells from elderly mice and link the aging-related reduction in Ca²⁺ signals to reductions of the main key players in the Ca²⁺ signaling pathway, further leading to coherent, concurrent functional alterations in Ca²⁺ homeostasis. Specifically, we found: 1) reduction of the expression of STIM and Orai proteins leads to reduced Ca²⁺ entry; 2) upregulation of PMCA4 additionally contributes to faster Ca²⁺ extrusion 3) less efficient TCR-induced [Ca²⁺]_int mobilization and 4) increased insensitivity to Ca²⁺ fluctuations during cytotoxic activity. These findings likely account for the observed reduced Ca²⁺ signals in CD8⁺ T cells from elderly mice and contribute to the declined T cell responses.

Generally, one would rather expect an overall reduction in expression of the key proteins involved in the Ca²⁺ homeostasis during aging. However, this is not what we observed in CD8⁺ T cells: whereas Orai1 and STIM1/2 proteins are downregulated, PMCA4b is in contrast upregulated. Both expression changes, however, result in reduced [Ca²⁺]_int. Thus, it can be speculated that there is a deliberate change or adjustment in CD8⁺ T cells to reduced Ca²⁺ signals during the aging process. The age-related reductions in CD8⁺ T cell Ca²⁺ signals observed in our elderly mice may not imperatively implicate flawed cellular pathways and functions. Hence, one of the fundamental questions is how CD8⁺ T cells may benefit from reduced Ca²⁺ entry in age?

For the lysis of target cells, CD8⁺ cytotoxic T Lymphocytes (CTL) require a sequence of programmed steps, including target cell binding (conjugate formation), delivery of the lethal hit, target cell lysis, and killer cell recycling [47–49]. Several of these steps in the CTL killing machinery require or are modulated by Ca²⁺ itself [13]. It is well conceivable that Ca²⁺ fluctuations may greatly influence target cell killing by CTL and NK cells, and CRAC channels are well-suited to modulate killing because their dissociation constant K_D for Ca²⁺ permeation is in the range of 0.84 and 1.17 mM [50, 51]. Recently, we analyzed the cytotoxicity of human CTL and NK cells against cancer cells [12]. CTLs showed Ca²⁺ dependent cytotoxicity with an optimum for cancer cell elimination at rather low free [Ca²⁺] concentrations. Downregulation of Orai1 in CTLs led to decreased Ca²⁺ signals and increased efficiency to eliminate cancer cells [12]. Alterations of the STIM:Orai stoichiometry might regulate the killing efficiency of CD8⁺ T cells by changing the cells Ca²⁺ signals to be closer to or further away from the Ca²⁺ optimum for target cell killing. In the context of aging, we were able to detect changes at the mRNA levels in untouched and stimulated CD8⁺ T cells but not all Orai and STIM are affected in the same way. While Orai3 has hardly changed, decreased mRNA levels for Orai1, STIM1 and STIM2 could also be confirmed at the protein levels (Figure 5, Supplementary Figure 4). Surprisingly, the data for STIM2 showed no tendency at mRNA levels for the untouched CD8⁺ T cells (Figure 5A) but a significant decrease at protein levels (Figure 5B). Interestingly, also for Orai1 we observed a slight but not significant increase on protein level (Figure 5B) while the mRNA levels were significantly decreased (Figure 5A). This discrepancy may indicate a change in the turnover rate [52], translational regulation [53] or posttranslational modification [54] of these proteins. The stoichiometry of the STIM:Orai ratio determines the current size and inactivation properties [34, 35, 55] and thus Ca²⁺ signals and cell-specific cellular responses. Therefore, small variations in external Ca²⁺ could significantly alter Ca²⁺ signals and Ca²⁺-dependent target cell killing. Surprisingly, CD8⁺ T cells of elderly mice were mostly unaffected by fluctuations in free [Ca²⁺]_ext during killing compared to the CD8⁺ T cells from adult mice (Figure 4C). This independence from external Ca²⁺ fluctuations could be an advantage for the per se impaired CD8⁺ T cells from elderly mice. This insusceptibility to the wide range in free [Ca²⁺]_ext may offer an excellent adaptation to constantly changing surroundings such as the tumor microenvironment [56].

