Cytochrome c oxidase dependent respiration is essential for T cell activation, proliferation and memory formation

doi:10.21203/rs.3.rs-4875322/v1

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Cytochrome c oxidase dependent respiration is essential for T cell activation, proliferation and memory formation

https://doi.org/10.21203/rs.3.rs-4875322/v1

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AAlthough glycolysis has received considerable attention in activated T cells, the utility and requirement of cytochrome c oxidase (COX) mediated mitochondrial respiration (MR) in T cell activation and function remain incompletely understood. To address this, we re-introduced MR via an alternative oxidase (AOX) from Ciona intestinalis into COX deficient T cells. Our findings reveal that MR serves as a critical safety valve for metabolic reprogramming during T cell activation, managing electron pressure and maintaining cellular redox balance. AOX-mediated MR restored mitochondrial function, reduced oxidative stress, and enhanced ATP production. This resulted in the elimination of the secondary effects of COX dysfunction, such as apoptosis and metabolic perturbations in glycolysis and the tricarboxylic acid cycle, thereby supporting robust effector and memory T cell generation and function. Our results underscore the importance of maintaining COX integrity for overall cellular health and emphasize the pivotal role of MR in T cell cellular proliferation and differentiation.

Biological sciences/Immunology

Biological sciences/Biochemistry

Metabolic reprogramming in activated T cells involves an upregulation of glycolysis to meet the bioenergetic and biosynthetic demands required for rapid proliferation^{1, 2}. While glycolysis has been emphasized in immunometabolism studies, it is important to note that oxidative phosphorylation (OXPHOS) also increases, with individual electron transport chain (ETC) complexes having unique roles. Seminal findings in genetic models of OXPHOS deficiency demonstrated the unique contributions of two of these ETC complexes. Coenzyme Q:cytochrome c oxidoreductase (complex III) was found to be involved in reactive oxygen species (ROS) production, which is essential for T cell activation³. Further work by our group revealed that cytochrome c oxidase (COX, complex IV) mediates cytochrome c-dependent apoptosis in T cells following activation, leading to immunodeficiency⁴.

Although COX is involved in various processes such as apoptosis, mtDNA maintenance, and mitochondrial transcription^{4, 5}, we hypothesized that all COX-dependent effects in T cells fundamentally stem from its role in mitochondrial respiration (MR). To test this directly, we generated mice lacking COX in T cells, concomitant with the expression of Ciona intestinalis alternative oxidase (AOX). AOX is a 37 kDa mitochondrial protein that functions upstream of complex III, to sustain the electron transport chain and ATP production^{6, 7, 8}. Similar to COX, AOX respires by transferring electrons from ubiquinol to molecular oxygen, reducing it to water. AOX-mediated MR restores many aspects of critical T cell functions, including proliferation and differentiation, particularly in memory T cells. Our findings suggest that the fundamental role of MR via COX is integral for proper T cell activation and function.

MR restoration impacts cellular dynamics

COX10, a protoheme:heme-O-farnesyl transferase is indispensable for the biosynthesis of heme a, an elemental component of COX. Deficiency of COX10 results in marked impairment of COX and cellular respiration⁹; the molecular pathology of our previously published model in T cells (TCox10^-/-). To restore MR specifically, we introduced a non-protonmotive ubiquinol oxidase (AOX) from Ciona intestinalis into TCox10^-/- T cells. We engineered this novel mouse model by breeding TCox10^-/- mice with counterparts constitutively expressing the Aox gene to generate TCox10^-/-/Aox mice (Fig. 1A), with PCR and qPCR analysis confirming the genotypes and expression of Cox10 in the progeny (Fig. 1B and 1C). As AOX is an electron acceptor upstream of complex III (Fig. 1D), AOX-expressing cells can resist the effects of sodium azide, a toxin for COX, and continue respiration unaffected^{7, 10, 11}. T cells derived from WT, Aox, TCox10^-/-, and TCox10^-/-/Aox mice were stimulated for 3 days with anti-CD3/CD28 and subjected to sodium azide. AOX-expressing cells maintained elevated oxygen consumption rates despite COX inhibition¹⁰, in contrast to the expected decline in WT and TCox10^-/- T cells (Fig. 1E).

To survey how AOX-mediated restoration of MR may impact cellular dynamics, we performed an RNAseq experiment using T cells from WT, Aox, TCox10^-/-, and TCox10^-/-/Aox mice. Each genotype showed significant changes in gene expression relative to WT, or in TCox10^-/-/Aox against TCox10^-/- (Extended Data Fig. 1A and Supplemental Table 1). As these comparisons suggested considerable changes in gene expression across the four genotypes, we performed a weighted gene coexpression network analysis (WGCNA) to simultaneously compare them. Hierarchical clustering grouped the genes into 18 modules (Extended Data Fig. 1B). Examining the correlation of each module’s eigengene intercepts allowed us to identify which modules had gene expression perturbed in TCox10^-/- and normalized by the introduction of AOX (Extended Data Fig. 1C). We selected four modules (turquoise, yellow, greenyellow, and midnightblue) that followed this pattern and used a heatmap of the normalized gene expression from each category to confirm the differences in expression across groups (Extended Data Fig. 1D). Overrepresentation analysis (ORA) of each of the modules indicated the main functions of the modules in these gene sets (Supplemental Table 2), which included cell cycle phase transition, apoptotic signaling, and T cell function (Fig. 1F). To further elucidate the functional implications of AOX-mediated restoration in MR, we proceeded to validate these gene expression changes through targeted biochemical and functional assays.

