Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α′) and two regulatory subunits (CK2β). CK2 promotes cancer progression by activating the NF-κB, PI3K/AKT/mTOR, and JAK/STAT pathways, and also is critical for immune cell development and function. The potential involvement of CK2 in CD8+ T cell function has not been explored. We demonstrate that CK2 protein levels and kinase activity are enhanced upon mouse CD8+ T cell activation. CK2α deficiency results in impaired CD8+ T cell activation and proliferation upon TCR stimulation. Furthermore, CK2α is involved in CD8+ T cell metabolic reprogramming through regulating the AKT/mTOR pathway. Lastly, using a mouse Listeria monocytogenes infection model, we demonstrate that CK2α is required for CD8+ T cell expansion, maintenance, and effector function in both primary and memory immune responses. Collectively, our study implicates CK2α as an important regulator of mouse CD8+ T cell activation, metabolic reprogramming, and differentiation both in vitro and in vivo.

The CD8+ T cells are critical in controlling infection caused by intracellular pathogens, including viruses and intracellular bacteria, and controlling cancer progression by cytolysis and recruitment and activation of other immune cells (1). In response to infection, naive CD8+ T cells differentiate into heterogeneous populations of pathogen-specific effector CD8+ T cells. Most effector cells undergo apoptosis and contract after resolution of the infection, whereas a small percentage of effector CD8+ T cells differentiate into memory cells and provide lasting protection against reinfection (24). Heterogeneous populations of effector CD8+ T cells can be distinguished by the expression of several markers, including KLRG1, IL-7Rα, CD27, CXCR3, and CD62L. For instance, in acute infection, effector CD8+ T cell pools can be distinguished as short-lived effector T cells (KLRG1hiIL-7Rαlo), which have a shorter lifespan and reduced proliferative capacity in response to secondary antigenic challenge. Conversely, memory precursor effector cells (KLRG1loIL-7Rαhi) differentiate into long-lived memory cells and proliferate vigorously in response to secondary challenge (5, 6). Current evidence suggests that multiple signals orchestrate CD8+ T cell fate decisions, including TCR, costimulatory signaling, cytokines, transcription factors, and changes in metabolism (3, 7).

Protein kinase CK2 is a highly conserved serine-threonine kinase that is expressed in all eukaryotic organisms. CK2 is often present as a tetrameric complex of two catalytic subunits (CK2α and/or CK2α′) and two regulatory subunits (CK2β) that phosphorylate serine and threonine as well as tyrosine residues on hundreds of substrates (8, 9). CK2 is involved in a wide range of biological processes and cellular functions, including cell growth, proliferation, differentiation, transcription, and translation (10, 11). Aberrant expression and high CK2 kinase activity are characteristic of many cancers, promoting tumor survival and growth, and CK2 is a promising therapeutic target for malignant diseases (1214). CK2 enhances the activity of several signaling pathways that are essential for cancer progression, including the NF-κB, PI3K/AKT/mTOR, JAK/STAT, and hypoxia inducible factor-1α (HIF-1α) pathways, which are also critical for immune cell development and function (15).

Emerging evidence further suggests that CK2 plays important roles in inflammatory responses and pathologies associated with inflammation (1620). Both Ulges et al. (16) and our group (17, 21) demonstrated that deletion of CK2 subunits, either CK2α or CK2β, resulted in significant protection in a model of multiple sclerosis, experimental autoimmune encephalomyelitis, by promoting differentiation of T regulatory cells (Tregs) and inhibiting Th17 cell differentiation. CK2α contributes to the pathogenesis of colitis by promoting CD4+ T cell proliferation and Th1 and Th17 responses (18). CK2 is also involved in the suppressive function of CD4+Foxp3+ Tregs against allergy-promoting Th2 cells (22). In addition, CK2 is critical for monocyte-derived dendritic cells to mature and produce cytokines that polarize effector T cells in response to chemicals related to allergic contact dermatitis (23). CK2α deficiency in myeloid cells increases inflammatory myeloid cell recruitment, activation, and resistance following systemic Listeria monocytogenes infection (19). However, the function of CK2 in CD8+ T cells is completely unknown.

In this study, we demonstrate that CK2 protein levels and kinase activity are enhanced upon CD8+ T cell activation and that CK2α is required for CD8+ T cell activation and proliferation upon TCR stimulation. Importantly, CK2α is involved in CD8+ T cell metabolic reprogramming during activation through regulating the AKT/mTOR signaling pathway. The function of CK2α was further explored in a L. monocytogenes infection model, and our findings indicate that CK2α is required for CD8+ T cell expansion, maintenance, and effector function in both primary and memory stages during L. monocytogenes infection. Taken together, our study demonstrates that CK2α regulates CD8+ T cell activation and differentiation both in vitro and in vivo.

CK2αfl/fl, CK2αfl/fldLck-Cre (CK2α−/−), and CD45.1 C57BL/6 mice were previously described (21). All mice were already backcrossed to C57BL/6 for at least 12 generations. CK2αfl/fl or CK2αfl/fldLck-Cre mice were further bred with OT-I mice to obtain OVA Ag-specific CD8+ T cells. All mice were maintained under specific pathogen-free conditions in the animal facility at the University of Alabama at Birmingham. Male and female mice between 8 and 12 wk old were used for all experiments. All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.

For the study of primary immune responses, CD45.1 C57BL/6 host mice were first i.v. transferred with 1 × 105 OT-I cells from either OT-I CK2αfl/fl or OT-I CK2αfl/fldLck-Cre (OT-I CK2α−/−) mice. At 1 d posttransfer, recipient mice were infected with 1 × 103 CFU of L. monocytogenes–expressing OVA (LM-OVA) (24) by i.v. injection. CD8+ T cell responses were determined at day 5 (early effector phase), day 7 (effector phase), and day 42 (early memory phase) postinfection (25). For analysis of recall immune responses, donor-derived memory CD8+ T cells were first sorted from CD45.1 host mice at the early memory phase (day 42 after primary infection). Then, OT-I CK2αfl/fl and OT-I CK2α−/− memory CD8+ T cells (5 × 103) were separately transferred into naive CD45.1 congenic recipient mice, which were then infected with 1 × 105 CFU of LM-OVA. At 7 d postinfection, CD8+ T cell responses in the spleen were analyzed.

Single-cell suspensions of spleen and lymph nodes (LNs) were prepared as previously described (18) and resuspended in R10 medium (RPMI 1640 with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 50 μM 2-ME).

For in vivo transfer, CD8+ T cells were first purified from the spleen using the EasySep mouse CD8α positive selection kit II (STEMCELL Technologies, Vancouver, BC, Canada), then naive OT-I cells were further sorted as CD8+Vβ5.1+CD44CD62L+ using FACSAria II (BD Bioscience, San Jose, CA). For memory CD8+ T cell transfer, memory OT-I cells were sorted as CD8+CD45.2+Vα2+CD44+CD62L+ on a FACSAria II, routinely to >98% purity. For in vitro activation and stimulation, naive CD8+ T cells were enriched from the spleen and peripheral LNs using the EasySep mouse naive CD8+ T cell isolation kit (STEMCELL Technologies, Vancouver, BC, Canada), routinely to 90–95% purity.

For polyclonal activation, naive CD8+ T cells were stimulated with plate-bound anti-CD3 (1 μg/ml) (clone 145-2C11, Bio X Cell, West Lebanon, NH) and soluble anti-CD28 (1 μg/ml) (clone 37.51, Bio X Cell, West Lebanon, NH) Abs for the indicated times. For Ag-specific T cell activation, splenocytes (1 × 106/ml) from OT-I CK2αfl/fl or OT-I CK2α−/− mice were cultured in R10 medium, incubated with OVA257–264 peptide (0.1 ng/ml) (InvivoGen, San Diego, CA), and then cells were harvested at the indicated time points.

For the polyclonal proliferation assay, naive CD8+ T cells were labeled with 5 μM CellTrace Violet (CTV), washed, and activated with plate-bound anti-CD3 (1 μg/ml) and soluble CD28 (1 μg/ml) Abs for 48 and 72 h. Cell proliferation indicated by CTV dilution was detected by flow cytometry. For the cell apoptosis assay, cells were stained with annexin V–allophycocyanin (BioLegend, San Diego, CA) according to the manufacturer’s protocol and analyzed by flow cytometry, as previously described (18). For the Ag-specific T cell proliferation assay, 5 μM CTV-labeled splenocytes (1 × 106/ml) from OT-I CK2αfl/fl or OT-I CK2α−/− mice were cultured in R10 medium and incubated with OVA257–264 peptide (0.1 ng/ml) for 48 h. CD8+ T cell proliferation was detected by CTV dilution.

