T Lymphocyte Integrated Endoplasmic Reticulum Ca2+ Store Signaling Functions Are Linked to Sarco/Endoplasmic Reticulum Ca2+-ATPase Isoform-Specific Levels of Regulation

Abstract

We explored the effects of altering expression levels of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) ion-transporting enzymes on key T lymphocyte signaling functions. In these studies, we have taken advantage of the Jurkat T cell line which provides a T lymphocyte model cell phenotype with a well-characterized T cell receptor (TCR)-activated signaling pathway, as well as offering a cellular system with a good understanding of the SERCA expression profile. These studies have been prompted by a strong imperative to gain a better understanding of the complex roles SERCA Ca²⁺ pumps play in the integrated TCR-activated signaling network, particularly given the central role of SERCA functions in regulating essential endoplasmic reticulum (ER) integrity. We find in this study that altering SERCA expression can significantly reconfigure ER Ca²⁺ stores, increasing or decreasing Ca²⁺ storage capacity depending on upregulation or downregulation of SERCA expression, and these effects are also associated with substantial changes in agonist-induced Ca²⁺ release and influx patterns. Not surprisingly, these fundamental changes in TCR-regulated Ca²⁺ signaling properties are associated with major alterations in T lymphocyte functions including regulation of growth patterns, cytokine secretion and energy utilization. Our study also describes additional evidence revealing intriguing functional distinctions between the major SERCA isoform-regulated Ca²⁺ stores in T lymphocytes. Our work thus serves to reinforce increasing efforts to target the SERCA pumps as a potential profitable strategy to produce novel engineered T lymphocytes in the rapidly growing field of T-cell immunotherapy.

Keywords: ER calcium stores; SERCA; T cell signaling; calcium homeostasis; calcium pumps; calcium signaling.

Figure 1

Overexpression of SERCA 2b and SERCA 3 Ca²⁺-ATPases Alters Ca²⁺ Signaling Properties in Jurkat T Lymphocytes. (A) Representative Western blot image of overexpression of SERCA 2b and SERCA 3 Ca²⁺-ATPase isoforms in Jurkat lymphocytes (see Section 4). The figure shows the approximate molecular weight of the SERCA proteins and β-actin protein bands as control. Additionally shown are the densitometry bar plots derived from band density quantification for SERCA 2b (green bars) and SERCA 3 (purple bars) compared to control sham transfected cells (black bars). (B,C) Jurkat T lymphocytes were loaded with Fura-2 and suspended in Ca²⁺-free media (balanced salt solution plus 2 mM EGTA) in cuvettes for use in the large cell-number configuration (1 × 10⁶ cells/mL). (B) Jurkat cell Ca²⁺ release responses in SERCA 2b_OE, SERCA 3_OE and control cells induced by the addition of TG (3 μM, arrow) as determined by the ratio of fluorescence changes at 340 and 380 nm (F340/380). (C) the same experiment as in B but with the application of ionomycin (3 μM, arrow). (D,E) Ca²⁺ measurements were acquired from Jurkat T lymphocytes in the small cell-number configuration (30–60 cells per field) using microscope photometry (Section 4). (D) Jurkat cell Ca²⁺ release and influx responses in SERCA 2b_OE, SERCA 3_OE and control cells induced by the addition of TG (3 μM, arrow); Ca²⁺ influx responses were determined by the addition of Ca²⁺ (2 mM) to the coverslips as indicated. (E) the same experiment as in D but with the application of ionomycin (3 μM, arrow). (F,G) Jurkat T lymphocytes were loaded with Fura-2 and suspended in Ca²⁺-free media (balanced salt solution plus 2 mM EGTA) in cuvettes for use in the large-cell number configuration (1 × 10⁶ cells/mL) and challenged with the low-dose SERCA blocker regimen. (F) Jurkat cell Ca²⁺ release responses in SERCA 2b_OE, SERCA 3_OE and control cells induced by the addition of low-dose TG (100 pM, arrow) as determined by the ratio of fluorescence changes at 340 and 380 nm (F340/380). (G) the same experiment as in F but with the application of low-dose tBHQ (1 μM, arrow). (H), Jurkat cell Ca²⁺ release and influx responses in SERCA 2b_OE, SERCA 3_OE and control cells induced by the addition of PHA (10 μg/mL, arrow); Ca²⁺ influx responses were determined by the addition of Ca²⁺ (2 mM) to the cuvettes as indicated. Fluorescence traces of Ca²⁺ experiments shown are representative of three to six separate experiments with significant differences assessed via use of student t test. One-way ANOVA followed by Tukey’s multiple comparison tests were used to analyze Western blot data for SERCA expression levels with n = 3 (Section 4). Asterisks denote statistical significance with * p < 0.05, ** p < 0.005, **** p < 0.0001 and ns is not significant p > 0.05.