MR via AOX restores multiple aspects of mitochondrial function

After confirming the activity of AOX in T cells, we subsequently concentrated on mitochondrial function, given that this cellular organelle is the primary site of its activity. Transcriptional profiling of TCox10^-/-/Aox mice revealed restoration of OXPHOS gene set enrichment, particularly gene sets associated with complex I (Extended Data Fig. 2A). Additionally, there were substantial enhancements in transcriptional signatures related to mtDNA metabolism and translation processes, as well as carbohydrate and nucleotide metabolism (Extended Data Fig. 2A and Supplemental Table 3). In TCox10^-/-/Aox T cells, mitochondrial number (MitoGreen, Fig. 2A) reverted to WT levels. However, mtDNA content by qPCR (Fig. 2B) remained increased, suggesting that cells were still compensating for COX deficiency. Indeed, COX has been demonstrated to modulate mitochondrial genomic homeostasis,⁵ a process which may be independent of MR, as indicated by our results.

Based on its position in the ETC, we hypothesized that COX-mediated MR functions as a regulatory mechanism to mitigate the build-up of electron pressure within the ETC by acting as a safety valve. This effectively reduces the formation of reactive oxygen species (ROS). TCox10^-/- T cells tend to have higher and more variable membrane potential (TMRE, Fig. 2C) suggesting an increase in electron build-up. TCox10^-/-/Aox T cells show a normalization of mitochondrial membrane potential, similar to WT levels. This relief of electron build-up is further highlighted in cellular ROS. In TCox10^-/- T cells, we observed elevated total cellular ROS (Fig. 2D), in the presence of diminished superoxide (Fig. 2E) and elevated hydrogen peroxide (Fig. 2F). These findings not only indicated oxidative stress, but also augmented superoxide dismutase activity (i.e., superoxide ® hydrogen peroxide). AOX, which bypasses complex III, a major source of ROS in T cells³, reduced ROS production, and hydrogen peroxide, indicating a restoration in redox balance. This was further supported by the AOX-mediated normalization of NAD⁺/NADH ratios, further signifying a restoration of cellular redox, and demonstrating the efficacy of AOX in alleviating electron pressure in the ETC (Fig. 2G).

We next turned to evaluating cellular respiration by extracellular flux analysis. OCR was markedly improved with AOX in CD4⁺ and CD8⁺ T cells with TCox10^-/-/Aox cells exceeding that of WT (Fig. 2H and Extended Data Fig. 2B). To demonstrate that the observed increase in OCR did not relate to COX activity, we used a targeted substrate (N′-tetramethyl-para-phenylene-diamine (TMPD)). COX-mediated respiration remained low in TCox10^-/-/Aox T cells, suggesting that AOX does not enhance COX activity but instead functions independently (Fig. 2I). AOX addition also resulted in the augmentation of total cellular ATP production, although not to WT levels (Fig. 2J). Therefore, reestablishing by AOX cellular respiration reduces oxidative stress, and improves ATP production and cellular redox balance, restoring mitochondrial homeostasis, ultimately revealing insights into the role of COX-dependent respiration in the T cell processes.

MR offloads upstream carriers of chemical energy during metabolic reprogramming

Based on our finding that MR reduces electron buildup and restores the cellular redox state (i.e., ROS and NAD⁺/NADH), we next asked whether this function also serves as a safety valve for upstream metabolic processes. The TCA cycle and glycolysis are integral to T cell metabolic reprogramming² and are dependent on the redox state of the cell. To better understand the role of MR in supporting upstream metabolic function, we employed stable isotope tracing experiments during metabolic reprogramming in 24 hour activated T cells, as above.

Glutamine is an anaplerotic amino acid in the TCA cycle, generating reducing equivalents that can drive OXPHOS^{12, 13}. Previously, we demonstrated that activated TCox10^-/- T cells develop glutamine addiction with increased incorporation of this amino acid into the TCA cycle⁴. In TCox10^-/-/Aox T cells, we observed that the incorporation of ¹³C-glutamine carbons into downstream metabolites (Fig. 3A), including fumarate (M + 4), malate (M + 4), and aspartate (M + 4), decreased, returning to WT levels (Fig. 3B) and indicating the resolution of glutamine addiction. Citrate (M + 4 and M + 2) concentrations were also improved, signaling a reactivation of cycling dynamics of the TCA (Fig. 3C).

