Zbtb20 Restrains CD8 T Cell Immunometabolism and Restricts Memory Differentiation and Antitumor Immunity

Zbtb20 modulates glycolytic and mitochondrial respiratory capacity in T_eff and memory CD8 T cells differentiated in vitro. Total splenocytes were harvested from WT OT-I mice and GZB-cre Zbtb20-fl/fl OT-I (KO) mice, then activated with SIINFEKL peptide for 48 h without exogenous IL-2. Activated cells were further cultured with 100 U/ml rhIL-2 or 50 μg/ml rmIL-15 for 7 d. Cultured cells were then analyzed using Seahorse XFe96 Analyzer. (A) Oxygen consumption profile showing mitochondrial respiration and (B) proton efflux rate profile showing glycolytic metabolism for IL-2–cultured cells. (C) Mitochondrial respiratory capacity of IL-2 cultured cells. (D) Glycolytic capacity of IL-2–cultured cells. (E) Mitochondrial and (F) glycolytic metabolic profiles for IL-15–cultured cells. (G) Mitochondrial respiratory capacity for IL-15–cultured cells. (H) Glycolytic capacity of IL-15–cultured cells. Each group consisted of at least four replicates, and each experiment was repeated three times. Each point represents data from an individual mouse. Statistics were performed with Student unpaired t test. Representative data from three experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

In T cells cultured with IL-15, we found that KO T_cm displayed higher basal mitochondrial respiration, higher maximal respiration as well as higher SRC when compared with WT T_cm (Fig. 1E, 1G). Zbtb20 KO T_cm displayed similar basal glycolysis and compensatory glycolysis but significantly lower SGC compared with WT CD8 T cells (Fig. 1F, 1H).

Collectively, these data show that Zbtb20 deletion increased spare capacity for both glycolysis and mitochondrial respiration in both T_eff and T_cm. Interestingly, we also found Zbtb20 deletion played opposite roles regulating basal and maximal mitochondrial respiration in in vitro–generated T_eff and T_cm, as it decreased those two parameters in T_eff but increased them in T_cm compared with WT. Therefore, Zbtb20 appears to regulate both glycolysis and mitochondrial respiration in CD8 T cells cultured in vitro.

Zbtb20-deficient memory CD8 T cells have increased mitochondrial mass

We wished to establish whether enhanced mitochondrial metabolism observed in Zbtb20-deficient T_eff or T_cm cells was accompanied by increased mitochondrial content. In vitro generated T_eff or T_cm CD8 T cells were fixed then stained to visualize the mitochondrial outer membrane. Examination by confocal microscopy and image analysis was used to quantify mitochondrial surface area and volume. This revealed that Zbtb20 KO T_eff had less mitochondrial surface area and volume than WT cells, whereas Zbtb20 KO T_cm had larger mitochondrial surface area and volume than WT cells (Fig. 2A–E).

FIGURE 2.

IL-15–differentiated memory CD8 T cells deficient in Zbtb20 have increased mitochondrial mass. Cells were cultured as described in Fig. 1, then analyzed by confocal microscopy at original magnification ×360. (A) Representative confocal image of KO OT-I T cells cultured with IL-2, (B) WT OT-I cells cultured with IL-2, (C) KO OT-I cells cultured with IL-15, (D) and WT OT-I cells cultured with IL-15. (E) Quantification of total mitochondrial surface area and volume in IL-2– or IL-15–treated groups. Quantification was determined on three-dimensionally (3D) reconstructed confocal images using Imaris software. Each point represents a single cell. Statistics were performed with Student unpaired t test. Combined data from three experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001.

Enhanced glycolysis and mitochondrial respiration in Zbtb20-deficient CD8 T cell responses ex vivo

Metabolic changes were observed in Zbtb20-deficient CD8 T cells activated in vitro, so next we tested whether similar changes were present ex vivo. We adoptively transferred either KO or WT OT-I cells into recipient mice subsequently infected with LM-actA–OVA. Splenocytes were harvested at day 7 (effector) or day 28 (memory) postinfection and OT-I cells assayed for mitochondrial respiratory and glycolytic rates. We found that both effector and memory Zbtb20 KO CD8 T cells had higher basal and maximal mitochondrial respiration compared with WT (Fig. 3A, 3C, 3E). Zbtb20 KO memory, but not T_eff, cells also had higher SRC compared with WT (Fig. 3A, 3C, 3E). In addition, both effector and memory Zbtb20 KO CD8 T cells exhibited higher basal and maximal glycolysis as well as SGC (Fig. 3B, 3D, 3F). Therefore, our data indicated that Zbtb20 KO effector and memory CD8 T cells directly taken from infected animals exhibited upregulated mitochondrial metabolism and glycolysis.

FIGURE 3.