Figure 2

CRISPR-Based Knockout of SERCA Isoforms Significantly Alters Jurkat T Lymphocyte Ca²⁺ Store Status, Revealing Heightened Ca²⁺ Influx Coupling. (A) Representative Western blot image of CRISPR-mediated knockout (KO) of SERCA 2b, SERCA 3 and SERCA 2b/SERCA 3 double-knockout (DKO) experiments in Jurkat lymphocytes (see Section 4). Figure shows the approximate molecular weight of the SERCA proteins and β-actin protein bands as control. Additionally shown are the densitometry bar plots derived from band density quantification for SERCA 2b KO (dark blue bars), SERCA 3 KO (light blue bars) and SERCA 2b/SERCA 3 DKO (red bars) compared to control sham transfected cells (black bars). (B,C), Jurkat T lymphocytes were loaded with Fura-2 and suspended in Ca²⁺-free media (balanced salt solution plus 2 mM EGTA) in cuvettes for use in the large cell-number configuration (1 × 10⁶ cells/mL). (B) Jurkat cell Ca²⁺ release responses in SERCA 2b_KO, SERCA 3_KO and control cells induced by the addition of TG (3 μM, arrow), as determined by the ratio of fluorescence changes at 340 and 380 nm (F340/380). (C) the same experiment as in B but with the application of low-dose TG (100 pM, arrow). (D,E) Ca²⁺ measurements were acquired from Jurkat T lymphocytes in the small cell-number configuration (30–60 cells per field) using microscope photometry (Section 4). (D) Jurkat cell Ca²⁺ release responses in SERCA 2b_KO, SERCA 3_KO and control cells induced by the addition of TG (3 μM, arrow). (E) the same experiment as in (D) but with the application of ionomycin (3 μM, arrow). (F,G) Jurkat T lymphocytes were loaded with Fura-2 and suspended in Ca²⁺-free media (balanced salt solution plus 2 mM EGTA) in cuvettes for use in the large-cell number configuration (approximately 1 × 10⁶ cells/mL). (F) Jurkat cell Ca²⁺ release and influx responses in SERCA 2b_KO, SERCA 3_KO and control cells induced by the addition of ionomycin (3 μM, arrow); Ca²⁺ influx responses were determined by the addition of Ca²⁺ (2 mM) to the cells suspended in cuvettes as indicated. (G) the same experiment as in (F) but with the application of PHA (10 μg/mL, arrow). Fluorescence traces of Ca²⁺ experiments shown are representative of three to five separate experiments with significant differences assessed via use of student t test. One-way ANOVA followed by Tukey’s multiple comparison tests were used to analyze Western blot data for SERCA expression levels with n = 3 (Section 4). Asterisks denote statistical significance with ** p < 0.005, **** p < 0.0001 and ns is not significant p > 0.05.

Figure 3

CRISPR-Mediated Double-Knockout of Jurkat Lymphocyte SERCA Pumps Severely Perturbs Ca²⁺ Stores and Further Clarifies Ca²⁺ Pool Agonist Sensitivity. (A–E) Jurkat T lymphocytes were loaded with Fura-2 and suspended in Ca²⁺-free media (balanced salt solution plus 2 mM EGTA) in cuvettes for use in the large cell-number configuration (approximately 1 × 10⁶ cells/mL). (A) Jurkat cell Ca²⁺ release responses in SERCA_DKO and control cells induced by the addition of TG (3 μM, arrow), as determined by the ratio of fluorescence changes at 340 and 380 nm (F340/380). (B) the same experiment as in (A) but with the application of ionomycin (3 μM, arrow). (C,D) Jurkat T lymphocytes were challenged with the low-dose SERCA blocker regimen. C, Jurkat cell Ca²⁺ release responses in SERCA_DKO and control cells induced by the addition of low-dose TG (100 pM, arrow), as determined by the ratio of fluorescence changes at 340 and 380 nm (F340/380). (D) the same experiment as in (C) but with the application of low-dose tBHQ (1 μM, arrow). (E) Jurkat cell Ca²⁺ release and influx responses in SERCA_DKO and control cells induced by the addition of PHA (10 μg/mL, arrow); Ca²⁺ influx responses were determined by the addition of Ca²⁺ (2 mM) to the cuvettes as indicated. Fluorescence traces of Ca²⁺ experiments shown are representative of three to six separate experiments with significant differences assessed via use of student t test.