TCox10^-/- T cells also demonstrated depressed glycolysis following activation⁴, driven in part by negative enrichment and downregulation of glycolytic pathway genes (Extended Data Fig. 2A). To study glycolysis, we first examined glucose transport, a process that is upregulated in T cells during metabolic reprogramming¹⁴. While glucose transport via 2-NDBG uptake was suppressed in activated TCox10^-/- T cells, this process was reestablished in TCox10^-/-/Aox CD4⁺ and CD8⁺ T cells (Fig. 3D). With enhanced glucose transport in TCox10^-/-/Aox T cells, we next focused on the fate of glucose carbons using stable isotope tracing with ¹³C-glucose (Fig. 3E). Glucose free media was supplemented with ¹³C-glucose and T cells were stimulated for 24 hours as above. TCox10^-/-/Aox T cells displayed a normalized uptake of ¹³C into pyruvate (M + 3) and lactate (M + 3), signaling a restoration of glycolytic activity (Fig. 3F). Interestingly, glycolytic gene expression is not restored in TCox10^-/-/Aox T cells (Extended Data Fig. 2A), suggesting that this enhancement occurs through post-transcriptional mechanisms. We next asked whether the conversion of glucose into the TCA cycle via pyruvate also returned. Indeed, we observed elevated ¹³C incorporation into citrate (M + 2) signifying enhanced influx of glucose-derived carbons into the TCA cycle (Fig. 3G). The enrichment of downstream TCA cycle intermediates—succinate, fumarate, malate, and aspartate (all M + 2)—further confirmed this restoration (Fig. 3H). An enhancement in the isotopic enrichment of citrate (M + 4) also highlighted the resumption of cycling of the TCA (Fig. 3I). Thus, by enabling the flow of electrons within the ETC, MR reduces the upstream electron pressure from glutaminolysis and glycolysis concomitantly maintaining TCA cycle function in T cells. Our results also suggest that a significant portion of glucose is eventually oxidized in the mitochondria during T cell activation.

MR abrogates apoptosis

In our previous study, we demonstrated that COX mediates apoptosis in activated T cells during the phase of proliferation⁴. We next asked whether the restoration of MR could restore cellular viability. Building on our previous findings, alterations in apoptosis were disentangled by mapping TCox10^-/-/Aox versus TCox10^-/- log₂ fold changes (L2FCs) from our RNAseq onto the KEGG apoptosis pathway (mmu04210) (Extended Data Fig. 3A). While multiple pro-apoptotic and pro-survival genes were upregulated in TCox10^-/-T Cells, interestingly, elevated expression of extrinsic activators Fas, FasL, Perf1, and Tradd was reversed by AOX. Apoptosis was measured in T cells by live/dead dye and Annexin V staining (Fig. 4A), revealing a reduction in apoptotic TCox10^-/-/Aox CD4⁺ and CD8⁺ T cells by approximately 50% (Fig. 4B). To investigate potential mechanisms underlying the reduction in apoptosis, we assessed caspase 3 activation, a common pathway for both intrinsic and extrinsic apoptosis. Caspase 3 activation was abnormally elevated in TCox10^-/- T cells and decreased in TCox10^-/-/Aox T cells (Fig. 4C). We also observed similar trends in specific apoptotic pathways, showing amelioration of both caspase 8 (i.e., extrinsic pathway, Fig. 4D) and caspase 9 (i.e., intrinsic pathway, Fig. 4E) activation in TCox10^-/-/Aox T cells. Since we observed more consistent increased expression of Caspase 8 and given our RNAseq results (Extended Data Fig. 3A and Supplemental Table 1), we further examined activation of the extrinsic pathway by quantifying Fas and FasL (Figs. 4F and 4G). Expression of both proteins were increased in TCox10^-/- T cells and reduced with AOX expression. To probe the activity of this pathway in vitro, we used anti-FasL antibodies on WT and TCox10^-/- CD8⁺ and CD4⁺ T cells. This intervention significantly increased the viability of TCox10^-/- T cells (Extended Data Fig. 3B) indicating a major role for this pathway in COX mediated apoptosis. However, blocking FasL did not improve proliferation as reflected by the retention of Cell Trace Violet (CTV, Extended Data Fig. 3C) underscoring the importance of maintaining MR. Therefore, these data support that COX-mediated apoptosis involves the external pathway and is secondary to MR dysfunction in T cells.

MR sustains T cell function in vitro

Significant deficiencies in T cell differentiation and function result from COX deficiency⁴. With a re-establishment of metabolic reprogramming and a suppression of apoptosis by AOX, we next evaluated core functions in TCox10^-/-/Aox T cells in vitro. Following metabolic reprogramming, T cells engage in robust proliferation. To examine the impact of MR on proliferation, we loaded T cells with CTV and activated for 3 days as above. TCox10^-/-/Aox T cell proliferation matched WT, even when challenged with sodium azide (Fig. 5A), indicating that the AOX-mediated MR could support proliferation. Flow cytometry analysis revealed that activated TCox10^-/- T cells displayed cell surface markers indicative of a heightened activation state (CD44, CD69, Fig. 5B) while TCox10^-/-/Aox cells more closely aligned to WT.

To evaluate the role of MR in T cell differentiation, we produced helper T cells (Th) in TCox10^-/-/Aox cells in vitro, following a standardized protocol (Fig. 5C). We found that TCox10^-/-/Aox T cells acquired differentiation capacities, particularly in Th1 and Th17 subsets, approximating the patterns observed in WT cells (Fig. 5D). Regulatory T cells (Foxp3⁺, T_reg), were decreased in AOX expressing cells, signifying a more complex relationship between mitochondrial function and their differentiation.