Zbtb20-deficient T_eff and memory CD8 T cells exhibit enhanced mitochondrial respiration and glycolytic capacity ex vivo. Naive CD8 T cells were harvested from CD45.1 OT-I mice (WT) or GZB-cre Zbtb20-fl/fl CD45.1 OT-I mice (KO). Naive OT-I cells were retro-orbitally injected into B6 recipients, which were then retro-orbitally infected with LM-actA–OVA 1 d later. On day 7 or day 28 postinfection, splenocytes were harvested from recipients, and OT-I cells were purified then subjected to mitochondrial and glycolytic metabolic analysis. (A) Oxygen consumption profile measuring mitochondrial respiration, (B) proton efflux rate measuring glycolytic metabolism for OT-I cells purified on day 7 postinfection. (C) Mitochondrial and (D) glycolytic metabolic profiles for OT-I cells purified on day 28 postinfection. (E) Quantitation of mitochondrial respiration in OT-I cells purified on either day 7 or day 28 postinfection. (F) Quantitation of glycolytic metabolism in OT-I cells purified on either day 7 or day 28 postinfection. Each point represents data from an individual mouse. Statistics were performed with Student unpaired t test. Representative data from three experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To determine the extent to which these observations extended to other, unrelated infections, we performed similar experiments infecting with MHV-68–OVA. Few changes were observed in the absence of Zbtb20 during the effector response to MHV-68–OVA (day 14) (Supplemental Fig. 2). In contrast, memory (day 28) CD8 T cells exhibited an increase in basal oxidative phosphorylation and elevation in all glycolytic parameters, similar to the changes observed after LM infection. Therefore, although the identity of the infection influences the phenotype, it is clear Zbtb20 regulates both mitochondrial and glycolytic metabolism in two distinct infection models.

Effector and memory CD8 T cells lacking Zbtb20 possess increased ATP content and higher mitochondrial mass

Elevated mitochondrial and glycolytic metabolism observed ex vivo in the absence of Zbtb20 implies these cells produce ATP at a higher rate. Therefore, we measured the ATP content in WT and Zbtb20-deficient CD8 T cells. WT or KO OT-I cells purified from recipient mice at day 7 or day 28 postinfection were used in a luminescence-based ATP detection assay. We found ex vivo–purified effector and memory Zbtb20 KO CD8 T cells consistently had higher ATP content than WT cells (Fig. 4A).

FIGURE 4.

Zbtb20-deficient T_eff and memory CD8 T cells possess a higher ATP concentration and greater mitochondrial mass ex vivo. Naive CD8 T cells were harvested from CD45.1 OT-I mice (WT) or GZB-cre Zbtb20-fl/fl CD45.1 OT-I mice (KO). Cells were retro-orbitally injected into B6 recipients, which were then retro-orbitally infected with LM-actA–OVA 1 d later. (A) On day 7 and day 28 postinfection, splenocytes were harvested from recipients, and OT-I cells were purified by magnetic positive selection then analyzed by an ATP detection assay. On day 7 (B) or day 28 (C) postinfection, splenocytes were harvested from recipients, stained with MitoTracker Green (MT-G) to quantify total mitochondrial mass then analyzed by flow cytometry. Representative histograms and quantification are shown. Shaded histogram—WT, empty histogram—Zbtb20 KO. Statistics were performed with Student unpaired t test. Data are representative of three experiments. *p < 0.05, ***p < 0.001.

As our in vitro data indicated higher mitochondrial volume in the absence of Zbtb20 under in vitro conditions that skewed to memory, we measured mitochondrial mass ex vivo. Accurate confocal analysis of these cells was not possible because of the smaller size of effector and memory CD8 T cells directly ex vivo when compared with in vitro–generated cells. We therefore measured mitochondrial content by staining with the mitochondrial dye MitoTracker Green. We found that Zbtb20 KO OT-I cells had no significant difference in mitochondrial content at day 7 (Fig. 4B) but higher mitochondrial content at day 28 postinfection (Fig. 4C).

Increased mitochondrial fuel flexibility in the absence of Zbtb20

As we observed increased mitochondrial respiration in the absence of Zbtb20 in memory cells, we tested whether there were differences in the reliance on different mitochondrial fuels. We generated T_cm in vitro by culture with IL-15, then treated with inhibitors of different mitochondrial fuel sources (etomoxir for long-chain fatty acid, UK5099 for pyruvate, and BPTES for l-glutamine), followed by conventional Seahorse mitochondrial stress tests. The maximal respiratory capacity in each condition was normalized to values without inhibitor treatment. We found that WT T_cm cells displayed reduced mitochondrial activity when they could not use pyruvate or l-glutamine (Fig. 5). In contrast, Zbtb20 KO T_cm cells only displayed reduced mitochondrial activity when they could not use both pyruvate and l-glutamine. Overall, Zbtb20 KO T_cm cells were more flexible with regard to mitochondrial fuel sources compared with WT T_cm, which suggests that Zbtb20 KO T_cm cells could have survival and/or functional advantages over WT T_cm cells in nutrient-limiting environments.

FIGURE 5.

IL-15–differentiated ZBTB20 KO memory CD8 T cell had increased mitochondrial fuel flexibility. Splenocytes were harvested from OT-I mice and GZB-cre ZBTB20-fl/fl OT-I (KO) mice, then activated with SIINFEKL peptide for 48 h without exogenous IL-2. Activated cells were further cultured with 50 μg/ml rmIL-15 for 7 d. Cultured cells were then analyzed using Seahorse XFe96 Analyzer. Cells were treated with no inhibitor or combinations of different inhibitors that prevented the use of different mitochondrial fuel source, followed by a Seahorse Agilent Mito Stress Test. The maximal respiratory capacity of each condition was normalized to the group without inhibitor treatment. Each group consisted of at least four replicates and each experiment was repeated two times. Statistics were performed using Student unpaired t test. *p < 0.05, ***p < 0.001, ****p < 0.0001. ^&p < 0.05, ^&&&p < 0.001, statistical significance between inhibitor-treated ZBTB20 KO cells versus no inhibitor–treated ZBTB20 KO cells. ^###p < 0.001, ^####p < 0.0001, statistical significance between inhibitor-treated WT cells versus no inhibitor–treated WT cells.