Cell surface staining and intracellular staining were performed as previously described (18). For cytokine production analysis, cells were stimulated with 50 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) and 750 ng/ml ionomycin (Sigma-Aldrich, St. Louis, MO), or 50 ng/ml OVA257–264 peptide, in the presence of GolgiStop (BD Biosciences, San Jose, CA) for 4 h. After surface staining, cells were fixed and permeabilized using Cytofix/Cytoperm fixation/permeabilization solution kits (BD Biosciences). For intracellular staining of transcription factors or CK2α, cells were fixed and permeabilized using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (eBioscience, Grand Island, NY), as previously described (21). For phosphorylated protein detection, cells were fixed and permeabilized using Cytofix/Cytoperm fixation/permeabilization solution kits. For glucose uptake, cells were incubated with 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; 50 μg/ml) for 30 min at 37°C, washed, and stained for viability and surface markers (17). For MitoTracker Green (MTG) staining, cells were stained for viability and surface markers first, then washed and incubated with MTG (50 nM) for 30 min at 37°C. Samples were acquired on an LSR II flow cytometer using FACSDiva (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo software (Tree Star, Ashland, OR). The following Abs were used in this study (all BioLegend except where noted otherwise): anti-CD3ε PerCP-Cy5.5/PE-Cy7/allophycocyanin (clone 145-2C11); anti-CD8 Pacific Blue/Alexa Fluor 488/PE-Cy7/BUV395 (clone 53-6.7); anti-TCR Vβ5.1/5.2 PerCP/Cyanine5.5 (clone MR9-4); anti-CD45.1 Alexa Fluor 488/PerCP-Cy5.5 (clone A20); anti-CD45.2 Alexa 647/allophycocyanin-Cy7 (clone 104); anti-CD44 Alexa Fluor 488/allophycocyanin/BV786 (clone IM7); anti-CD25 Alexa Fluor 647/PE-Cy7 (clone PC61.5); anti-CD69 PE (clone H1.2F3); anti-CD62L PE/Brilliant Violet 650 (clone MEL-14); anti-CD127 (IL-7Rα) Brilliant Violet 605 (clone A7R34); anti-KLRG1 Brilliant Violet 421 (clone 2F1/KLRG1); anti-CD98 Alexa Fluor 647 (clone RL388); anti-CD71 Brilliant Violet 421 (clone C2); anti–IFN-γ Pacific Blue/PE-Cy7 (clone XMG1.2); anti–granzyme B Pacific Blue (clone GB11); anti–IL-2 Brilliant Violet 421 (clone JES6-5H4); anti-Bcl2 PE (clone 3F11, BD Biosciences); anti-Tcf7 Brilliant Violet 421 (clone S33-966, BD Biosciences), anti-Bcl6 Alexa Fluor 647 (clone K112-91, BD Biosciences); phospho-ERK1/2 (pT202/pY204) Alexa Fluor 488 (clone 20A, BD Biosciences); phospho-Akt (Ser473) allophycocyanin (clone D9E, Cell Signaling Technology); phospho-S6 ribosomal protein (Ser235/236) Pacific Blue (clone D57.2.2E, Cell Signaling Technology); c-Myc rabbit mAb (D84C12, CST); HIF-1α rabbit mAb (CST); anti-CSNK2A1 (CK2α) (Abcam); and Alexa Fluor 488 anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories).

CD8+ T cells were lysed in buffer containing 1% Triton X-100 (Sigma-Aldrich), and protein lysates were separated by electrophoresis, transferred to a nitrocellulose membrane, and then blotted with CK2α (ab76040), CK2β (ab76025) (Abcam, Cambridge, MA), CK2α′ (sc-514403) (Santa Cruz Biotechnology, Dallas, TX), and β-actin (Sigma-Aldrich, St. Louis, MO) Abs, as previously described (26).

The casein kinase 2 assay kit (Millipore) was used to assess CK2 kinase activity. Cells were lysed, and both catalytic subunits (CK2α and CK2α′) were immunoprecipitated. The lysates were assayed for CK2 kinase activity as previously described (17).

Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) measurements were performed using the Seahorse XF96 analyzer (Agilent Technologies, Santa Clara, CA). Naive CK2αfl/fl or CK2α−/− CD8+ T cells were activated with plate-bound anti-CD3 (1 μg/ml) and soluble CD28 (1 μg/ml) Abs for 24, 48, and 72 h. For the glycolytic stress test (ECAR), naive and activated cells were plated at 2 × 105 cells/well into a XF96 plate coated with Cell-Tak (Corning Life Sciences) and resuspended in assay medium (Seahorse basic DMEM supplemented with 1 mM pyruvate, 2 mM glutamine, and 5 mM HEPES [pH 7.4]). The plated cells were maintained in a non-CO2 incubator at 37°C for 1 h prior to the assay. The glycolysis stress test was conducted by subsequent injections of glucose (10 mM), oligomycin (1 μg/ml), antimycin A (10 μM), and 2-deoxyglucose (2-DG; 50 mM) as described in Hills et al. (27). The values of the indicated parameters were calculated as follows: glycolysis = ECARpostglucose ECARbasal and glycolytic capacity = ECARpostantimycin A/rotenone (AA) − ECARpost–2-DG). For the mitochondrial stress test (OCR), cells were plated at 2 × 105/well into a XF96 plate coated with Cell-Tak, and switched to XF media (DMEM supplemented with 5.5 mM glucose, 1 mM pyruvate, 2 mM glutamine, and 5 mM HEPES [pH 7.35]). OCR was measured under basal conditions and upon the sequential addition of oligomycin (1 μg/ml), fluoro-carbonyl cyanide phenylhydrazone (FCCP; 2 μM), and antimycin A (10 μM) to determine the mitochondrial respiration of cells. The value of the indicated parameters were calculated as follows: basal OCR = OCRinitial − OCRpost-AA; maximal respiration = OCRpostFCCP − OCRpost-AA; ATP-linked respiration = OCRbasal − OCRpostoligomycin; proton leak = OCRpostoligomycin − OCRpost-AA; and reserve capacity = OCRmax − OCRbasal (28). For the ex vivo assay, OT-I CK2αfl/fl and OT-I CK2α−/− CD8+ T cells were sorted from the spleen at day 7 postinfection, and then the mitochondrial stress test was performed as described above.

Total DNA was extracted from CD8+ T cells. The mitochondrial small fragment (mitochondrial DNA [mtDNA]) and nuclear 18S DNA were amplified using real-time SYBR Green PCR master mix (Fisher Scientific) in an ABI 7500. The primer sequences used for mtDNA were mt forward (5′-CCCCAGCCATAACACAGTATCAAAC-3′) and mt reverse (5′-GCCCAAAGAATCAGAACAGATGC-3′). The primer sequences for nuclear DNA were 18S forward (5′-AAACGGCTACCACATCCAAG-3′) and 18S reverse (5′-CAATTACAGGGCCTCGAAAG-3′). mtDNA copy number was normalized to amplification of an 18S nuclear amplicon (29).

RNA sequencing (RNA-seq) was performed as previously described (17). Briefly, OT-I CK2αfl/fl and OT-I CK2α−/− CD8+ T cells were sorted from the spleen at day 7 postinfection. Total RNA was extracted from FACS-sorted CD8+ T cells using the miRNeasy mini kit (Qiagen, Venlo, the Netherlands) according to the manufacturer’s protocols and submitted to GENEWIZ (South Plainfield, NJ) for RNA-seq and bioinformatics analysis. RNA-seq data were submitted to the Gene Expression Omnibus under accession number GSE175941 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE175941). Genes with a false discovery rate <0.05 with a fold change >1.5 were considered as differentially expressed genes. Further pathway analysis was performed by gene set enrichment analysis available through the Broad Institute.

For quantitative RT-PCR analysis, 500–1000 ng of RNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega) as previously described (17, 18). cDNA was subjected to quantitative RT-PCR using TaqMan primers (Thermo Fisher Scientific). Relative gene expression was calculated according to the ΔΔCt method.

Levels of significance for comparisons between two groups were determined by Student t test distribution. Multiple comparisons were performed by ordinary one-way ANOVA. The p values are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All error bars represent mean ± SD. All statistical analyses (excluding RNA-seq, described above) were performed using Prism software (GraphPad Software).