We next evaluated T cell effector (T_eff) and memory (T_mem) differentiation in vitro, essential processes for sustained immune protection (Fig. 5E). In TCox10^-/- T cells, memory and effector cell differentiation were previously unachievable due to overwhelming apoptosis and death. The re-introduction of MR facilitated the generation of both T_eff and T_mem. These cells exhibited improvements in the expression of phenotypic markers of differentiation for their respective cell types, albeit incompletely (Fig. 5F). To assess the functionality of T_eff cells, we stained for the expression of granzyme, an essential molecule for cytolytic activity. Granzyme levels in TCox10^-/-/Aox matched those in WT and Aox controls, and their killing activity was similarly robust, confirming that the cytotoxic capabilities were fully present (Figs. 5G and 5H). Given the lack of a robust in vitro assay for memory function, we performed RNAseq analysis on TCox10^-/-/Aox differentiated memory T cells (Fig. 5I and Supplemental Table 4). Despite significant differences in their transcriptional profiles, the expression of core genes involved in effector memory (T_em) and central memory (T_cm) differentiation was largely similar between TCox10^-/-/Aox and WT cells, with the notable exception of Eomes. This indicates that while TCox10^-/-/Aox memory T cells exhibit broad transcriptional changes, essential pathways for memory differentiation mostly remain intact. All things considered, our findings in TCox10^-/-/Aox T cells underscore that maintaining MR, the core function of COX, is essential for T cell differentiation. Following these encouraging results in T_eff and T_mem, it became imperative to further assess the functional capabilities of these cells in a physiologically relevant setting (i.e., in vivo).

MR is critical for T cells in vivo

T cells orchestrate and execute the immune response through cytokine production or direct cellular interactions, serving as both regulators and effectors. A core function of T cells is maintaining immune memory, ensuring a rapid and effective response to previously encountered antigens. To evaluate how MR may support these diverse abilities, we studied TCox10^-/-/Aox T cell development and function in vivo. As a secondary lymphoid organ, the spleen serves as a microenvironment for immune interactions, providing a specialized niche where immune cells such as T cells, B cells, and macrophages coordinate responses. In the spleens of both TCox10^-/- and TCox10^-/-/Aox mice, we observed a tendency for elevated B cells and macrophages, signifying imbalances between splenic resident cells (Figs. 6A and 6B). Despite the AOX, decreased quantities of both CD4⁺ and CD8⁺ T cells persisted in the spleens of TCox10^-/-/Aox mice (Fig. 6C). This reduction signals that AOX, while improving some aspects of T cell function, cannot fully restore T cell populations to normal levels. With these numeric perturbations in splenic populations, we next measured T-dependent B cell responses through immunization with 2,4,6-Trinitrophenyl-Chicken Gamma Globulin (TNP-CGG). The results were promising: unlike TCox10^-/-, TCox10^-/-/Aox mice mounted effective primary (2 weeks) and secondary (5 weeks) B cell responses, illustrating that MR enhances the supportive role of T cells (Fig. 6D).

Mitochondria are integral to the development of T cell memory by providing the necessary energy and signaling pathways that support their long-term survival and rapid response capabilities¹⁵. To assess the requirement of MR for development of memory T cells, we conducted an in vivo challenge with influenza virus (Fig. 6E). Mice were first immunized with influenza A/X31 (X31, H3N2) followed with influenza A/PR/8 (PR8, H1N1) challenge at 5 weeks. Both immunization and challenge were conducted using inhalation. Our experimental design, based on a switch in viral isotypes (X-31→PR8), eliminates the memory humoral responses, allowing us to focus on T cells. To demonstrate the generation of influenza-specific memory, we stained T cells with tetramers against two T cell antigenic determinants, the nucleoprotein (NP_366–374, H2Db) and the acid polymerase (PA_224–233, H2Db)^{16, 17}. The primary CD8⁺ T cell response to both strains is dominated by naïve T-cell recognition of both determinants. However, the NP_366–374 response dominates the secondary response in X-31→PR8 isotype switch challenge. TCox10^-/- mice showed a limited ability to generate memory cells, while TCox10^-/-/Aox mice successfully generated memory T cells at levels comparable to WT (Fig. 6F). The most direct evidence of functional recovery came from viral load assessments, where TCox10^-/-/Aox mice exhibited viral loads not significantly different from WT, significantly lower than those seen in TCox10^-/- mice (Fig. 6G). This dramatic reduction in viral load highlights the restored antiviral efficacy of T cells in AOX-expressing mice. Lastly, to ascertain whether the improvements were cell-autonomous, we conducted adoptive transfer experiments using bone marrow from WT, Aox, TCox10^-/-, and TCox10^-/-/Aox mice. We found that mice reconstituted with TCox10^-/-/Aox bone marrow could produce added viral specific T cells (Fig. 6H) and reduce viral load to levels similar to WT (Fig. 6I), demonstrating that the benefits of AOX were indeed cell-autonomous.

In this study, we introduced an AOX from Ciona intestinalis into TCox10^-/- T cells to investigate the role of MR in T cell function. Our findings reveal that MR serves as a safety valve to manage electron pressure from metabolic reprogramming during T cell activation. Restoring MR reinstates the cellular redox state and eliminates the secondary effects of COX dysfunction: apoptosis and metabolic perturbations. By providing this metabolic adaptability, MR ensures robust T cell activation and function, emphasizing the importance of maintaining COX integrity.

Ubiquinol, the reduced form of ubiquinone (coenzyme Q), is vital in mammalian cells, facilitating electron transfer from complexes I and II to complex III in the ETC, essential for ATP production and maintaining mitochondrial membrane potential. Moreover, it is integral to redox regulation, balancing oxidized and reduced states central to cellular signaling, metabolic processes, and stress-induced apoptosis^{18, 19}. The job of ubiquinol in cell growth is also notable, as its oxidation supports the TCA cycle and de novo pyrimidine synthesis, essential for cell proliferation⁸. COX interacts with the ubiquinol pool by ensuring the final transfer of electrons to oxygen in the ETC. This step is indispensable for the continuous oxidation of ubiquinol, thereby maintaining the flow of electrons through the ETC and supporting mitochondrial functions.