Zbtb20 is induced in activated CD8 T cells

To dissect the expression pattern of Zbtb20 in CD8 T cells, we took advantage of a Zbtb20 reporter mouse strain in which GFP is expressed from the Zbtb20 promoter. Naive Zbtb20-GFP OT-I cells were adoptively transferred into recipient mice, which were then i.v. infected with LM-actA–OVA. Initial experiments showed little GFP expression during the peak of the response, so we focused on early postinfection and memory. Splenocytes were harvested from recipient mice on days 2, 3, 4, and 28 postinfection for analysis. We found Zbtb20 was expressed in approximately half of the OT-I population on day 2 postinfection (Fig. 6A, 6B). By day 3, the proportion of positive cells decreased and was very low by day 4 postinfection. However, by day 28, Zbtb20 reporter was again detectable in a small proportion of cells. To identify T cell populations expressing Zbtb20 during the steady-state in the polyclonal T cell repertoire in a naive mouse, we harvested splenocytes from unmanipulated Zbtb20-GFP mice. We found the phenotype with the highest proportion of Zbtb20-expressing cells was T_cm (defined as CD44⁺CD62L⁺) (Fig. 6C–E). Naive CD8 T cells (defined as CD44⁻CD62L⁺) also contained a small proportion of Zbtb20-expressing cells. However, CD44⁺CD62L⁻ and CD44⁻CD62L⁻ CD8 T cells contained very low proportions of cells expressing Zbtb20.

FIGURE 6.

Kinetics of Zbtb20 expression in vivo. Naive CD8 T cells were purified from CD45.1 OT-I Zbtb20-GFP mice. Cells were retro-orbitally transferred into CD45.2 B6 recipients, which were then retro-orbitally infected with LM-actA–OVA 1 d later. Splenocytes were harvested from recipients and analyzed by flow cytometry. Naive Zbtb20-GFP mice were used for the naive time point. (A) Representative histograms for GFP expression at the times indicated postinfection and (B) quantification. (C) Representative dot plot for CD44 and CD62L staining in naive Zbtb20-GFP mice, (D) histograms showing corresponding GFP expression from each quadrant, shaded histogram—B6 negative control, empty histogram—Zbtb20 GFP. (E) Quantification of data shown in (D). Each point represents data from an individual mouse. Each group used at least four mice and each experiment was repeated three times. Statistics were performed with Student unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

To determine the Zbtb20 expression profile among polyclonal virus-specific CD8 T cells, we infected Zbtb20-GFP reporter mice on a B6 background intranasally with MHV-68. Their splenocytes were harvested before infection and on day 10, day 14, and day 28 postinfection, then stained with an MHC/peptide tetramer folded with the dominant epitope derived from the ORF61 protein. Reporter expression within the tetramer-positive population was then measured within populations defined by CD44 and CD62L expression (Supplemental Fig. 3A–C). Consistent with data from naive mice, Zbtb20 reporter expression was detected mostly in T_cm (CD44⁺CD62L⁺) and naive (CD44⁻CD62L⁺) populations (Supplemental Fig. 3D). PCR analysis on FACS-purified GFP⁺ and GFP⁻ cells showed GFP faithfully represented Zbtb20 mRNA expression in reporter mice (Supplemental Fig. 3E).

Given Zbtb20 expression at the early stages of effector differentiation and in a subset of central memory phenotype cells, we tested how Zbtb20 deficiency affected effector and memory differentiation in vivo.

Zbtb20 deletion enhances cytokine production and favors memory precursor differentiation

We sought to determine how Zbtb20 deletion affected CD8 T cell clonal expansion, accumulation, function, and differentiation. Naive OT-I cells from KO or WT donor mice were adoptively transferred into recipient mice, which were then i.v. infected with LM-actA–OVA. Splenocytes were harvested for analysis on day 7 or day 14 postinfection. We found the number of transferred OT-I cells recovered from the spleens of recipient to be the same at day 7, which measures the peak CD8 T cell response against LM, and day 14, which is during the contraction phase (Fig. 7A, gating strategy Supplemental Fig. 1C). Examining the phenotype of responding OT-I T cells, we found that on both day 7 and day 14 postinfection, Zbtb20 KO OT-I cells were more skewed toward memory precursors (defined as KLRG-1⁻CD127⁺) and less toward terminally differentiated effectors (defined as KLRG-1⁺CD127⁻) (Fig. 7B). In addition, cytokine production profiles revealed that a higher proportion of Zbtb20 KO OT-I cells could produce IFN-γ or TNF-α as well as both IL-2 and IFN-γ simultaneously (Fig. 7C, 7D). Production of IL-2 is a characteristic of memory cells, consistent with memory precursor skewing, and we detected a higher proportion of KO cells expressing CD27 that is preferentially expressed on T_cm cells (Fig. 7E). We also found a higher proportion of Zbtb20 KO T_eff cells expressed CXCR3 during the contraction phase (Fig. 7F), an important chemokine receptor that drives T_eff cell to sites of inflammation. Taken together, our data suggested that Zbtb20 KO T_eff cells had a phenotype indicative of increased memory potential and enhancements in cytokine production.

FIGURE 7.