We first determined the expression patterns of CK2α and CK2α′, the catalytic subunits of CK2, and CK2β, the regulatory subunit of CK2, in naive and activated primary murine CD8+ T cells. Expression of CK2α, CK2α′, and CK2β was induced after activation with anti-CD3 and anti-CD28 Abs, and increased with time (Fig. 1A). The expression of CK2α was confirmed by flow cytometry at 24 h (Fig. 1B). Most importantly, CK2 kinase activity was enhanced upon activation with anti-CD3 and anti-CD28 Abs (Fig. 1C). These results demonstrate the induction of CK2 expression and kinase activity upon CD8+ T cell activation, which indicates a potential role of CK2 in CD8+ T cell responses.

FIGURE 1.

CK2 subunit protein expression and kinase activity are induced in CD8+ T cells upon activation, and CK2α is required for CD8+ T cell activation and proliferation. Naive CD8+ T cells were enriched from the spleen of C57BL/6 mice and activated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for the indicated time points. (A) Expression of CK2 subunits CK2α, CK2α′, and CK2β was detected by immunoblotting at the indicated time points. Actin served as a loading control. (B) CK2α expression was detected by flow cytometry at 24 h under unstimulated conditions (UN) and with anti-CD3 and anti-CD28 Abs treatment. (C) CK2 kinase activity was measured at 48 h by a casein kinase 2 assay kit. (D) Naive CD8+ T cells from CK2αfl/fl and CK2α−/− mice were activated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for 24, 48, and 72 h. Expression of CD44, CD25 and CD69 was detected by flow cytometry. Representative line graphs of 24 h are shown. (E) Percentages of CD44-, CD25-, and CD69-positive cells at the indicated time points are shown. (F) Naive CD8+ T cells were labeled with 5 μM CTV, then activated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) Abs for 48 and 72 h, and proliferation was assessed by CTV dilution. Representative line graphs are shown. (G) Dead and apoptotic cells were assessed by annexin V staining at 48 h. Representative line graphs are shown. n = 3 in each group. Each experiment was performed two times with three biological replicates per experiment. Bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

FIGURE 1.

CK2 subunit protein expression and kinase activity are induced in CD8+ T cells upon activation, and CK2α is required for CD8+ T cell activation and proliferation. Naive CD8+ T cells were enriched from the spleen of C57BL/6 mice and activated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for the indicated time points. (A) Expression of CK2 subunits CK2α, CK2α′, and CK2β was detected by immunoblotting at the indicated time points. Actin served as a loading control. (B) CK2α expression was detected by flow cytometry at 24 h under unstimulated conditions (UN) and with anti-CD3 and anti-CD28 Abs treatment. (C) CK2 kinase activity was measured at 48 h by a casein kinase 2 assay kit. (D) Naive CD8+ T cells from CK2αfl/fl and CK2α−/− mice were activated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for 24, 48, and 72 h. Expression of CD44, CD25 and CD69 was detected by flow cytometry. Representative line graphs of 24 h are shown. (E) Percentages of CD44-, CD25-, and CD69-positive cells at the indicated time points are shown. (F) Naive CD8+ T cells were labeled with 5 μM CTV, then activated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) Abs for 48 and 72 h, and proliferation was assessed by CTV dilution. Representative line graphs are shown. (G) Dead and apoptotic cells were assessed by annexin V staining at 48 h. Representative line graphs are shown. n = 3 in each group. Each experiment was performed two times with three biological replicates per experiment. Bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

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To study the function of CK2α in T cells, we generated transgenic mice in which CK2α was specifically deleted in both CD4+ and CD8+ T cells, as described previously (21). CK2α protein was undetectable in CK2α−/− CD8+ T cells upon TCR stimulation (Supplemental Fig. 1A). Although CK2α−/− mice have normal immune phenotypes with no signs of systemic or organ-specific inflammation (21), there was a decrease in the percentage of total leukocytes and absolute number of CD8+ T cells in the spleen but not LNs compared with CK2αfl/fl mice (Supplemental Fig. 1B). In addition, CK2α−/− mice had an increase in naive CD8+ T cells (CD44CD62L+) in the spleen, no change in the LNs, and a decrease in central memory CD8+ T (Tcm) cells (CD44+CD62L+), but no change of effector memory CD8+ T cells (CD44+CD62L) in the spleen and LNs compared with CK2αfl/fl mice (Supplemental Fig. 1C, 1D).

Next, we sought to determine the contribution of CK2α to CD8+ T cell activation. Upon anti-CD3 and anti-CD28 stimulation, CK2α−/− CD8+ T cells exhibited significantly lower expression levels of CD44, CD25, and CD69 compared with CK2αfl/fl CD8+ T cells (Fig. 1D, 1E). We next examined the role of CK2 in proliferation and cell survival in vitro. CD8+ T cells from CK2αfl/fl and CK2α−/− mice were labeled with CTV dye followed by anti-CD3 and anti-CD28 activation. Interestingly, CK2α−/− CD8+ T cells exhibited a significant decrease in proliferation, as determined by the frequency of cells undergoing more than two divisions at both 48 and 72 h (Fig. 1F). When we analyzed the survival of CK2αfl/fl and CK2α−/− CD8+ T cells by annexin V staining, no difference was observed (Fig. 1G). These findings indicate that CK2α regulates CD8+ T cell activation and proliferation but does not affect cell survival.

Following TCR activation, T cells undergo substantial metabolic reprogramming, specifically increases in glucose uptake and metabolism and mitochondrial function (30). To determine whether CK2α is involved in CD8+ T cell glycolytic and mitochondrial metabolic switch during activation, we first examined the function of CK2α on glycolysis. mRNA levels of glucose transporter 1 (Scl2a1), glucose transporter 3 (Scl2a3), and hexokinase 2 (Hk2) were upregulated in CK2αfl/fl CD8+ T cells upon TCR stimulation, although expression levels of these genes were significantly lower in CK2α−/− CD8+ T cells (Fig. 2A). Consistent with these findings, CK2α−/− CD8+ T cells had a decreased ability to uptake glucose as measured by 2-NBDG (Fig. 2B). Furthermore, we measured the glycolytic metabolism of naive and activated CD8+ T cells via extracellular flux analysis. After glucose supplementation, activated CK2αfl/fl CD8+ T cells promptly engaged glycolysis, as manifested by a sharp increase in the ECAR, which was further enhanced by adding oligomycin and antimycin A. Activated CK2α−/− CD8+ T cells had significantly lower responses than CK2αfl/fl CD8+ T cells (Fig. 2C, 2D, Supplemental Fig. 2A, 2B). These findings indicate that activated CK2α−/− CD8+ T cells show less glycolytic capacity than activated CK2αfl/fl CD8+ T cells, whereas there was no difference between naive CK2αfl/fl and CK2α−/− CD8+ T cells.

FIGURE 2.

CK2α is required for CD8+ T cell glycolysis and mitochondrial respiration in vitro. Naive CD8+ T cells from CK2αfl/fl and CK2α−/− mice were activated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for 24 or 48 h. (A) Gene expression levels of Glut 1 (Slc2a1), Glut 3 (Slc2a3), and Hk2 were detected by qPCR at 48 h. (B) Glucose update was measured by 2-NBDG uptake. n = 3–4 in each group. (C) Glycolysis was measured using extracellular acidification rate (ECAR) with injections of glucose (10 mM), oligomycin (1 μg/ml), antimycin A (10 μM), and 2-DG (50 mM). (D) ECAR values were pooled from two independent glycolysis stress tests (glycolysis = ECARpostglucose − ECARbasal; glycolytic capacity = ECARpost-AA − ECARpost–2-DG). (E) Oxidative phosphorylation profiles were measured by the oxygen consumption rate (OCR) with injections of oligomycin (1 μg/ml), FCCP (2 μM), and antimycin A/rotenone (AA) (10 μM/1 μM). (F) Calculation of basal OCR, ATP-linked, proton leak, maximal OCR, and reserve respiration capacity. (G) Energy map of CD8+ T cells at basal OCR and ECAR condition. Data were pooled from three independent mitochondrial stress tests. (H) Mitochondrial content of CD8+ T cells was assessed by flow cytometry with MitoTracker Green (MTG); n = 3 in each group. Mean fluorescence intensity (MFI) was normalized to corresponding untreated controls. (I) Mitochondrial DNA copy numbers relative to genomic DNA measured using qPCR; n = 3 in each group. Each experiment was performed two times with three to four technical replicates per experiment. Bars represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.

FIGURE 2.