In TCox10^-/-/Aox T cells, AOX becomes a crucial component in the mitochondrial ETC, providing an azide-resistant pathway that bypasses complexes III and IV. By oxidizing ubiquinol to ubiquinone and reducing oxygen to water, AOX effectively prevents the over-reduction of the ubiquinone pool and mitigates ROS formation, thus protecting cells from oxidative stress²⁰. The activity of AOX is tightly regulated by the redox state of the ubiquinone pool, becoming significantly active when the pool is more than 35-40% reduced^{21, 22}. This regulation allows AOX to function as an "energy overflow" mechanism, facilitating the continuous shunting of excess electrons when the cytochrome pathway is saturated or inhibited, as in TCox10^-/- T cells. Similarly, COX plays a vital role in maintaining the redox state of the ubiquinol pool and preventing the over-reduction that leads to ROS formation. By ensuring that the ubiquinone pool remains properly balanced, COX prevents the accumulation of excess electrons, which can result in ROS and cellular damage. COX acts as an overflow mechanism, much like AOX, ensuring efficient electron flow within the electron transport chain and protecting cells from oxidative stress.

T cells undergo metabolic reprogramming to meet the demands of activation and function, dynamically shifting between glycolysis, the TCA cycle, FAO and OXPHOS. Notably, the Warburg effect, a shift to glycolysis even under aerobic conditions upon activation, ensures rapid energy production. However, mitochondrial dysfunction, as seen in COX-deficient T cells, can severely impair these metabolic pathways, affecting T cell responses and leading to immunodeficiency due to compromised metabolic functionality⁴. By providing this offloading pathway for reducing equivalents, MR by AOX ensures more efficient operation of both glycolysis and the TCA cycle. Consequently, AOX enhances cellular energy production and reduces oxidative stress, playing a pivotal role in restoring metabolic functionality, cellular energy balance and stress adaptation^{23, 24}.

COX has multiple functions, however the transfer of electrons from cytochrome c to molecular oxygen sustains the ETC²⁵. This process facilitates the translocation of protons from the mitochondrial matrix to the intermembrane space, creating an electrochemical gradient necessary for ATP synthesis^{26, 27}. The inability to perform this core function leads to release of cytochrome c from mitochondria into the cytoplasm, triggering apoptotic pathways²⁸. In TCox10^-/- T cells, overwhelming apoptosis leads to a T cell mediated immunodeficiency⁴. While apoptosis is a significant outcome of COX deficiency, by introducing the AOX, we show that its primary role is in MR. All other pathologies of COX deficiency stem from the loss of MR. Besides mediating apoptosis and acting as a metabolic safety valve as discussed above, MR also modulates mtDNA and nDNA transcription and maintenance of mtDNA⁵ as supported by our RNAseq and mtDNA studies. The impact on pathways related to the cell cycle, proliferation, and lymphocyte differentiation, migration, and activation in TCox10^-/-/Aox T cells demonstrates the broad effects of MR. It should be noted however, that COX deficiency, and resultant impairment of MR, is not just limited to genetic models. Inhibition of COX is mediated through diverse mechanisms which can occur in immune niches including chemical inhibition by drug complexes, ionic competition, and physiological regulatory molecules like nitric oxide and ATP^{29, 30, 31, 32}, making our findings broadly applicable.

By demonstrating the compensatory role of AOX, we establish the primary function of COX in T cells as MR. The prevention of ROS overproduction, metabolic perturbations, apoptosis, and T cell dysfunction by AOX emphasizes that these are secondary pathologies of COX deficiency. Our results underscore the importance of maintaining COX integrity for overall cellular health, highlighting the pivotal role of MR in energy production, transcriptional regulation, and cellular proliferation and differentiation.

Mouse Lines

C57Bl/6J (WT, B6) (strain #000664), Tg(Cd4-cre)1Cwi/BfluJ (Cd4-cre) (strain #017336), and C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-1) (strain #003831) mice were purchased from Jax. B6.129X1-Cox10^tm1Ctm/J (Cox10^fl/fl) were a kind gift from C. Morales and F.Diaz (University of Miami). Rosa26-targeted Ciona intestinalis alternative oxidase expressing (Aox) mice were a generous gift from Drs. Marten Szibor and Howard T. Jacobs³³. All procedures involving animals were in compliance with the Animal Care and Use Committee of the National Human Genome Research Center under an established protocol (G-11-3).

Cell Culture

Bulk T cell preparations were isolated from spleen with Pan-T cell Separation kit (Miltenyi Biotec) and stimulated ex vivo for 24 or 72 hours with anti-CD3/CD28 (BioXCell) before collection. For effector and memory T cell preparation, splenocytes from mice crossed with OT1 transgenic mice were isolated, treated ex vivo for three days with OVA peptide, followed by treatment for three days with IL-2 or IL-15 before collection as indicated in the text. Pan T cells, CD8⁺ T or CD4⁺ cells were enriched using isolation kits (Miltenyi Biotec). Purity of T cells was > 95% in all cases. T cells were stimulated with plate-bound anti-CD3 (5 µg/ml) and anti-CD28 (0.5 µg/ml). Cells were treated sodium azide with varying concentrations as described in the results.