Figure 4

SERCA Modulators and Altered SERCA Expression Levels Exert Sensitive Regulatory Influence on Jurkat T Lymphocyte Growth Responses. For (A–C), Jurkat T lymphocytes were grown in the presence of the indicated concentrations of SERCA modulators for 72 h to determine levels of cell proliferation (Section 4). (A), Effects on cell growth for cells incubated with the indicated concentrations of CDN1163 (beige bars) relative to untreated controls (black bar). (B), Effects on cell growth for cells incubated with the indicated concentrations of TG (purple bars) relative to untreated controls (black bar). (C), Effects on cell growth for cells incubated with the indicated concentrations of tBHQ (green bars) relative to untreated controls (black bar). (D), Effects of SERCA overexpression, SERCA 2b_OE (green bar) and SERCA 3_OE (purple bar) on Jurkat T lymphocyte growth responses relative to untreated controls (black bar). Additionally depicted are the effects of SERCA knockout, SERCA 2b_KO (dark blue bar), SERCA 3_KO (light blue bar) and SERCA_DKO (red bar) on Jurkat T lymphocyte growth responses relative to untreated controls (black bar). For (E,F), Jurkat cell growth responses were determined in the presence of T cell mitogens PHA and Con A for the indicated time intervals. (E), Effects on cell growth for cells incubated with the indicated concentrations of PHA (pink bars) and Con A (yellow bars) relative to untreated controls (black bar) for 72 h. (F), Effects on cell growth for cells incubated with the indicated concentrations of PHA (pink bars) and Con A (yellow bars) relative to untreated controls (black bar) for 96 h. For (G,H), Jurkat T lymphocyte growth responses to PHA stimulation were measured in cells with altered SERCA expression levels. G, Effects of SERCA 2b_OE on PHA-stimulated (pink bar) Jurkat lymphocyte growth responses compared to unstimulated SERCA 2b_OE (green bar) and sham transfected PHA-stimulated (pink bar) and unstimulated controls (black bar). Additionally depicted are the effects of SERCA 3_OE on PHA-stimulated (pink bar) Jurkat lymphocyte growth responses compared to unstimulated SERCA 3_OE (purple bar) and sham transfected PHA-stimulated (pink bar) and unstimulated controls (black bar). (H), Effects of SERCA 2b_KO on PHA-stimulated (pink bar) Jurkat lymphocyte growth responses compared to unstimulated SERCA 2b_KO (dark blue bar) and sham transfected PHA-stimulated (pink bar) and unstimulated controls (black bar). Additionally depicted are the Effects of SERCA 3_KO on PHA-stimulated (pink bar) Jurkat lymphocyte growth responses compared to unstimulated SERCA 3_KO (light blue bar) and sham transfected PHA-stimulated (pink bar) and unstimulated controls (black bar). Data are expressed as the mean ± SD, n = 3 replicates. One-way ANOVA followed by Dunnett’s multiple comparison test was used to calculate p values (A–F), except for (D), where Tukey’s multiple comparisons test were used. Two-way ANOVA employing Tukey’s multiple comparisons test was used to calculate p values (G–H). Asterisks denote statistical significance with * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001, and ns is not significant p > 0.05.