CK2α is required for CD8+ T cell glycolysis and mitochondrial respiration in vitro. Naive CD8+ T cells from CK2αfl/fl and CK2α−/− mice were activated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for 24 or 48 h. (A) Gene expression levels of Glut 1 (Slc2a1), Glut 3 (Slc2a3), and Hk2 were detected by qPCR at 48 h. (B) Glucose update was measured by 2-NBDG uptake. n = 3–4 in each group. (C) Glycolysis was measured using extracellular acidification rate (ECAR) with injections of glucose (10 mM), oligomycin (1 μg/ml), antimycin A (10 μM), and 2-DG (50 mM). (D) ECAR values were pooled from two independent glycolysis stress tests (glycolysis = ECARpostglucose − ECARbasal; glycolytic capacity = ECARpost-AA − ECARpost–2-DG). (E) Oxidative phosphorylation profiles were measured by the oxygen consumption rate (OCR) with injections of oligomycin (1 μg/ml), FCCP (2 μM), and antimycin A/rotenone (AA) (10 μM/1 μM). (F) Calculation of basal OCR, ATP-linked, proton leak, maximal OCR, and reserve respiration capacity. (G) Energy map of CD8+ T cells at basal OCR and ECAR condition. Data were pooled from three independent mitochondrial stress tests. (H) Mitochondrial content of CD8+ T cells was assessed by flow cytometry with MitoTracker Green (MTG); n = 3 in each group. Mean fluorescence intensity (MFI) was normalized to corresponding untreated controls. (I) Mitochondrial DNA copy numbers relative to genomic DNA measured using qPCR; n = 3 in each group. Each experiment was performed two times with three to four technical replicates per experiment. Bars represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.

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Activated T cells show a considerable increase in mitochondrial respiration (31). Next, we sought to test whether CK2α was involved in mitochondrial respiration in CD8+ T cells by analyzing the OCR. Activated CK2α−/− CD8+ T cells showed significantly lower levels of basal OCR, ATP-linked, proton leak, maximal, and reserve capacity OCR compared with activated CK2αfl/fl CD8+ T cells, whereas no significant difference was observed between naive CK2αfl/fl and CK2α−/− CD8+ T cells (Fig. 2E, 2F, Supplemental Fig. 2C, 2D). CK2α−/− CD8+ T cells failed to complete metabolic reprogramming to achieve the aerobic glycolytic phenotype characteristic of T cell activation, as shown by the energy map (Fig. 2G, Supplemental Fig. 2E). Mitochondrial mass and mtDNA also increase during T cell activation (32). We next sought to define whether CK2α regulated mitochondrial mass and mtDNA during activation. Our data demonstrated that activated CK2α−/− CD8+ T cells had significantly less mitochondrial mass as indicated by staining with MTG compared with activated CK2αfl/fl CD8+ T cells (Fig. 2H). Also, CK2α−/− CD8+ T cells had less mtDNA compared with activated CK2αfl/fl CD8+ T cells (Fig. 2I). These findings support the involvement of CK2α in CD8+ T cell metabolic reprogramming during TCR activation.

Our previous studies showed that CK2α regulated AKT/mTOR activity in CD4+ T cells (17, 21). mTOR is a protein kinase that acts as a central integrator of various environmental cues and is able to regulate multiple cellular processes accordingly (33). As the mTOR pathway is a major pathway regulating T cell metabolism (33), we sought to test whether CK2α regulated CD8+ T cell metabolism through mTOR signaling. Expression levels of mTORC1-dependent cell surface markers CD71 and CD98 were significantly decreased in CK2α−/− CD8+ T cells compared with CK2αfl/fl CD8+ T cells upon activation (Fig. 3A, 3B). In addition, phosphorylation of the downstream target of mTORC1, ribosomal protein S6, was decreased in CK2α−/− CD8+ T cells compared with CK2αfl/fl CD8+ T cells (Fig. 3C). CK2α−/− CD8+ T cells also exhibited a significant decrease in phosphorylation of Akt at S473, which is essential for Akt activity (34) (Fig. 3D). It is well known that mTOR signaling pathways modulate T cell metabolism through controlling HIF-1α and c-Myc (35, 36). HIF-1α and c-Myc promote and regulate glycolysis and mitochondrial biogenesis (35). CK2α−/− CD8+ T cells showed significant decreases in HIF-1α and c-Myc compared with CK2αfl/fl CD8+ T cells upon activation (Fig. 3E, 3F). Furthermore, phosphorylation levels of ERK1/2 in CK2α−/− CD8+ T cells were significantly lower compared with CK2αfl/fl CD8+ T cells (Fig. 3G), which indicates that CK2 may control CD8+ T cell activation by regulating ERK1/2 activation. Altogether, these findings suggest that CK2α plays an important role in regulating CD8+ T cell metabolism reprogramming through promoting AKT/mTOR and ERK1/2 signaling and regulating HIF-1α and c-Myc expression in CD8+ T cells upon activation.

FIGURE 3.

CK2α controls CD8+ T cell metabolic reprogramming by regulating the AKT/mTOR pathway. Naive CD8+ T cells from CK2αfl/fl and CK2α−/− mice were activated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for 24 h. (A) CD71 and (B) CD98 expression as detected by flow cytometry is shown. (C) Phosphorylated S6 ribosomal protein S235/236 and (D) AKT S473 were detected by flow cytometry. (E) HIF-1α, (F) c-Myc, and (G) ERK1/2pT202/pY204 expression was detected by flow cytometry. n = 3–5 in each group. Each experiment was performed two times with two to three biological replicates per experiment; each dot indicates one mouse. Bars represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

CK2α controls CD8+ T cell metabolic reprogramming by regulating the AKT/mTOR pathway. Naive CD8+ T cells from CK2αfl/fl and CK2α−/− mice were activated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for 24 h. (A) CD71 and (B) CD98 expression as detected by flow cytometry is shown. (C) Phosphorylated S6 ribosomal protein S235/236 and (D) AKT S473 were detected by flow cytometry. (E) HIF-1α, (F) c-Myc, and (G) ERK1/2pT202/pY204 expression was detected by flow cytometry. n = 3–5 in each group. Each experiment was performed two times with two to three biological replicates per experiment; each dot indicates one mouse. Bars represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

To determine the function of CK2α in regulating CD8+ T cell responses to infection, we crossed CK2α−/− mice to OT-I TCR transgenic mice, whose TCR recognizes the SIINFEKL epitope from chicken OVA (37). Similar to the phenotype observed in polyclonal CD8+ T cells, CK2α−/− OT-I cells exhibited significantly lower expression levels of CD44, CD25, and CD69 compared with CK2αfl/fl OT-I cells upon OVA257–263 peptide stimulation (Supplemental Fig. 3A, 3B). In addition, CK2α deficiency impaired OT-I cell proliferation (Supplemental Fig. 3C).

We transferred CD45.2 CK2αfl/fl or CK2α−/− OT-I CD8+ T cells into CD45.1 congenic C57BL/6 recipients, followed by infection with LM-OVA, and analyzed CD8+ T cell responses at day 7 postinfection (Fig. 4A). The frequencies of CK2α−/− OT-I cells in the peripheral blood and spleen were significantly lower than CK2αfl/fl OT-I cells, as were the absolute numbers of CK2α−/− OT-I cells in the spleen compared with CK2αfl/fl OT-I cells (Fig. 4B, 4C). We next examined whether CK2α deficiency affected CD8+ T cell differentiation. CD62L and KLRG1 expression was used to classify different subsets of CD8+ T cells into CD62L+ Tcm cells, CD62LKLRG1 effector cells, and KLRG1+ terminally differentiated effector (TD) cells (3). We observed a decrease in TD KLRG1+ cells and an increase in CD62L+ Tcm cells in CK2α−/− OT-I cells compared with CK2αfl/fl OT-I cells at day 7 postinfection (Fig. 4D). No change was observed in effector cells (Fig. 4D). Expression of the cytotoxic-related molecule granzyme B was significantly reduced in CK2α−/− OT-I cells (Fig. 4E). Furthermore, upon restimulation with OVA peptide, CK2α−/− OT-I cells produced similar levels of IFN-γ, but increased IL-2 production compared with CK2αfl/fl OT-I cells (Fig. 4F). In particular, there was an increase in IFN-γ+IL-2+–producing cells in CK2α−/− OT-I cells, which suggests that CK2α−/− OT-I cells have more potential to develop into memory cells (38).

FIGURE 4.