Stable Isotopes

Mouse T cells were stimulated for 24 hr with plate-bound anti-CD3/CD28. Labeling experiments were performed essentially as described previously^{34, 35}. All labeling experiments were performed with 1 million cells/mL cultured in RPMI containing 11 mM glucose and 2 mM glutamine, with one nutrient or the other replaced by a uniformly ¹³C-labeled analog (i.e., [U−¹³C] glucose or [U−¹³C] glutamine; Cambridge Isotope Laboratories). Cells were rinsed in phosphate-buffered saline, then replenished with labeling medium at time 0. Culture proceeded for 24 hr, then the cells were briefly rinsed in cold saline, pelleted, and lysed in cold 50% methanol. The lysates were subjected to at least three freeze-thaw cycles, then centrifuged to remove debris. The supernatants were evaporated to dryness, methoximated and derivatized by tert-butyl dimethylsilylation. One µL of the derivatized material was injected onto an Agilent 6970 gas chromatograph equipped with a fused silica capillary GC column (30 m length, 0.25 mm diameter) and networked to either an Agilent 5973 or a 5975 Mass Selective Detector. Retention times of all metabolites of interest were validated using pure standards. The measured distribution of mass isotopomers was corrected for natural abundance of ¹³C.³⁶

RNAseq

T cells (10⁶ cells) were lysed in TRIzol (Thermo Fisher). Total RNA was isolated and purified with RNeasy kit (Qiagen) according to manufacturer’s protocol. RNAseq was performed by an outside commercial laboratory (Novogene, Sacramento, CA). Messenger RNA was purified using poly-T oligo-attached magnetic beads. First strand cDNA was synthesized with random hexamer primers, followed by second strand cDNA synthesis using dTTP. Libraries then underwent end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Sequencing was performed on the Illumina NovaSeq 6000 with 150bp paired-end reads. Reads were aligned to reference genome mm10 with Hisat2 v2.0.5, and raw read counts were determined using FeatureCounts v1.5.0-p3. Raw count normalization and differential expression analysis was performed using DESeq2 1.42.0³⁷. Hierarchical clustering was performed for outlier detection (Extended Data Fig. 4A). Volcano plots were prepared using EnhancedVolcano 1.20 with apeglm fold change shrinkage³⁸. For subsequent analysis, genes were considered significant if padj < 0.05.

GSEA

Gene sets were ranked by t-stat determined by DESeq2 prior to enrichment analysis. GSEA and visualization was performed with clusterProfiler 4.11.0³⁹. MitoCarta pathways were derived from a subset of mouse MitoCarta3.0 MitoPathways⁴⁰, with the addition of GO:0006098 (pentose-phosphate shunt) and GO:0061621 (canonical glycolysis). Visualization of KEGG pathway “apoptosis” (mmu04210) was performed with pathview 1.42.0 and KEGGREST 1.42.0.

Statistical analysis and visualization

Statistical significance was determined using GraphPad Prism v10 or R 4.3.2. All flow cytometry data were analyzed with FlowJo v10. Statistical tests used and n for each experiment are indicated in each figure legend. Sample sizes used are similar to our previous publication⁴. Experiments and data analysis were not performed blinded to genotype. Data distribution was assumed to be normal. Data was plotted and visualized with GraphPad Prism v10, or R packages clusterProfiler 4.11.0, ggplot2 3.5, pheatmap 1.0.12, or ComplexHeatmap 2.18.0⁴¹. Figures were prepared in Adobe Illustrator.

Influenza Infection

Mouse adapted human influenza virus A/PR/8/34 (PR8) and A/X/31 (X31) and X31 were used for infection. Mice were exposed to aerosolized (Glas-Col) 500 TCID or PR8 in 7mL of saline. Details and time points of infection are outlined in the text. Expression of viral hemagglutinin (HA) in the lungs of infected mice was determined by real time PCR.

BM Transfer

Bone marrow cells (CD45.2, 5 × 10⁶ cells) were injected intraorbitally into 8–12 week old irradiated B6 mice (CD45.1) (950 rad). Mice were then euthanized at time points as defined in the results.

Flow Cytometry

Single-cell suspensions of tissues were prepared. Anti- CD4, CD8, CD44, B220, CD69, CD247, IgM, CXCR5, PD-1, Foxp3 antibody were purchased from BD Biosciences or Thermofisher scientific. Data were acquired on a Cytoflex S flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star). Labeled tetramers (NIH tetramer core facility) were used to identify viral specific T cells. MitoTracker Green (Thermofisher), Total Reactive Oxygen Species (ROS) assay kit (Thermofisher) and TMRE (Abcam), 2-NBDG uptake kit (Abcam), MitoPY1 kit (Tocris Bioscience), ATP-Red (Millipore Sigma), were used according to manufacturer instructions. Apoptosis analysis was measured by Annexin V staining (ebioscience). Substrate cleavage by caspases were measured with caspase substrates PhiPhilus-G1D2, CaspaLux9- M2D2, CaspaLux8-L1D2 (OncoImmunin) according to the manufacturer instructions. Cells were loaded with 5 µM Cell Trace Violet (CTV) (ThermoFisher Scientific) and proliferation were estimated on day 3 by FACS. Gating strategy for CD4+ and CD8+ T cells is shown in Extended Data Fig 4B.