Zbtb20 deletion affects T_eff cell differentiation during acute LM infection. Naive CD8 T cells were harvested from CD45.1 OT-I mice (WT) or GZB-cre Zbtb20-fl/fl CD45.1 OT-I mice (KO). Naive OT-I cells were retro-orbitally injected into B6 recipients, which were then retro-orbitally infected with LM-actA–OVA 1 d later. Splenocytes were harvested on day 7 or 14 postinfection and analyzed by flow cytometry. (A–F) All plots were gated on transferred OT-I cells. (A) Total cell counts for transferred OT-I cells from the spleen of each recipient. (B) Representative dot plot showing KLRG-1 and CD127 staining to measure memory precursor cells (KLRG-1⁻CD127⁺) and terminal effector cells (KLRG-1⁺CD127⁻) and quantification. (C) Representative dot plot showing TNF-α and IFN-γ staining and quantification. (D) Representative dot plot showing IL-2 and IFN-γ staining and quantification. (E) Representative dot plot showing CD27 and CD8 staining and quantification. (F) Representative dot plot showing CXCR3 and CD8 staining and quantification. Each point represents data from an individual mouse. Each group used at least four mice, and each experiment was repeated three times. Numbers in dot plot quadrants show percentage of events in the relevant quadrant. Statistics were performed with Student unpaired t tests. *p < 0.05, **p < 0.01, ***p < 0.001.

Zbtb20 deletion affects the memory CD8 T cell phenotype and cytokine production

Using the OT-I transfer and LM-actA–OVA infection model described above, we tracked Zbtb20 KO and WT OT-I cells until later times postinfection, which allowed us to study the role of Zbtb20 in CD8 T cell memory. On day 28 and day 60, we found the number of splenic Zbtb20 KO memory OT-I cells to be the same as WT OT-I cells (Fig. 8A). Consistent with earlier times postinfection, Zbtb20 KO OT-I cells were more skewed toward memory precursors than effector cells on day 28 (Fig. 8B). In addition, a higher proportion of Zbtb20 KO memory OT-I cells could produce IFN-γ or TNF-α (Fig. 8C) as well as both IL-2 and IFN-γ simultaneously (Fig. 8D). Moreover, a larger percentage of Zbtb20 KO memory OT-I cells expressed CXCR3 and CD27 on day 28 (Fig. 8E, 8F). Data summarizing phenotypic marker expression and cytokine production over time postinfection are shown in Supplemental Fig. 1D. Data obtained from memory timepoints therefore showed skewing toward memory CD8 T cells was consistent with earlier times postinfection.

FIGURE 8.

Zbtb20 deletion affects CD8 T cell differentiation to memory precursors and T_eff cells. Samples from the experiment described in Fig. 7 were used to measure cytokine production and memory precursor or effector differentiation on day 28 or day 60 postinfection. (A) Cell counts for transferred OT-I cells from the spleen of each recipient. (B) Representative dot plot showing KLRG-1 and CD127 staining and the percentage of memory precursors (KLRG-1⁻CD127⁺) and terminal effector cells (KLRG-1⁺CD127⁻) and quantification. (C) Representative dot plot showing TNF-α and IFN-γ staining and quantitation. (D) Representative dot plot showing IL-2 and IFN-γ staining and quantitation. (E) Representative dot plot showing CXCR3 and CD8 staining and quantitation. (F) Representative dot plot showing CD27 and CD8 staining and quantitation. Each point represents data from an individual mouse. Each group consisted of at least four mice, and each experiment was repeated three times. Numbers in dot plot quadrants show percentage of events in the relevant quadrant. Statistics were performed with Student unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Modulation of key transcription factors in the absence of Zbtb20

A network of transcription factors tightly orchestrates differentiation of effector and memory CD8 T cells. These regulate the expression of crucial cytokine receptors, proapoptotic and antiapoptotic factors, cellular metabolism, and other critical functions. Interrogation of transcription factor expression revealed that Zbtb20 KO T_eff cell expressed higher levels of Bcl-6 and lower levels of Blimp-1 on day 7, whereas on day 14, KO CD8 T cell expressed lower Bcl-6 and higher Blimp-1 compared with WT (Fig. 9A, 9B). At the memory timepoint (day 28), both Bcl-6 and Blimp-1 were lower in KO CD8 T cells. We found Zbtb20 KO CD8 T cells had lower expression of Eomes, a transcription factor necessary for memory CD8 T cell differentiation, on day 7 and day 28 postinfection, but not on day 14 (Fig. 9C). T-bet, a transcription factor important for T_eff cell differentiation, was expressed at a lower level in Zbtb20 KO CD8 T cells at day 14 and day 28 but did not reach statistical significance at day 7 postinfection (Fig. 9D). Collectively, our data suggested that Zbtb20 affects expression of several transcription factors important for effector and memory CD8 T cell differentiation. Consistent with these data, we also found Zbtb20 KO memory cells expressed lower levels of Bcl-6, EOMES, and T-bet in the MHV-68 infection model (Supplemental Fig. 4).

FIGURE 9.