CK2α promotes CD8+ T cell effector responses during LM-OVA infection. (A) Naive CD8+ T cells (1 × 105) (CD45.2+) from OT-I CK2αfl/fl or OT-I CK2α−/− mice were adoptively transferred into age- and sex-matched CD45.1 C57BL/6 mice. Twenty-four hours later, recipients were infected with 1 × 103 CFU of LM-OVA by i.v. injection. Donor CD8+ T cells from the blood and spleen were analyzed by flow cytometry at day 7. (B) Percentages of OT-I CK2αfl/fl- or OT-I CK2α−/−-derived CD8+ T cells in total leukocytes in the blood of recipients at day 7 postinfection. (C) Percentages and numbers of donor CD8+ T cells in the spleen at day 7 postinfection. n = 12 in each group. (D) Central memory T (Tcm) cells, effector (Eff) cells, and terminally differentiated effector (TD) cells were detected by CD62L and KLRG1 staining of donor CD8+ T cells. (E) Granzyme B expression was detected in donor CD8+ T cells by intracellular staining. (F) IFN-γ and IL-2 expression was detected in donor CD8+ T cells by intracellular staining. Quantitation of IFN-γIL-2+, IFN-γ+IL-2+, IFN-γ+IL-2, and IFN-γIL-2 is shown. n = 4–6 in each group. (G) RNA-seq of OT-I CK2αfl/fl and OT-I CK2α−/− CD8+ T cells from the spleen at day 7 postinfection. A summary of genes differentially regulated by CK2α using the following cutoffs is shown: p < 0.05, fold change >1.5. (H) Heatmap shows relative gene expression. (I) Real-time PCR analysis of the indicated genes. n = 4 in each group. Each experiment was performed at least two times with two to four biological replicates per experiment; each dot indicates one mouse. Bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

CK2α promotes CD8+ T cell effector responses during LM-OVA infection. (A) Naive CD8+ T cells (1 × 105) (CD45.2+) from OT-I CK2αfl/fl or OT-I CK2α−/− mice were adoptively transferred into age- and sex-matched CD45.1 C57BL/6 mice. Twenty-four hours later, recipients were infected with 1 × 103 CFU of LM-OVA by i.v. injection. Donor CD8+ T cells from the blood and spleen were analyzed by flow cytometry at day 7. (B) Percentages of OT-I CK2αfl/fl- or OT-I CK2α−/−-derived CD8+ T cells in total leukocytes in the blood of recipients at day 7 postinfection. (C) Percentages and numbers of donor CD8+ T cells in the spleen at day 7 postinfection. n = 12 in each group. (D) Central memory T (Tcm) cells, effector (Eff) cells, and terminally differentiated effector (TD) cells were detected by CD62L and KLRG1 staining of donor CD8+ T cells. (E) Granzyme B expression was detected in donor CD8+ T cells by intracellular staining. (F) IFN-γ and IL-2 expression was detected in donor CD8+ T cells by intracellular staining. Quantitation of IFN-γIL-2+, IFN-γ+IL-2+, IFN-γ+IL-2, and IFN-γIL-2 is shown. n = 4–6 in each group. (G) RNA-seq of OT-I CK2αfl/fl and OT-I CK2α−/− CD8+ T cells from the spleen at day 7 postinfection. A summary of genes differentially regulated by CK2α using the following cutoffs is shown: p < 0.05, fold change >1.5. (H) Heatmap shows relative gene expression. (I) Real-time PCR analysis of the indicated genes. n = 4 in each group. Each experiment was performed at least two times with two to four biological replicates per experiment; each dot indicates one mouse. Bars represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

To identify CK2α-dependent transcriptional programs in CD8+ T cells, CK2αfl/fl and CK2α−/− OT-I cells from recipient mice were sorted at day 7 after LM-OVA infection and subjected to RNA-seq. Using a setting of p < 0.05 and fold change >1.5 to define significant differences in differentially expressed genes, 153 genes were upregulated and 240 genes downregulated as a result of CK2α deficiency (Fig. 4G). CK2α−/− OT-I cells showed decreased expression of effector function–related molecules, including Klgr1, Prf1, Gzmb, and IL12rb2 (Fig. 4H). These data are consistent with the flow staining analyses (Fig. 4D, 4E). Furthermore, CK2α−/− OT-I cells showed a memory-like transcriptional signature (39), as evidenced by the downregulation of genes that regulate effector cell differentiation, including Zeb2, Prdm1, Id2, and Bcl2, and upregulation of genes that control memory cell development, including Id3, Nsg2, and Tcf7 (Fig. 4H). The expression of selected genes was validated by quantitative PCR (qPCR) (Fig. 4I).

We noticed that CK2α−/− OT-I cells contain twice as many CD62L+ (Tcm) and fewer KLGR1+ (TD) cells compared with CK2αfl/fl OT-I cells (Fig. 4D). To rule out the possibility that the RNA-seq data were only reflective of population differences, we verified certain key genes identified in (Fig. 4H at day 5 postinfection (Supplemental Fig. 4), which is the early effector phase with fewer KLRG1+ cells. Consistent with day 7, the frequencies and absolute number of CK2α−/− OT-I cells in the spleen were significantly lower than CK2αfl/fl OT-I cells at day 5 postinfection (Supplemental Fig. 4B). Importantly, expression of the activation marker CD25 and effector marker KLRG1 was significantly lower on CK2α−/− OT-I cells compared with CK2αfl/fl OT-I cells (Supplemental Fig. 4C, 4D). Expression of granzyme B was significantly reduced in CK2α−/− OT-I cells (Supplemental Fig. 4E), whereas IFN-γ–producing cells were comparable between CK2α−/− OT-I and CK2αfl/fl OT-I cells (Supplemental Fig. 4F). Moreover, transcription factors Tcf7 and Bcl6, which control memory cell development, were significantly increased in CK2α−/− OT-I cells (Supplemental Fig. 4G, 4H). Taken together, our data verified that CK2α is also critical for the CD8+ T cell response in the early effector phase, and it is more likely our RNA-seq data reflect the key gene differences derived from T cell programing rather than population changes.

Furthermore, expression of the prosurvival protein Bcl2 in CK2αfl/fl OT-I and CK2α−/− OT-I cells was detected by intracellular staining at day 7 postinfection, showing that the expression level of Bcl2 in CK2α−/− OT-I cells was significantly lower compared with CK2αfl/fl OT-I cells (Supplemental Fig. 4I). We used annexin V and Live/Dead costaining to detect live, early apoptotic, and late apoptotic cells. The percentage of late apoptotic cells among CK2α−/− OT-I cells was significantly higher compared with CK2αfl/fl OT-I cells (Supplemental Fig. 4J). These data indicate that CK2 controls CD8+ T cell cellularity by regulating cell survival. Furthermore, gene set enrichment analysis based on the RNA-seq data revealed that differentially expressed genes are associated with mitochondrial proteins, including components of the respiratory chain and oxidoreductase complexes (Supplemental Fig. 4K). It is known that memory CD8+ T cells exhibit enhanced oxidative phosphorylation and fatty acid oxidation compared with effector CD8+ T cells (40). Our extracellular flux assays show that CK2α−/− OT-I cells have a higher basal and ATP-linked OCR compared with CK2αfl/fl OT-I cells (Supplemental Fig. 4L), which suggests that CK2α regulates CD8+ T cell function in a manner that engages mitochondrial metabolism during infection. Taken together, our findings demonstrate that deletion of CK2α in CD8+ T cells affects cell expansion, differentiation, and effector functions in response to L. monocytogenes infection.

To investigate the role of CK2α in the control of memory T cell differentiation, we assessed the CD8+ T cell response in the spleen at day 42 postinfection (Fig. 5A). The percentage and absolute number of CK2α−/− OT-I cells were significantly lower compared with CK2αfl/fl OT-I cells (Fig. 5B). Interestingly, no difference was detected in the differentiation of CD8+ T cell subsets (Fig. 5C). As for cytokine production capacity, IFN-γ production by CK2α−/− OT-I cells was significantly lower than by CK2αfl/fl OT-I cells upon restimulation with OVA peptide (Fig. 5D), but no difference was observed in IL-2 production (data not shown). Expression of Bcl2 was significantly decreased in CK2α−/− OT-I cells compared with CK2αfl/fl OT-I cells (Fig. 5E). These findings suggest that CK2α is required for maintenance of the memory CD8+ T cell pool.

FIGURE 5.