Figure 5

Altered SERCA Expression Levels Regulate Tonic and Stimulated Jurkat T Lymphocyte IL-2 Secretion. (A), Levels of IL-2 were measured in PHA-stimulated and unstimulated Jurkat T lymphocyte culture supernatants (Section 4). SERCA 2b_OE and SERCA 3_OE Jurkat cells were treated with PHA (10 μg/mL, yellow bars), and IL-2 levels were compared to unstimulated SERCA 2b_OE and SERCA 3_OE (black bars) cells. IL-2 levels from SERCA 2b_OE and SERCA 3_OE cells were also compared to untransfected control Jurkat lymphocytes from either the unstimulated (black bar) or PHA-stimulated (yellow bar) condition. (B), SERCA 2b_KO, SERCA 3_KO and SERCA_DKO Jurkat cells were treated with PHA (10 μg/mL, pink bars), and IL-2 levels were compared to unstimulated SERCA 2b_KO, SERCA 3_KO and SERCA_DKO (black bars) cells. IL-2 levels from SERCA 2b_KO, SERCA_KO and SERCA_DKO cells were also compared to untransfected control Jurkat lymphocytes from either the unstimulated (black bar) or PHA-stimulated (pink bar) condition. (C), Similar to (B), measuring IL-2 levels in SERCA knockout Jurkat lymphocytes but with testing the effects of preincubation with CDN1163 (10 μM, 24 h) in both PHA-stimulated and unstimulated conditions. IL-2 levels were determined in SERCA 2b_KO, SERCA 3_KO and SERCA_DKO cells stimulated with PHA (10 μg/mL, pink bars) and compared with levels observed in PHA-treated cells preincubated with CDN1163 (10 μM, 24 h, grey bars). The same experiment was conducted in the SERCA knockout cells with (yellow bars) or without (black bars) CDN1163 pretreatment. Levels of IL-2 in the SERCA knockout lymphocytes were also compared to the same experimental conditions performed on untransfected Jurkat lymphocytes, denoted as the control condition in the figure. Data are expressed as the mean ± SD, n = 3 replicates. Two-way ANOVA followed by Tukey’s multiple comparisons test was used to calculate p values. Asterisks denote statistical significance with **** p < 0.0001 and ns is not significant p > 0.05.

Figure 6

Altered Expression Levels Reveal SERCA Isoform-Specific Regulation on Jurkat T Lymphocyte Functional Parameters Spanning Glucose Uptake and Glutathione Antioxidant Production. (A), Glucose uptake was measured in Jurkat T lymphocytes with altered SERCA expression levels (Section 4). Glucose uptake in SERCA 2b_OE (green bar), SERCA 3_OE (purple bar), SERCA 2b_KO (dark blue bar), SERCA 3_KO (light blue bar) and SERCA_DKO (red bar) lymphocytes was measured and compared to untransfected control Jurkat lymphocytes (black bar). (B), similar to A but including PHA-stimulated effects on glucose uptake. Yellow bars denote glucose uptake, respectively, in SERCA 2b_OE (green bar), SERCA 3_OE (purple bar), SERCA 2b_KO (dark blue bar), SERCA 3_KO (light blue bar), SERCA_DKO (red bar) and control unstransfected (black bar) PHA-stimulated Jurkat lymphocytes. (C), Representative Western blot image of expression levels of the Glucose Transporter 3 (Glut3) protein in SERCA 2b_OE, SERCA 3_OE, SERCA 2b_KO and SERCA 3_KO Jurkat T lymphocytes. Figure shows the approximate molecular weight of the Glut3 protein and β-actin protein bands as control. Additionally shown are the densitometry bar plots derived from band density quantification for SERCA 2b_OE (green bar), SERCA 3_OE (purple bar), SERCA 2b_KO (dark blue bar) and SERCA 3_KO (light blue bar) cells compared to control sham transfected cells (black bars). (D), Glutathione (GSH/GSSG) levels were measured in Jurkat T lymphocytes with altered SERCA expression levels (Section 4). Glutathione levels in SERCA 2b_OE (purple bar), SERCA 3_OE (yellow bar), SERCA 2b_KO (grey bar), SERCA 3_KO (blue bar) and SERCA_DKO (orange bar) lymphocytes were measured and compared to untransfected control Jurkat lymphocytes (black bar). Data are expressed as the mean ± SD, n = 3 replicates. One-way ANOVA followed by Tukey’s multiple comparison test were used to calculate p values (A,C,D). Two-way ANOVA employing Tukey’s multiple comparisons test were used to calculate p values (B). Asterisks denote statistical significance with * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001 and ns is not significant p > 0.05.