Zbtb20 deletion changes expression of key transcription factors in CD8 T cells during acute infection and in memory. Splenocytes from mice treated as described in Fig. 7 were stained for expression of intranuclear transcription factors on days 7, 14, and 28 postinfection. Representative histograms from day 7 are shown, together with quantification for all timepoints for (A) Bcl-6, (B) Blimp-1, (C) EOMES, and (D) T-bet staining. Shaded histogram—WT, empty histogram—Zbtb20 KO. Each point represents data from an individual mouse. Each group comprised at least four mice, and each experiment was repeated three times. Statistics were performed using Student unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Single-cell transcriptomic analysis shows enrichment in metabolic and memory pathways in the absence of Zbtb20

Many studies have shown there is substantial heterogeneity in the CD8 T cell response with respect to the potential to differentiate into memory cells. To conduct transcriptomic analyses that could capture this heterogeneity, we performed single-cell RNAseq analysis on OT-I cells during the primary response. Using the OT-I transfer, LM-actA–OVA infection model described, WT and Zbtb20 KO CD8 T cells were purified and CITEseq performed with oligonucleotide-labeled Abs against KLRG-1, CD127, and CD62L to orient gene expression patterns with known effector/memory markers.

UMAP plots showed some overlaps in clusters occupied by WT and KO cells (Fig. 10A–C); however, there were also regions in which there was little overlap. In particular, a higher proportion of WT cells were in clusters 1 and 2, whereas clusters 0 and 3 were more highly represented in conditional KO cells (Fig. 10B–D). Analysis of gene representation in these clusters showed that clusters 1 and 2 were enriched for genes and proteins associated with effector T cells (Zeb2, granzyme A, and KLRG-1 staining) (Fig. 10E–G). In contrast, memory-associated genes and proteins (IL-7R, Cd27, and CD62L staining) were not present in these clusters, and instead seen preferentially in clusters 0, 3, and 5 (Fig. 10H–J), where the majority of KO cells were located. Examination of a wider array of genes expressed in these clusters showed preferential expression of genes associated with effector activity in clusters 1 and 2 (Zeb2, CX3CR1, Klrg1, GZB, and granzyme A) (26–30) (Supplemental Fig. 5A, left panel). Comparison of genes differentially regulated between WT and KO samples showed KO cells expressed higher levels of Pkm and mt-Nd3, necessary for pyruvate synthesis in glycolysis and mitochondrial NADH dehydrogenase, respectively (Supplemental Fig. 5A, right panel). An extended list of metabolism-associated genes that were differentially expressed is shown in Supplemental Fig. 5C.

FIGURE 10.

Gene- and pathway-level, single-cell RNAseq KO and WT comparison. Mice received naive OT-I or Zbtb20-deficient OT-I cells and were then infected with LM-actA–OVA. Spleen cells were harvested during the effector response, OT-I cells purified, and CITEseq/RNAseq performed as described. (A) UMAP embeddings of merged KO and WT profiles at day 10, colored by KO and WT status. (B and C) UMAP embeddings colored by expression cluster and displaying distribution of KO and WT cells within each expression cluster. KO and WT cells per cluster are denoted in (C) as percentages (i.e., the number of KO or WT cells divided by the total number of cells in the cluster). (D) The distribution of clusters across all KO cells examined and the distribution of clusters across all WT cells are displayed as pie charts. (E–J) UMAP embeddings displaying expression of effector and memory function genes and the cell surface protein expression of the KLRG-1 and CD62L markers. (K–R) UMAP embeddings of merged KO and WT profiles colored by cell-level pathway enrichment scores for gene sets in the Hallmark and C7 pathway collections in the Molecular Signature Database (MSigDB). Activity of pathways enriched in WT cells is displayed in (K)–(N), whereas activity of pathways enriched in KO cells is displayed in (O)–(R).

Pathway-level analyses were performed using the novel VAM (23) method that was recently developed to compute cell-level gene-set scores visualized in the UMAP plots. Differentially active pathways were also computed using a rank-sum test. Cluster 2 was associated with gene sets previously shown to be upregulated in effector T cells in addition to gene sets from proinflammatory conditions such as allograft rejection and the IFN-γ response (Fig. 10K–N). Gene sets associated with oxidative phosphorylation and glycolysis were preferentially associated with clusters 0 and 3, where the majority of KO cells were located (Fig. 10O, 10P). A similar pattern of association with clusters 0 and 3 was seen with gene sets previously shown to be downregulated in T_eff cells relative to memory or memory precursor cells (Fig. 10Q, 10R). An extended list of pathways differentially expressed in the various clusters is shown in Supplemental Fig. 5B (left panel) and was consistent with effector-associated pathway enrichment in clusters 1 and 2, and memory, glycolysis, and mitochondrial metabolism-associated pathway enrichment in clusters 0 and 3. Comparison of pathways enriched in KO versus WT samples (Supplemental Fig. 5B, right panel) showed glycolysis and mitochondrial metabolism-associated pathways enriched in KO samples. Pathways upregulated in memory cells when compared with either effector or naive cells were also enriched KO compared with WT samples. In contrast, effector-associated pathways were enriched in WT samples.

These data clearly confirm our flow cytometric and Seahorse data, showing in the absence of Zbtb20, the CD8 T cell response skews toward the memory phenotype, with enhancement of both glycolytic and mitochondrial metabolism.