Additionally, there is no doubt about the importance of Ca²⁺ for cell proliferation not only for immune cells but also in the context of cancer cells [57–60]. Increased Ca²⁺ levels lead to an increased proliferation rate and SOCE plays a central role in its regulation [61]. However, the role of Orai channels in proliferation seems to be more complex considering the different results for different mouse models used for the investigation on this matter [62, 63]. Deletion of Orai [62] as well as STIM1 in T cells did not alter the proliferation, while T cells lacking both STIM1 and STIM2 proliferate to a much lesser extent [64]. This data support the hypothesis that the threshold of [Ca²⁺]_int necessary for T cell proliferation seems to be low and proliferation does not require high [Ca²⁺]_int levels [65]. However, we did observe declined proliferative capacity in the first three days during stimulation which has been lifted from day four. Similar observations of declined proliferation have already been reported for CD4⁺ and CD8⁺ T cells from elderly mice and humans [4, 26, 61, 65–67]. Maybe, this decelerated proliferation benefits the cytotoxic capacity of CD8⁺ T cells and represents an elaborate adaptation during aging process.

Apoptotic deletion of activated T cells is an essential physiological process to terminate the immune response and the control of the overall number of immunocompetent cells [68, 69]. Ca²⁺ signaling plays a critical role for the initiation and effectuation of cell death [70–73]. Studies about apoptosis in aging are controversial and have shown increased apoptosis in T cells while others have observed a decrease or no effect [74–78]. Beside STIM- and Orai-mediated Ca²⁺ signals being crucial for T cell cytotoxicity and proliferation, excessive or prolonged Ca²⁺ entry can also lead to cell death [62]. Since it has been shown that Orai1 deficiency renders T cells resistant to death upon long-term exposure to anti-CD3 and anti-CD28 [62], the decreased Ca²⁺ influx in CD8⁺ T cells from elderly individuals could provide potential protection against increased apoptosis.

Although the signaling machinery in T cells is extremely complicated and many steps remain to be clarified, age-related changes in Ca²⁺ entry may be one important cause of cell-mediated immune response decline with aging. In this study we were able to demonstrate the contribution of Orai, STIM and PMCA in this multifaceted network of channels and pumps in Ca²⁺ homeostasis at molecular levels. Additional studies are required to clarify the influence of the reduced Ca²⁺ signaling in the context of CD8⁺ T cells cytotoxicity as one of the main killers in our body.

MATERIALS AND METHODS

Abs and reagents

If not mentioned otherwise, chemicals were purchased from Sigma Aldrich and antibodies from Biolegend. Antibodies used in our experiments include PerCP-labeled anti-mouse CD3 (145-2C11), Pacific Blue-labeled anti-mouse CD4 (RM4-5), FITC-labeled anti-mouse CD8a (53-6.7), PE-labeled anti-mouse/human CD44 (IM7), APC-labeled anti-mouse CD62L (MEL-14) and LEAF™ Purified anti-mouse CD3ε (1452C11).

Mice

C57BL6/J mice were bread in our own colony from stock purchased from Charles River Laboratories. Only female mice were used between 12 and 24 weeks (adult mice) or between 78 and 102 weeks (elderly mice). Mice with splenomegaly (spleen to body weight ratio above 0.6) or macroscopically visible tumors were excluded from the studies. Mice were housed under specific pathogen-free conditions. At the designated times, animals were sacrificed by cervical dislocation and their spleens were harvested. Splenocytes were isolated by pushing the spleen through a 70 μm cell strainer (Corning®) and depleting the erythrocytes by a hypoosmolar solution. All animals used for this study were sacrificed and their organs harvested in compliance with the German Animal Protection Law (Tierschutzgesetz, §11, Abs.1 Nr.1).