CK2α is required for CD8+ T cell memory maintenance and function. (A) Naive CD8+ T cells (1 × 105) (CD45.2+) from OT-I CK2αfl/fl or OT-I CK2α−/− mice were adoptively transferred into age- and sex-matched CD45.1 C57BL/6 mice. Twenty-four hours later, recipients were infected with 1 × 103 CFU of LM-OVA by i.v. injection. Donor CD8+ T cells from the spleen were analyzed by flow cytometry at day 42. (B) Percentages and numbers of donor CD8+ T cells in the spleen at day 42 postinfection. (C) Central memory T (Tcm) cells, effector (Eff) cells, and terminally differentiated effector (TD) cells were detected by CD62L and KLRG1 staining of donor CD8+ T cells. (D) IFN-γ production was detected in donor CD8+ T cells by intracellular staining. (E) Bcl2 expression was detected in donor CD8+ T cells by intracellular staining. n = 5–6 in each group. (F) Memory CD8+ T cells (5 × 103) (CD8+CD45.2+Vα2+CD44+CD62L+) from OT-I CK2αfl/fl or OT-I CK2α−/− mice were adoptively transferred into CD45.1 C57BL/6 mice. One day later, recipients were infected with 1 × 105 CFU of LM-OVA by i.v. injection. Secondary expansion of memory CD8+ T cells in the spleens was detected at day 7. (G) Percentages and absolute numbers of OT-I CK2αfl/fl or OT-I CK2α−/− CD8+ T cells in the spleen were analyzed by flow cytometry at day 7. (H) Granzyme B, (I) IFN-γ, and (J) Bcl2 expression in OT-I CK2αfl/fl or OT-I CK2α−/− CD8+ T cells was detected by intracellular staining. n = 6–8 in each group. Each experiment was performed at least two times with three or four biological replicates per experiment; each dot indicates one mouse. Bars represent mean ± SD. *p < 0.05, ***p < 0.001, ****p < 0.0001. ns, not significant.

FIGURE 5.

CK2α is required for CD8+ T cell memory maintenance and function. (A) Naive CD8+ T cells (1 × 105) (CD45.2+) from OT-I CK2αfl/fl or OT-I CK2α−/− mice were adoptively transferred into age- and sex-matched CD45.1 C57BL/6 mice. Twenty-four hours later, recipients were infected with 1 × 103 CFU of LM-OVA by i.v. injection. Donor CD8+ T cells from the spleen were analyzed by flow cytometry at day 42. (B) Percentages and numbers of donor CD8+ T cells in the spleen at day 42 postinfection. (C) Central memory T (Tcm) cells, effector (Eff) cells, and terminally differentiated effector (TD) cells were detected by CD62L and KLRG1 staining of donor CD8+ T cells. (D) IFN-γ production was detected in donor CD8+ T cells by intracellular staining. (E) Bcl2 expression was detected in donor CD8+ T cells by intracellular staining. n = 5–6 in each group. (F) Memory CD8+ T cells (5 × 103) (CD8+CD45.2+Vα2+CD44+CD62L+) from OT-I CK2αfl/fl or OT-I CK2α−/− mice were adoptively transferred into CD45.1 C57BL/6 mice. One day later, recipients were infected with 1 × 105 CFU of LM-OVA by i.v. injection. Secondary expansion of memory CD8+ T cells in the spleens was detected at day 7. (G) Percentages and absolute numbers of OT-I CK2αfl/fl or OT-I CK2α−/− CD8+ T cells in the spleen were analyzed by flow cytometry at day 7. (H) Granzyme B, (I) IFN-γ, and (J) Bcl2 expression in OT-I CK2αfl/fl or OT-I CK2α−/− CD8+ T cells was detected by intracellular staining. n = 6–8 in each group. Each experiment was performed at least two times with three or four biological replicates per experiment; each dot indicates one mouse. Bars represent mean ± SD. *p < 0.05, ***p < 0.001, ****p < 0.0001. ns, not significant.

Close modal

We next sought to assess the function of CK2α in the quality of CD8+ T cell memory in secondary responses. We transferred the same number of sorted CK2αfl/fl OT-I or CK2α−/− OT-I memory cells at day 42 postinfection into naive CD45.1 congenic recipients (Fig. 5F). After infection with 1 × 105 CFU of LM-OVA, secondary proliferation of CK2α−/− OT-I cells was impaired in the spleen (Fig. 5G). Expression of granzyme B was decreased in CK2α−/− OT-I cells compared with CK2αfl/fl OT-I cells (Fig. 5H), whereas no difference was observed in IFN-γ production (Fig. 5I). Bcl2 expression in CK2α−/− OT-I cells was significantly lower compared with CK2αfl/fl OT-I cells (Fig. 5J). These data suggest that CK2α is critical for the fitness of memory CD8+ T cells, not only in the maintenance of cell number but also for their expansion to secondary challenge.

In this study, we identify a critical role for CK2α in regulating CD8+ T cell activation, proliferation, differentiation, and function both in vitro and in vivo. CK2 kinase activity is induced in CD8+ T cells upon TCR stimulation and is required for CD8+ T cell activation and proliferation in vitro. To explore the function of CK2 in CD8+ T cell responses in vivo, we introduced a mouse model to abrogate CK2α expression in Ag-specific CD8+ T cells in response to LM-OVA infection. Our findings demonstrate that CK2α−/− mice develop significantly less effector and memory T cells, and they have impaired memory responses against bacterial rechallenge.

Our previous findings identified that treatment with CX-4945, a small molecule inhibitor of CK2, results in a shift from a CD4+ Th17-associated glycolytic metabolic gene expression profile to expression of genes associated with oxidative metabolism, preferred by Tregs in vitro (17). These findings suggest that CK2α controls CD4+ T cell fate by regulating the metabolic switch enabling aerobic glycolysis (17). In this study, we demonstrate that CK2α is involved in CD8+ T cell metabolic reprogramming upon TCR stimulation by regulating both glycolysis and mitochondrial respiration. CK2α regulates CD8+ T cell glycolysis by controlling the expression of glucose transporters and key enzymes of the glycolytic pathway, which are important for glucose uptake. Mechanistically, CK2α controls metabolic reprogramming in CD8+ T cells through regulating the activity of AKT/mTOR, ERK1/2, and c-Myc, which are the primary regulators of early metabolic changes associated with T cell activation and differentiation (35, 36, 41). However, the ex vivo flux assay showed that CK2α-deficient CD8+ T cells exhibited enhanced mitochondrial function. The explanation for this inconsistency in CK2 function on CD8+ T cell metabolism between in vitro and ex vivo is not clear. One possibility is the fact that the population of cells generated in vivo was exposed to asynchronous stimulation over a different time course compared with in vitro activation. Although the exact role of CK2α in CD8+ T cell metabolism needs to be explored further, our findings suggest that CK2α is involved in T cell metabolic reprogramming, which is important for CD8+ T cell function and differentiation.

Previous studies identified a transcriptional network regulating effector to memory CD8+ T cell development. Expression or activation of T-bet, Blimp-1, Id2, and STAT4 drives CD8+ T cell terminal differentiation, while the expression of Eomes, Bcl-6, Id3, and STAT3 promotes memory CD8+ T cell formation (3, 4246). In our study, we found that CD8+ T cells developed into more central memory CD8+ T cells in the absence of CK2α, accompanied by a decrease in granzyme B expression and an increase in IL-2 production. These findings indicate that CK2α regulates CD8+ T cell effector and memory differentiation. To characterize transcriptional features in CD8+ T cells in the presence and absence of CK2α, we performed gene expression profiling in CK2αfl/fl and CK2α−/− CD8+ T cells at day 7 postinfection with L. monocytogenes. Consistent with previous studies (4345), expression levels of Id2 and Blimp1 decreased and the level of Id3 increased in CK2α−/− CD8+ T cells. Based on gene expression profiling, CK2α−/− CD8+ T cells exhibited a memory signature. These findings suggest that CK2α may regulate CD8+ T cell effector and memory differentiation through controlling these key transcription factors. However, the precise mechanism of how CK2α regulates these transcription factors and their activity will be further studied.

Our study has demonstrated an essential role for CK2α in maintaining the CD8+ T cell memory pool and regulating the recall response, which is vital in the immune response against infection. Although CK2α−/− CD8+ T cells at day 7 postinfection exhibited more potential to develop into memory cells, CK2α−/− CD8+ T cells had compromised memory cell numbers at day 42 postinfection compared with CK2αfl/fl CD8+ T cells. These findings do not necessarily contradict each other. One explanation is that the initial total number of memory precursor cells is still lower in CK2α−/− mice compared with CK2αfl/fl mice at day 7 postinfection, which eventually results in less memory CK2α−/− CD8+ T cells. Another explanation is linked with cell survival. Our data demonstrated that the expression of the prosurvival gene Bcl2 was significant lower in CK2α−/− memory CD8+ T cells, which indicates that CK2α may control CD8+ T cell responses by regulating cell survival. Although our in vitro data suggested that CK2α is not critical for CD8+ T cell survival upon TCR stimulation, for the CD8+ T cell response against infection in vivo, this was not the case. A possible explanation is that additional CK2-dependent survival signals are required for CD8+ T cell survival in vivo but not in vitro. Moreover, our data are consistent with findings that using a CK2 inhibitor downregulates Bcl2 expression in a leukemia cell line, whereas overexpression of CK2 induces Bcl2 expression (47). However, whether CK2 directly regulates Bcl2 expression is still unclear.