Zbtb20 KO memory CD8 T cells mount a more efficient secondary response and confer better antitumor immunity

As the previous data indicated the absence of Zbtb20-enhanced differentiation toward memory CD8 T cells, we next tested the capacity of Zbtb20 KO and WT memory CD8 T cells to accumulate following secondary antigenic challenge. Using the same OT-I transfer and LM-actA–OVA infection model, we i.v. challenged groups of recipient mice on day 29 or day 81 postinfection with MHV-68–OVA. Fig. 11A shows numbers of OT-I cells both before and after challenge. The secondary infection was insufficient to induce a detectable recall response from WT memory cells; however, Zbtb20 KO memory CD8 T cells expanded robustly upon rechallenge at both timepoints (Fig. 11A, 11B). Both Zbtb20 KO and WT OT-I cells cleared the MHV-68–OVA to undetectable levels within 5 d after rechallenge.

FIGURE 11.

Zbtb20 deletion enhances the recall response of memory CD8 T cells and increases protection against MC38 tumor. (A–D) OT-I cells were adoptively transferred and infected as described in Fig. 7. On day 29 or day 81 postinfection, recipient mice were challenged with MHV-68–OVA retro-orbitally. Splenocytes were harvested after rechallenge for flow cytometric analysis. (A and B) Cell counts for transferred OT-I cells from the entire spleen of recipients challenged on (A) day 28 or (B) day 80 postinfection. Each point represents data from an individual mouse. Each group comprised at least four mice, and each experiment was repeated three times. Statistics were performed with Student unpaired t test. (C and D) On day 80 postinfection, splenocytes were harvested from recipient mice, and OT-I cells were purified. A total of 1 × 10⁶ memory OT-I cells were transferred into MC38-OVA, tumor-bearing mice (day 4 after s.c. inoculation of MC38-OVA), and tumor area was measured. (C) Plot showing tumor growth in individual animals. Arrow represents time of T cell injection. Numbers at bottom right indicate the proportion in each group that did not grow measurable tumor (baseline on graph). (D) Time to end point curve, with the end point defined as the tumor area exceeding 400 mm². n = 9 for KO group, n = 10 for WT and no T cell transfer groups. Data are representative of three replicate experiments. Statistics were performed with two-way ANOVA (C) or a Mantel–Cox log-rank test (D). **p < 0.01, ****p < 0.0001.

To test whether Zbtb20 KO memory CD8 T cells had enhanced protective capability, we tested their ability to inhibit growth of MC38-OVA tumors. Naive OT-I cells were transferred into recipient mice that were subsequently infected with LM-actA–OVA. At 80 d postinfection, memory OT-I cells were purified then adoptively transferred into recipient mice bearing s.c. MC38-OVA tumors that had been growing for 4 d. In mice that received no CD8 T cells, tumors developed quickly in all mice (Fig. 11C). Tumor growth was significantly slower in recipients of WT memory cells; however, the majority of mice developed tumors (Fig. 11D). In contrast, most mice that received Zbtb20 KO memory OT-I cells did not develop measurable tumors, and those that were measurable grew only to a small size then resolved (Fig. 11C, 11D). Therefore, memory CD8 T cells lacking Zbtb20 conferred superior protection against tumors compared with memory cells in which Zbtb20 was intact.

Taken together, our data show Zbtb20 restrains mitochondrial metabolism and glycolysis together with differentiation toward memory precursor cells. This results in memory CD8 T cells with a higher capacity for cytokine production and a more potent ability to mount secondary and antitumor responses.

Discussion

Based on phenotypic, functional, and metabolic techniques, in conjunction with transcriptional profiling, it is clear that the absence of Zbtb20 skews CD8 T cell differentiation toward the generation of memory. Interestingly, it seems not all KO memory precursor cells survived, as we did not consistently see a larger memory population in KO mice. Bias away from an effector-type profile was particularly evident in our single-cell RNAseq analyses, which also showed enrichment for genes sets associated with memory. A particularly novel finding is that both glycolytic and mitochondrial metabolism were enhanced, whereas typically perturbations that promote memory differentiation enhance mitochondrial metabolism at the expense of glycolytic metabolism (31–34).

Previous studies have shown a critical role for Zbtb20 in hippocampal development and the correct development of neuronal layers in the cerebral cortex (35–38). Consistent with this, patients with certain mutations in Zbtb20 develop Primrose syndrome (39), which includes intellectual disability, macrocephaly, and increased height and weight (40–45). Detailed study of patients with Primrose syndrome revealed metabolic changes, including reduced glucose tolerance, with prevalence of amino acid and fatty acid catabolism, ketogenesis, and gluconeogenesis (46). This indicates impairment in the normal pathway from glucose to pyruvate and then into the citric acid cycle. Instead, amino acids and fatty acids are converted to glucose and ketone bodies, similar to the processes that occur in diabetes and during prolonged fasting. This indicates Zbtb20 regulates genes associated with glucose and fatty acid metabolism in humans. Consistent with this, data from Zbtb20 KO mice showed disrupted glucose homeostasis, and dysregulation of genes associated with glucose metabolism in the liver (21). These mice had severe growth defects and decreased survival, not living beyond 12 wk of age; however, restoration of Zbtb20 selectively in the liver markedly improved survival. Later work using liver-specific Zbtb20 deletion showed Zbtb20 regulates genes associated with glycolysis and de novo lipogenesis (47), and β cell–specific Zbtb20 deletion lead to aberrant glucose metabolism and altered expression of glycolysis-associated genes (48). To our knowledge, this article is the first to describe a role for Zbtb20 in metabolic control in the immune system. Our single-cell RNAseq data suggest several genes central to glycolysis and mitochondrial metabolism are regulated by Zbtb20, and future studies will confirm whether these genes represent direct or indirect targets of Zbtb20.