Real-Time PCR

RNA was extracted from the tissues using Pure link RNA mini kit (Thermo Fisher Scientific) and was reverse transcribed to cDNA (iScript, Bio-Rad) according to the manufacturer’s instructions. Reactions were cycled and quantitated with an CFX96 Real Time Biorad PCR System (Applied Biosystems).

In Vitro Differentiation

Naive (CD4⁺ (PerCP/Cy5.5, clone RM4–5) CD44^low (APC, clone IM7) CD62L^hi (eFluor 450, clone MEL-14) CD25^neg (PE, clone PC61.5) T cells) were purified by cell sorting. Purity was greater than 99% purity. Sorted naive CD4⁺ T cells (2 × 10⁵) were co-cultured at a ratio of 1:5 with mitomycin-treated T-depleted splenocytes as APCs in 48-well plates under various differentiation conditions for 3 days. Th1 conditions included 40 ng/ml IL-12 and anti-IL-4.Th17 used 20 ng/ml of IL-6, 5 ng/ml of TGF-β1, anti-IL-4, anti-IFN-γ, and anti-IL-12. Treg used 100 U/ml hIL-2, 5 ng/ml TGF-β1 with 1µg anti-CD3, and 10 µg/ml of each anti-IL-4, anti-IFN-γ, and anti-IL-12 antibodies. Antibodies were purchased from BioXcell unless otherwise indicated.

OCR and ECAR Measurement

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XF^e96 analyzer (Agilent). CD4⁺ or CD8⁺ T cells from mice activated for 24 hr with anti CD3 and anti CD28 were attached with Cell-Tak (Corning) according to manufacturer’s instructions at concentration 0.2 million cells/well in Seahorse BASE media with proprietary additives. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using the Mitostress kit (Agilent) according to the manufacturer’s standard protocol. OCR and ECAR were calculated and recorded by the Seahorse XF^e96 software. Complex IV activity (COX) was measured according to published methods⁴² using tetramethyl-p-phenylenediamine (TMPD) as an electron donor that is specific for complex IV with OCR as the readout.

Immunization and Serum Analysis

Mice were immunized with 50 µg of TNP-CGG in Imject Alum (Pierce Chemical) and re-immunized with TNP-CGG alone in 28 days. Sera were tested by ELISA for TNP reactivity. Briefly, plates were coated with TNP-BSA or (10 µg/ml; Biosearch Technologies), and bound immunoglobulins were detected by alkaline phosphatase-conjugated detection antibodies to specific mouse isotypes (Southern Biotechnology Associates).

ACKNOWLEDGEMENTS:

We would like to acknowledge Ms. Stacie Anderson and Ms. Martha Kirby for their assistance with cell sorting and flow cytometry (NHGRI Flow Cytometry Core).We would also like to thank Dr. Howard T. Jacobs (Tampere University) for the Mit-AOX mouse and Dr. Afshin Beheshti for advice on RNAseq analyses.

Funding:

National Institutes of Health grant ZIA HG200381-12 (PJM)

Author contributions:

Conceptualization: TT, PJM

Methodology: TT, EW, AF, BS, JMF, MSZ

Investigation: TT, EW, AF, BS, JMF

Visualization: TT, EW

Funding acquisition: PJM

Project administration: TT, EW, PJM

Supervision: PJM

Writing – original draft: TT, EW, PJM

Writing – review & editing: TT, EW, AF, BS, JMF, MSZ, PJM

Competing interests:

MSZ is a co-founder of a start-up company founded to develop therapeutics based on AOX.

Data and materials availability:

All data, code, and materials used in the analysis will be made available via paper figures or supplements for purposes of reproducing or extending the analysis. RNAseq data is available at GEO under accession GSE269797.