T cell culture and stimulation

CD8⁺ T cells were negatively isolated from murine splenocytes by using the Dynabeads™ Untouched™ Mouse CD8 Cells Kit (ThermoFisher). Murine CD8⁺ T cells were cultured in AIM V medium, supplemented with 10 % FCS, 100 U/ml IL-2, and 50 μM β-Mercaptoethanol, without stimulus. For the stimulation, the above mentioned medium was supplemented with Dynabeads™ Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation (ThermoFisher). The stimulated CD8⁺ T cells were kept in the medium with a 5:4 cell-to-bead ratio at 37 °C and 5 % CO₂ for up to 4 days.

Flow cytometry and cell sorting

Splenocytes and CD8⁺ T cells of adult and elderly mice were stained with the antibodies listed in ‘Abs and reagents’ and incubated for 20 min in the dark at room temperature. Stained samples were acquired using a FACSVerse™ (BD Biosciences) flow cytometer, and acquired data were analyzed with FlowJo software (FlowJo, LLC). For the sorted subtypes, stained 72 h stimulated CD8⁺ T cells were sorted on a FACSAria™III (BD Biosciences) sorter and left to rest from the sorting procedure for at least 4 h.

Quantitative real-time PCR

Total RNA from CD8⁺ T cells was extracted by phenol-chloroform extraction. The concentration of intact total RNA was measured with a BioPhotometer (Eppendorf). Real-time PCR was performed with a Real Time System CFX 96/Thermal Cycler C1000 (BioRad). Relative expression levels were calculated using the ΔCq method (2^-ΔCq). Expression of STIMs and Orais was normalized to the average mRNA levels of the housekeeping genes HPRT1 and TBP. Primers for QuantiTect Primer Assays were purchased from QIAGEN.

QuantiTect primers

Target gene	Product	Cat. No.
Orai1	Mm_Orai1_1_SG	QT00285775
Orai2	Mm_Orai2_1_SG	QT00304738
Orai3	Mm_Orai3_1_SG	QT00255598
STIM1	Mm_Stim1_1_SG	QT00105119
STIM2	Mm_Stim2_1_SG	QT00289009
PMCA1	Mm_Atp2b1_1_SG	QT01072106
PMCA4	Mm_Atp2b4_2_SG	QT01076271
HPRT1	Mm_Hprt_1_SG	QT00166768
TBP	Mm_Tbp_1_SG	QT00198443

Western blot analysis

CD8⁺ T cells were collected directly after isolation (untouched) or after 72 h of stimulation (stimulated). Equivalent amounts of proteins were separated by 12–14 % SDS-PAGE and transferred to nitrocellulose membrane using a transblot electrophoresis transfer cell (Fisherbrand). Primary antibodies against Orai1, STIM1 and 2 were purchased from Proteintech. Primary antibody for GAPDH as reference was purchased from Cell Signaling. Secondary anti-rabbit antibody was purchased from GE Healthcare. ECL reagent (Amersham) was used for immunoblot detection. Densitometric quantification of Western blot data was done with Quantity one software (Bio-Rad).

Fluorescence-based Ca²⁺-imaging

Ca²⁺-Imaging was performed according to Alansary, Kilch et al. paper [79]. Briefly, murine CD8⁺ T cells were loaded in AIM V medium with 1 μM Fura2-AM for 30 min at room temperature and allowed to attach to polyornithine-coated glass coverslips for 15 min. All experiments were carried out at room temperature in self-built perfusion chambers with low volume and high solution exchange rate. The external Ca²⁺ Ringer solution contained (in mM): 155 NaCl, 2 MgCl₂, 10 glucose, 5 HEPES and 0.5 or 2 CaCl₂ (0.5 / 2 Ca²⁺ Ringer) or no CaCl₂, but 1 EGTA and 3 MgCl₂ instead (0 Ca²⁺ Ringer). The pH was adjusted to 7.4 with NaOH. Images were analyzed with VisiView software (Visitron). Quantification of the trace shows the ratio (340 nm/380 nm) corresponding to Ca²⁺ influx peak and plateau and Ca²⁺ influx and efflux rate as ratio (340 nm/380 nm)s^-1. Parameters analyzed were the influx peak, as maximal Ca²⁺ signal reached after TG application after (re-)adding of Ca²⁺, and the average plateau. The plateau mirrors the balance between influx and efflux and was analyzed before the application of 0 mM Ca²⁺ (Ca²⁺ removal).