It is clear that activated and quiescent T cells have distinct bioenergetic and biosynthetic demands (49). Activation, proliferation, epigenetic, cytotoxic functions, and differentiation of T cells are directed by dynamic changes of their metabolism (50). These changes are evident both in mitochondrial structure and in the choice of predominantly mitochondrial or glycolytic metabolism used by the T cell. Mitochondria have a highly compartmentalized structure, and their morphology can be very dynamic. Mitochondrial morphology is critical for DNA sequestration, reactive oxygen species regulation, oxidative phosphorylation, and calcium homeostasis (51). Interconnected mitochondria are linked to increased demand for ATP or nutrient starvation (13, 14, 52–55), whereas globular and fragmented mitochondria are linked to nutrient excess, lower demand for ATP, or severe cellular stress (56, 57). Mitochondria can adapt their morphology under different cellular activation states in T cells, macrophages, and mast cells (16, 58, 59). Rapidly proliferating T_eff cells possess globular mitochondria, whereas memory CD8 T cells contain highly interconnected, tubular mitochondria (16). As memory CD8 T cells rely upon mitochondrial respiration for their energy demands, elongated mitochondria with well-ordered cristae are thought to hold components of the electron transport chain in a more efficient configuration (60). Our data indicate that mitochondria in Zbtb20 KO memory CD8 T cells have a larger volume and surface area compared with WT cells, which is consistent with enhanced oxidative phosphorylation observed in these cells. Interestingly, mitochondrial content was lower in Zbtb20 KO in vitro–derived T_eff cells. This is consistent with the observed lower basal and maximal oxidative phosphorylation. Nevertheless, KO effector cells did not exhibit impairments in cytokine production or proliferation, presumably because of the enhanced glycolytic metabolism we observed that provided the necessary ATP and biosynthetic intermediates.

Our Seahorse assays clearly showed Zbtb20 deficiency modulates T cell metabolism; however, there were some subtle differences observed between in vitro– and ex vivo–generated effector and memory cells. Basal and maximal glycolysis and oxidative phosphorylation were uniformly increased in ex vivo T_eff and memory CD8 T cells. Although IL-15–generated memory cells also displayed elevated basal and maximal oxidative phosphorylation, glycolytic parameters were similar to WT cells. T_eff cells generated with IL-2 had elevated basal, but not maximal glycolysis, but depressed basal and maximal oxidative phosphorylation. Several factors may be responsible for these discrepancies. CD8 T cells responding to an infection in lymph nodes or the spleen are exposed to a variety of proinflammatory mediators, cytokines, and activated APCs that are not faithfully replicated by standard in vitro culture conditions. In addition, concentrations of key nutrients such as glucose and glutamate are in excess in vitro and likely more limiting in vivo (61). A recent study found in vitro–derived effector cells operated at their maximal glycolytic capacity, whereas ex vivo–derived cells had larger spare energetic capacity (61). Ex vivo cells also displayed greater oxidative metabolism and switched more easily between mitochondrial and glycolytic pathways. Therefore, it is possible the increased metabolic flexibility in Zbtb20 KO cells, possibly in addition to exposure to inflammatory factors present uniquely in vivo, results in the metabolic changes in these cells being better revealed in vivo.

T_eff cells heavily rely on glycolysis and have high rates of glucose uptake (25), whereas memory CD8 T cells rely on mitochondrial respiration (49). It is clear that the substrate used in the mitochondrial citric acid cycle also influences CD8 T cell function, differentiation, and longevity (50). Glutamine metabolism has been reported to be crucial for survival, proliferation, and effector function of CD4 T cells upon activation (62). Fatty acid oxidation has been linked to superior mitochondrial capacity and longevity of memory CD8 T cells (8, 63). In addition, instead of obtaining fatty acids from their external environment, memory CD8 T cell synthesize their own triacylglycerol using glucose-derived carbon (63, 64). Concomitantly, memory CD8 T cell also upregulate expression of the glycerol channel, aquaporin 9, to facilitate the uptake of glycerol required for triacylglycerol synthesis and storage (64). Subsequent studies showed that medium or short chain fatty acids such as acetate also play important roles as mitochondrial fuels in memory CD8 T cells (65–67). Our studies regarding mitochondrial fuel sources show inhibition of glutaminolysis or glycolysis markedly impair mitochondrial respiratory activity in WT CD8 T_cm cells. However, Zbtb20-deficient memory CD8 T cells tolerated inhibition of either fuel source without significant diminution of mitochondrial respiration, and only when both pathways were inhibited was there a significant reduction. Availability of glucose and glutamate are limiting in many growing tumors, creating an environment not conducive for protective T cell responses. Limited flexibility with respect to mitochondrial fuel sources may restrict the protective capacity of WT CD8 T cells, and increased flexibility on the part of Zbtb20-deficient memory cells may partially explain their increased protective capacity. Future studies will determine the underlying mechanism for this increased fuel flexibility and whether it leads to better accumulation and/or effector activity in tumors after T cell infusion.

SRC is thought to be an important factor contributing to enhanced secondary responses by memory CD8 T cells in response to antigenic rechallenge (8). Therefore, it is likely that the larger SRC we observed in Zbtb20-deficient memory CD8 T cells is at least partly responsible for the greater secondary expansion following virus rechallenge. Improved protective capacity from Zbtb20 KO memory cells was demonstrated by superior ability to protect against MC38-OVA tumors. Although enhanced expansion of memory cells is no doubt important in this protection, a higher proportion of cells expressing effector cytokines such as IFN-γ and TNF-α, and CXCR3, which may promote homing to the tumor site, may also have contributed to antitumor activity.