As noted above, CK2α−/− naive and memory CD8+ T cells exhibited compromised expansion capacity and expressed less granzyme B during primary and secondary challenge, respectively. However, the biological function of CK2α in CD8+ T cells, particularly CD8+ T cell cytotoxic function, remains to be explored. A recent study showed that CK2 is enhanced after SARS-CoV-2 infection (48), and CX-4945, an inhibitor of CK2, is being tested in a phase II clinical trial for COVID-19 patients, potentially targeting infected cells. Our data suggest that CK2 is vital for CD8+ T cell function, which may affect bacterial and viral clearance. Conversely, LysMcreCK2αfl/fl mice, in which CK2α is specifically deleted in myeloid cells, are more resistant to systemic L. monocytogenes infection (19), which indicates that CK2α has a divergent function in myeloid cells compared with CD8+ T cells related to pathogen infection. Therefore, systematic CK2 inhibition may compromise the function of CD8+ T cells, while enhancing the function of myeloid cells, with the ultimate biological consequence uncertain. Our study highlights the complexity of CK2 biology with respect to CD8+ T cell function and underlines caution for therapeutic use of CK2 inhibitors.

We thank the Comprehensive Flow Cytometry Core at the University of Alabama at Birmingham for assistance with flow cytometry.

This work was supported by National Institutes of Health Grants R01NS057563 and R01CA194414 (to E.N.B.), R21AI142641 and R01DK125870 (to L.E.H.), R01AG050886R01 (to J.Z. and V.M.D.-U.), and National Multiple Sclerosis Society Grant RG-1606-24794 (to H.Q.).

The online version of this article contains supplemental material.

The RNA sequencing data presented in this article have been submitted to the Gene Expression Omnibus under accession number GSE175941.

Abbreviations used in this article:

AA

antimycin A/rotenone

CTV

CellTrace Violet

2-DG

2-deoxyglucose

ECAR

extracellular acidification rate

FCCP

fluoro-carbonyl cyanide phenylhydrazone

HIF-1α

hypoxia inducible factor-1α

LM-OVA

L. monocytogenes-expressing OVA

LN

lymph node

MTG

MitoTracker Green

2-NBDG

2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose

OCR

oxygen consumption rate

qPCR

quantitative PCR

RNA-seq

RNA sequencing

Tcm

central memory T

TD

terminally differentiated effector

Treg

T regulatory cell

1.
Zhang
N.
,
M. J.
Bevan
.
2011
.
CD8+ T cells: foot soldiers of the immune system.
Immunity
35
:
161
168
.
2.
Ahmed
R.
,
D.
Gray
.
1996
.
Immunological memory and protective immunity: understanding their relation.
Science
272
:
54
60
.
3.
Kaech
S. M.
,
W.
Cui
.
2012
.
Transcriptional control of effector and memory CD8+ T cell differentiation.
Nat. Rev. Immunol.
12
:
749
761
.
4.
Williams
M. A.
,
M. J.
Bevan
.
2007
.
Effector and memory CTL differentiation.
Annu. Rev. Immunol.
25
:
171
192
.
5.
Kaech
S. M.
,
J. T.
Tan
,
E. J.
Wherry
,
B. T.
Konieczny
,
C. D.
Surh
,
R.
Ahmed
.
2003
.
Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells.
Nat. Immunol.
4
:
1191
1198
.
6.
Joshi
N. S.
,
W.
Cui
,
A.
Chandele
,
H. K.
Lee
,
D. R.
Urso
,
J.
Hagman
,
L.
Gapin
,
S. M.
Kaech
.
2007
.
Inflammation directs memory precursor and short-lived effector CD8+ T cell fates via the graded expression of T-bet transcription factor.
Immunity
27
:
281
295
.
7.
Zhang
L.
,
P.
Romero
.
2018
.
Metabolic control of CD8+ T cell fate decisions and antitumor immunity.
Trends Mol. Med.
24
:
30
48
.
8.
Litchfield
D. W.
2003
.
Protein kinase CK2: structure, regulation and role in cellular decisions of life and death.
Biochem. J.
369
:
1
15
.
9.
Dominguez
I.
,
G. E.
Sonenshein
,
D. C.
Seldin
.
2009
.
Protein kinase CK2 in health and disease: CK2 and its role in Wnt and NF-κB signaling: linking development and cancer.
Cell. Mol. Life Sci.
66
:
1850
1857
.
10.
Duncan
J. S.
,
D. W.
Litchfield
.
2008
.
Too much of a good thing: the role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2.
Biochim. Biophys. Acta
1784
:
33
47
.
11.
Singh
N. N.
,
D. P.
Ramji
.
2008
.
Protein kinase CK2, an important regulator of the inflammatory response?
J. Mol. Med. (Berl.)
86
:
887
897
.
12.
Rabalski
A. J.
,
L.
Gyenis
,
D. W.
Litchfield
.
2016
.
Molecular pathways: emergence of protein kinase CK2 (CSNK2) as a potential target to inhibit survival and DNA damage response and repair pathways in cancer cells.
Clin. Cancer Res.
22
:
2840
2847
.
13.
Borgo
C.
,
M.
Ruzzene
.
2021
.
Protein kinase CK2 inhibition as a pharmacological strategy.
Adv. Protein Chem. Struct. Biol.
124
:
23
46
.
14.
Silva-Pavez
E.
,
J. C.
Tapia
.
2020
.
Protein kinase CK2 in cancer energetics.
Front. Oncol.
10
:
893
.
15.
Husain
K.
,
T. T.
Williamson
,
N.
Nelson
,
T.
Ghansah
.
2021
.
Protein kinase 2 (CK2): a potential regulator of immune cell development and function in cancer.
Immunol. Med.
44
:
159
174
.
16.
Ulges
A.
,
E. J.
Witsch
,
G.
Pramanik
,
M.
Klein
,
K.
Birkner
,
U.
Bühler
,
B.
Wasser
,
F.
Luessi
,
N.
Stergiou
,
S.
Dietzen
, et al
2016
.
Protein kinase CK2 governs the molecular decision between encephalitogenic TH17 cell and Treg cell development.
Proc. Natl. Acad. Sci. USA
113
:
10145
10150
.
17.
Gibson
S. A.
,
W.
Yang
,
Z.
Yan
,
Y.
Liu
,
A. L.
Rowse
,
A. S.
Weinmann
,
H.
Qin
,
E. N.
Benveniste
.
2017
.
Protein kinase CK2 controls the fate between Th17 cell and regulatory T cell differentiation.
J. Immunol.
198
:
4244
4254
.
18.
Yang
W.
,
S. A.
Gibson
,
Z.
Yan
,
H.
Wei
,
J.
Tao
,
B.
Sha
,
H.
Qin
,
E. N.
Benveniste
.
2020
.
Protein kinase 2 (CK2) controls CD4+ T cell effector function in the pathogenesis of colitis.
Mucosal Immunol.
13
:
788
798
.
19.
Larson
S. R.
,
N.
Bortell
,
A.
Illies
,
W. J.
Crisler
,
J. L.
Matsuda
,
L. L.
Lenz
.
2020
.
Myeloid cell CK2 regulates inflammation and resistance to bacterial infection.
Front. Immunol.
11
:
590266
.
20.
Gibson
S. A.
,
E. N.
Benveniste
.
2018
.
Protein kinase CK2: an emerging regulator of immunity.
Trends Immunol.
39
:
82
85
.
21.
Gibson
S. A.
,
W.
Yang
,
Z.
Yan
,
H.
Qin
,
E. N.
Benveniste
.
2018
.
CK2 controls Th17 and regulatory T cell differentiation through inhibition of FoxO1.
J. Immunol.
201
:
383
392
.
22.
Ulges
A.
,
M.
Klein
,
S.
Reuter
,
B.
Gerlitzki
,
M.
Hoffmann
,
N.
Grebe
,
V.
Staudt
,
N.
Stergiou
,
T.
Bohn
,
T. J.
Brühl
, et al
2015
.
Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo.
Nat. Immunol.
16
:
267
275
.
23.
de Bourayne
M.
,
Y.
Gallais
,
Z.
El Ali
,
P.
Rousseau
,
M. H.
Damiens
,
C.
Cochet
,
O.
Filhol
,
S.
Chollet-Martin
,
M.
Pallardy
,
S.
Kerdine-Römer
.
2017
.
Protein kinase CK2 controls T-cell polarization through dendritic cell activation in response to contact sensitizers.
J. Leukoc. Biol.
101
:
703
715
.
24.
Harrington
L. E.
,
K. M.
Janowski
,
J. R.
Oliver
,
A. J.
Zajac
,
C. T.
Weaver
.
2008
.
Memory CD4 T cells emerge from effector T-cell progenitors.
Nature
452
:
356
360
.
25.
Kim
M. V.
,
W.
Ouyang
,
W.
Liao
,
M. Q.
Zhang
,
M. O.
Li
.
2013
.
The transcription factor Foxo1 controls central-memory CD8+ T cell responses to infection.
Immunity
39
:
286
297
.
26.
Zheng
Y.
,
B. C.
McFarland
,
D.
Drygin
,
H.
Yu
,
S. L.
Bellis
,
H.
Kim
,
M.
Bredel
,
E. N.
Benveniste
.
2013
.
Targeting protein kinase CK2 suppresses prosurvival signaling pathways and growth of glioblastoma.
Clin. Cancer Res.
19
:
6484
6494
.
27.
Hill
B. G.
,
G. A.
Benavides
,
J. R.
Lancaster
Jr.
,
S.
Ballinger
,
L.
Dell’Italia
,
Z.
Jianhua
,
V. M.
Darley-Usmar
.
2012
.
Integration of cellular bioenergetics with mitochondrial quality control and autophagy.
Biol. Chem.
393
:
1485
1512
.
28.
Dranka
B. P.
,
G. A.
Benavides
,
A. R.
Diers
,
S.
Giordano
,
B. R.
Zelickson
,
C.
Reily
,
L.
Zou
,
J. C.
Chatham
,
B. G.
Hill
,
J.
Zhang
, et al
2011
.
Assessing bioenergetic function in response to oxidative stress by metabolic profiling.
Free Radic. Biol. Med.
51
:
1621
1635
.
29.
Quiros
P. M.
,
A.
Goyal
,
P.
Jha
,
J.
Auwerx
.
2017
.
Analysis of mtDNA/nDNA ratio in mice.
Curr. Protoc. Mouse Biol.
7
:
47
54
.
30.
Chang
C. H.
,
J. D.
Curtis
,
L. B.
Maggi
Jr.
,
B.
Faubert
,
A. V.
Villarino
,
D.
O’Sullivan
,
S. C.
Huang
,
G. J.
van der Windt
,
J.
Blagih
,
J.
Qiu
, et al
2013
.
Posttranscriptional control of T cell effector function by aerobic glycolysis.
Cell
153
:
1239
1251
.
31.
Almeida
L.
,
M.
Lochner
,
L.
Berod
,
T.
Sparwasser
.
2016
.
Metabolic pathways in T cell activation and lineage differentiation.
Semin. Immunol.
28
:
514
524
.
32.
Desdín-Micó
G.
,
G.
Soto-Heredero
,
M.
Mittelbrunn
.
2018
.
Mitochondrial activity in T cells.
Mitochondrion
41
:
51
57
.
33.
Chi
H.
2012
.
Regulation and function of mTOR signalling in T cell fate decisions.
Nat. Rev. Immunol.
12
:
325
338
.
34.
Manning
B. D.
,
A.
Toker
.
2017
.
AKT/PKB signaling: navigating the network.
Cell
169
:
381
405
.
35.
Pollizzi
K. N.
,
J. D.
Powell
.
2014
.
Integrating canonical and metabolic signalling programmes in the regulation of T cell responses.
Nat. Rev. Immunol.
14
:
435
446
.
36.
Wang
R.
,
C. P.
Dillon
,
L. Z.
Shi
,
S.
Milasta
,
R.
Carter
,
D.
Finkelstein
,
L. L.
McCormick
,
P.
Fitzgerald
,
H.
Chi
,
J.
Munger
,
D. R.
Green
.
2011
.
The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation.
Immunity
35
:
871
882
.
37.
Hogquist
K. A.
,
S. C.
Jameson
,
W. R.
Heath
,
J. L.
Howard
,
M. J.
Bevan
,
F. R.
Carbone
.
1994
.
T cell receptor antagonist peptides induce positive selection.
Cell
76
:
17
27
.
38.
Kahan
S. M.
,
R. K.
Bakshi
,
R.
Luther
,
L. E.
Harrington
,
R. C.
Hendrickson
,
E. J.
Lefkowitz
,
C. T.
Weaver
,
A. J.
Zajac
.
2017
.
IL-2 producing and non-producing effector CD8 T cells phenotypically and transcriptionally coalesce to form memory subsets with similar protective properties.
J. Immunol.
198
(
1 Suppl
):
212.6
.
39.
Best
J. A.
,
D. A.
Blair
,
J.
Knell
,
E.
Yang
,
V.
Mayya
,
A.
Doedens
,
M. L.
Dustin
,
A. W.
Goldrath
;
Immunological Genome Project Consortium
.
2013
.
Transcriptional insights into the CD8+ T cell response to infection and memory T cell formation.
Nat. Immunol.
14
:
404
412
.
40.
Buck
M. D.
,
D.
O’Sullivan
,
E. L.
Pearce
.
2015
.
T cell metabolism drives immunity.
J. Exp. Med.
212
:
1345
1360
.
41.
Rathmell
J. C.
,
C. J.
Fox
,
D. R.
Plas
,
P. S.
Hammerman
,
R. M.
Cinalli
,
C. B.
Thompson
.
2003
.
Akt-directed glucose metabolism can prevent Bax conformation change and promote growth factor-independent survival.
Mol. Cell. Biol.
23
:
7315
7328
.
42.
Intlekofer
A. M.
,
N.
Takemoto
,
E. J.
Wherry
,
S. A.
Longworth
,
J. T.
Northrup
,
V. R.
Palanivel
,
A. C.
Mullen
,
C. R.
Gasink
,
S. M.
Kaech
,
J. D.
Miller
, et al
2005
.
Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. [Published erratum appears in 2006 Nat. Immunol. 7: 113.]
Nat. Immunol.
6
:
1236
1244
.
43.
Cannarile
M. A.
,
N. A.
Lind
,
R.
Rivera
,
A. D.
Sheridan
,
K. A.
Camfield
,
B. B.
Wu
,
K. P.
Cheung
,
Z.
Ding
,
A. W.
Goldrath
.
2006
.
Transcriptional regulator Id2 mediates CD8+ T cell immunity.
Nat. Immunol.
7
:
1317
1325
.
44.
Yang
C. Y.
,
J. A.
Best
,
J.
Knell
,
E.
Yang
,
A. D.
Sheridan
,
A. K.
Jesionek
,
H. S.
Li
,
R. R.
Rivera
,
K. C.
Lind
,
L. M.
D’Cruz
, et al
2011
.
The transcriptional regulators Id2 and Id3 control the formation of distinct memory CD8+ T cell subsets.
Nat. Immunol.
12
:
1221
1229
.
45.
Kallies
A.
,
A.
Xin
,
G. T.
Belz
,
S. L.
Nutt
.
2009
.
Blimp-1 transcription factor is required for the differentiation of effector CD8+ T cells and memory responses.
Immunity
31
:
283
295
.
46.
Ichii
H.
,
A.
Sakamoto
,
Y.
Kuroda
,
T.
Tokuhisa
.
2004
.
Bcl6 acts as an amplifier for the generation and proliferative capacity of central memory CD8+ T cells.
J. Immunol.
173
:
883
891
.
47.
Kim
J. S.
,
J. I.
Eom
,
J. W.
Cheong
,
A. J.
Choi
,
J. K.
Lee
,
W. I.
Yang
,
Y. H.
Min
.
2007
.
Protein kinase CK2α as an unfavorable prognostic marker and novel therapeutic target in acute myeloid leukemia.
Clin. Cancer Res.
13
:
1019
1028
.
48.
Bouhaddou
M.
,
D.
Memon
,
B.
Meyer
,
K. M.
White
,
V. V.
Rezelj
,
M.
Correa Marrero
,
B. J.
Polacco
,
J. E.
Melnyk
,
S.
Ulferts
,
R. M.
Kaake
, et al
2020
.
The global phosphorylation landscape of SARS-CoV-2 infection.
Cell
182
:
685
712.e19
.

The authors have no financial conflicts of interest to declare.

Supplementary data