Patch-clamp measurements

Patch-clamp experiments were performed in the tight-seal whole-cell configuration at RT. Voltage ramps of 50 ms duration spanning a range of –100 to +100 mV were delivered from a holding potential of 0 mV at a rate of 0.5 Hz. All voltages were corrected for a liquid junction potential of 10 mV. Currents were filtered at 2.9 kHz and digitized at 100 μs intervals. Capacitive currents were determined and corrected before each ramp. Statistical errors of averaged data are given as means ± SEM with n determinations. Standard external solutions were as follows (in mM): 120 NaCl, 2.8 KCl, 2 MgCl₂, 10 CaCl₂, 10 CsCl, 10 HEPES, 10 glucose, pH 7.2 with NaOH, 305 mOsm. Standard internal solutions were as follows (in mM): 120 Cs-glutamate, 10 Cs-BAPTA, 3 MgCl₂, 0 CaCl₂, 10 HEPES, pH 7.2 with CsOH, 298 mOsm.

Real-time killing assay

The real-time killing assays were carried out as described in Kummerow et al. 2014 [80]. Briefly, P815 mastocytoma cells were loaded with 500 nM calcein-AM in AIM V medium containing 10 mM HEPES at room temperature for 15 minutes. The loaded P815 cells were then settled at 2.5×10⁴ cells per well into black 96-well plates with clear-bottoms (353219, Corning, Amsterdam, Netherlands). CD8⁺ T cells were pulsed with 2 μg/ml anti-CD3ε antibody and subsequently added onto the P815 cells at a 20:1 effector to target ratio. Target lysis was measured either in an M200 Infinite plate reader (Tecan, Crailsheim, Germany) or a Genios Pro (Tecan) using bottom reading function at 37°C. The quantification of free Ca²⁺ concentration in AIM V medium supplemented with different amounts of Ca²⁺ or EGTA was done as previously described in Zhou et al. 2018 [12].

Statistical analysis

All values are given as mean ± SEM or SD. Data were analyzed using VisiView (Visitron), Microsoft Excel (Microsoft), Igor Pro (Wavemetrics), Image Lab™ (Bio-Rad) and GraphPad (GraphPad Software Inc.). Rate constants (k values) of iso-cells were calculated for each trace by exponential decay analysis after removal of external Ca²⁺. Significances of data were calculated with an unpaired two-sided Student’s t-test if Gaussian distribution was given. If no Gaussian distribution was given, data were analyzed with the nonparametric Mann-Whitney test. For multi-parameter analysis data were analyzed with ANOVA. Degrees of significance were set at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Abbreviations

AM

acetoxymethyl ester

ATP

adenosine triphosphate

BAPTA

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

Ca²⁺

calcium

[Ca²⁺]_int

internal Ca²⁺ concentration

[Ca²⁺]_ext

external Ca²⁺ concentration

CTL

cytotoxic T lymphocyte

EGTA

ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

ER

endoplasmic reticulum

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

HEPES

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

HPRT1

hypoxanthine phosphoribosyltransferase 1

IP₃

inositol 1,4,5-trisphosphate

PMCA

plasma membrane Ca²⁺ ATPase

SDS-PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SERCA

sarco/endoplasmic reticulum Ca²⁺ ATPase

SOCE

store operated Ca²⁺ entry

STIM

stromal interaction molecule

TG

thapsigargin

TRPM

transient receptor potential melastatin