Our data indicate that Zbtb20 is expressed in the first 2–3 d following CD8 T cell activation and is important in shaping the phenotypic, metabolic, and functional evolution of the antimicrobial response. Expression then declines rapidly, but re-emerges in a small subset of memory CD8 T cells. This may indicate that Zbtb20 exerts its effects during the first few days of the T cell response, then is subsequently active in a defined population of memory cells. Early Zbtb20 activity may exert a sustained effect in part through modulation of the network of other transcription factors critical for T cell differentiation. Blimp-1 suppresses T_eff cell proliferation and drives their terminal differentiation, whereas Bcl-6 promotes proliferation, survival, and memory differentiation of CD8 T cells (68). Eomesodermin induces expression of several effector molecules in T cells, such as IFN-γ, perforin, and GZB (69), but also promotes homeostatic self-renewal of memory cells through inducing expression of the IL-15R (70). Reduced expression of Blimp-1 and Eomes at day 7 may contribute to the skewing away from terminally differentiated effector cells and toward memory precursors. Expression of these molecules change during the contraction phase (day 14); however, this could be a reflection of the altered proportions of effector and memory cells during contraction, as effectors die off and the proportion of memory precursors enlarges. We also observed elevated Bcl-6 expression at day 7, which is consistent with promotion of memory precursor development. However, a key function of Bcl-6 is to directly repress genes involved in the glycolysis pathway, including Slc2a1, Slc2a3, Hk2, and Pkm2 (71). As we observed increased glycolytic metabolism in the absence of Zbtb20, the effects of elevated Bcl-6 were likely mitigated by other transcription factors or cofactors.

Although most experiments focused on the CD8 T cell response to Listeria infection, we also tested the extent to which they extended to a different, unrelated infection. Murine gammaherpesvirus infection is a different class of pathogen (virus versus intracellular bacteria), and unlike Listeria, it establishes a persistent infection (72). Although we detected changes in T cell metabolism and altered expression of key transcription factors in both infections, there were important differences. Glycolysis was increased in Zbtb20-deficient CD8 T cells in both infections. Basal and maximal mitochondrial respiratory capacity and SRC were all enhanced in KO memory cells in Listeria infection; however, these changes were of smaller magnitude in MHV-68 infection. The pattern of expression of Bcl-6, Eomes, and T-bet were consistent in memory cells in both infections; however, they differed at the acute timepoints. There are a number of factors that may be responsible for these differences, including Ag persistence, engagement of different pattern recognition receptors, and cellular tropism. Despite these differences, however, it is clear Zbtb20 affects both immunometabolism and the transcriptional network during CD8 T cell differentiation across infection types.

In conclusion, data presented in this study identify Zbtb20 as an important regulator of effector and memory CD8 T cell differentiation and metabolism. Given our data showing improved protection from tumors and the known superiority of memory cells in adoptive T cell therapy, deletion or inhibition of Zbtb20 may provide a promising novel strategy for antitumor immunotherapy.

Acknowledgements

We thank Dr. Randy Noelle (Dartmouth College) for useful discussions and supplying breeding stocks of Zbtb20-GFP and Zbtb20-fl/fl mice and Drs. Eyal Amiel (University of Vermont) and Juan Cubillos-Ruiz (Weill Cornell Medical School) for discussions regarding analysis of metabolic data. We thank Dr. Mary Jo Turk (Dartmouth College) for kindly providing OT-I breeding stocks.

Footnotes

This work was supported by National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases Grant R01 AI122854 (to E.J.U.), NIH/U.S. National Library of Medicine Grant K01 LM012426 (to H.R.F.), and NIH/National Institute of General Medical Sciences Centers of Biomedical Research Excellence Grant P20 GM130454 (to H.R.F.), which supported the Center for Quantitative Biology Single Cell Genomics Core and the Genomics and Molecular Biology Shared Resource at Dartmouth. The Center for Quantitative Biology Single Cell Genomics Core and the Genomics and Molecular Biology Shared Resource at Dartmouth is also supported by NIH/National Cancer Institute Cancer Center Support Grant 5P30CA023108-37. Imaging studies were performed at the Dartmouth Institute for Biomolecular Targeting, supported by NIH Grant P20 GM113132.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ADT: Ab-DNA tag
BPTES: bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide
BTB-ZF: broad complex, tramtrack, bric-à-brac, and zinc finger
CITEseq: cellular indexing of transcriptomes and epitopes by sequencing
FCCP: 4-(trifluoromethoxy)phenyl)carbonohydrazonoyl dicyanide
GZB: granzyme B
KLRG-1: killer cell lectin-like receptor G1
KO: knockout
MHV-68: murine gammaherpesvirus-68
MHV-68–OVA: murine gammaherpesvirus-68 encoding OVA
rhIL-2: recombinant human IL-2
rmIL-15: recombinant mouse IL-15
RNAseq: RNA sequencing
SGC: spare glycolytic capacity
SRC: spare respiratory capacity
T_cm: central memory CD8 T
T_eff: effector CD8 T
UMAP: Uniform Manifold Approximation and Projection
VAM: variance-adjusted Mahalanobis
WT: wild-type.

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