Peng, M. et al. Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism. Science 354,481-484 (2016).
Wang, R.N. et al. The Transcription Factor Myc Controls Metabolic Reprogramming upon T Lymphocyte Activation. Immunity 35,871-882 (2011).
Sena, L.A. et al. Mitochondria Are Required for Antigen-Specific T Cell Activation through Reactive Oxygen Species Signaling. Immunity 38,225-236 (2013).
Tarasenko, T.N. et al. Cytochrome c Oxidase Activity Is a Metabolic Checkpoint that Regulates Cell Fate Decisions During T Cell Activation and Differentiation. Cell Metab 25,1254-1268 e1257 (2017).
Miranda, M., Bonekamp, N.A. & Kühl, I. Starting the engine of the powerhouse: mitochondrial transcription and beyond. Biological Chemistry 403,779-805 (2022).
Moore, A.L. & Siedow, J.N. The regulation and nature of the cyanide-resistant alternative oxidase of plant mitochondria. Biochim Biophys Acta 1059,121-140 (1991).
Rasmusson, A.G., Fernie, A.R. & van Dongen, J.T. Alternative oxidase: a defence against metabolic fluctuations? Physiol Plant 137,371-382 (2009).
Martínez-Reyes, I. et al. Mitochondrial ubiquinol oxidation is necessary for tumour growth. Nature 585,288-+ (2020).
Mogi, T., Saiki, K. & Anraku, Y. Biosynthesis and functional role of haem O and haem A. Mol Microbiol 14,391-398 (1994).
Hakkaart, G.A., Dassa, E.P., Jacobs, H.T. & Rustin, P. Allotopic expression of a mitochondrial alternative oxidase confers cyanide resistance to human cell respiration. EMBO Rep 7,341-345 (2006).
Rajendran, J. et al. Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy. Embo Mol Med 11 (2019).
Le, A. et al. Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell Metab 15,110-121 (2012).
Balazs, R. Control of Glutamate Oxidation in Brain and Liver Mitochondrial Systems. Biochem J 95,497-508 (1965).
MacIver, N.J. et al. Glucose metabolism in lymphocytes is a regulated process with significant effects on immune cell function and survival. J Leukocyte Biol 84,949-957 (2008).
van der Windt, G.J.W. et al. CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability. P Natl Acad Sci USA 110,14336-14341 (2013).
Belz, G.T., Xie, W.D., Altman, J.D. & Doherty, P.C. A previously unrecognized H-2D-restricted peptide prominent in the primary influenza A virus-specific CD8 T-cell response is much less apparent following secondary challenge. J Virol 74,3486-3493 (2000).
Townsend, A.R., Gotch, F.M. & Davey, J. Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell 42,457-467 (1985).
Dallner, G. & Sindelar, P.J. Regulation of ubiquinone metabolism. Free Radic Biol Med 29,285-294 (2000).
Kelso, G.F. et al. Selective targeting of a redox-active ubiquinone to mitochondria within cells - Antioxidant and antiapoptotic properties. Journal of Biological Chemistry 276,4588-4596 (2001).
Umbach, A.L., Fiorani, F. & Siedow, J.N. Characterization of transformed Arabidopsis with altered alternative oxidase levels and analysis of effects on reactive oxygen species in tissue. Plant Physiol 139,1806-1820 (2005).
Dry, I.B., Moore, A.L., Day, D.A. & Wiskich, J.T. Regulation of Alternative Pathway Activity in Plant-Mitochondria - Nonlinear Relationship between Electron Flux and the Redox Poise of the Quinone Pool. Arch Biochem Biophys 273,148-157 (1989).
Hoefnagel, M.H.N. & Wiskich, J.T. Activation of the plant alternative oxidase by high reduction levels of the Q-Pool and pyruvate. Arch Biochem Biophys 355,262-270 (1998).
Weaver, R.J. Hypothesized Evolutionary Consequences of the Alternative Oxidase (AOX) in Animal Mitochondria. Integr Comp Biol 59,994-1004 (2019).
Millenaar, F.F. & Lambers, H. The alternative oxidase:: regulation and function. Plant Biology 5,2-15 (2003).
Thompson, D.A., Suarez-Villafane, M. & Ferguson-Miller, S. The active form of cytochrome c oxidase: effects of detergent, the intact membrane, and radiation inactivation. Biophys J 37,285-293 (1982).
Wikstrom, M.K. Proton pump coupled to cytochrome c oxidase in mitochondria. Nature 266,271-273 (1977).
Denis, M. Structure and function of cytochrome-c oxidase. Biochimie 68,459-470 (1986).
Kagan, V.E. et al. Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine. Free Radic Biol Med 37,1963-1985 (2004).
Hasinoff, B.B. & Davey, J.P. The Iron(Iii) Adriamycin Complex Inhibits Cytochrome-C Oxidase before Its Inactivation. Biochem J 250,827-834 (1988).
Vygodina, T.V., Mukhaleva, E., Azarkina, N.V. & Konstantinov, A.A. Cytochrome c oxidase inhibition by calcium at physiological ionic composition of the medium: Implications for physiological significance of the effect. Bba-Bioenergetics 1858,982-990 (2017).
Torres, J., Darley-Usmar, V. & Wilson, M.T. Inhibition of cytochrome c oxidase in turnover by nitric oxide: mechanism and implications for control of respiration. Biochem J 312 ( Pt 1),169-173 (1995).
Arnold, S. & Kadenbach, B. Cell respiration is controlled by ATP, an allosteric inhibitor of cytochrome-c oxidase. Eur J Biochem 249,350-354 (1997).
Szibor, M. et al. Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology. Dis Model Mech 10,163-171 (2017).
Cheng, T.L. et al. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells. P Natl Acad Sci USA 108,8674-8679 (2011).
Mullen, A.R. et al. Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature 481,385-U171 (2012).
Desrosiers, C., Fernandez, C.A., David, F. & Brunengraber, H. Reversibility of the Mitochondrial Isocitrate Dehydrogenase Reaction in the Perfused-Rat-Liver - Evidence from Isotopomer Analysis of Citric-Acid Cycle Intermediates. Journal of Biological Chemistry 269,27179-27182 (1994).
Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15,550 (2014).
Zhu, A., Ibrahim, J.G. & Love, M.I. Heavy-tailed prior distributions for sequence count data: removing the noise and preserving large differences. Bioinformatics 35,2084-2092 (2019).
Wu, T. et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation (Camb) 2,100141 (2021).
Rath, S. et al. MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations. Nucleic Acids Res 49,D1541-D1547 (2021).
Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32,2847-2849 (2016).
Salabei, J.K., Gibb, A.A. & Hill, B.G. Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis. Nat Protoc 9,421-438 (2014).

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Cytochrome c oxidase dependent respiration is essential for T cell activation, proliferation and memory formation

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