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. 2026 May 19;16:22767. doi: 10.1038/s41598-026-53892-8

Metabolic impairments in peripheral blood mononuclear cells after BNT162b2 vaccination in liver transplantation adolescents

Supranee Buranapraditkun 1,2,3,✉, Nakarin Kitkumthorn 4, Arkom Chaiwongkot 5,6, Pattarawat Thantiworasit 7, Palittiya Sintusek 2
PMCID: PMC13385713  PMID: 42156880

Abstract

The mRNA vaccine BNT162b2 was approved to prevent COVID-19 disease in adolescents. Currently, there are no data on cellular metabolism in adolescents receiving liver transplantation (LT) before and after SARS-CoV-2 vaccination. This study aimed to investigate the effects of the BNT162b2 mRNA vaccine on cellular metabolism in LT adolescents compared to healthy adolescents. The bioenergetics study was performed using the PBMCs of 15 LT adolescents and 15 healthy adolescents aged between 12 and 18 years. All participants received 30 µg of BNT162b2 on days 0 and 21. Blood samples were collected at 3 time points: before vaccination (day 0), on day 21 and on day 35 after the first dose. Mitochondrial respiration and glycolysis in the frozen PBMCs were measured by using a Seahorse XFp analyzer. The results showed that LT adolescents had a lower oxygen consumption rate (OCR) during mitochondrial respiration than healthy adolescents at all time points. Additionally, LT adolescents exhibited reduced glycolysis, as indicated by a lower extracellular acidification rate (ECAR), throughout the study period. These findings suggest that impairments in mitochondrial and glycolytic metabolism observed in PBMCs from LT adolescents after BNT162b2 vaccination may contribute to the early suppression of the immune response. Future studies should evaluate which immune cells are critical for metabolic dysfunctions.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-53892-8.

Keywords: Cellular metabolism, Liver transplantation, BNT162b vaccine, Adolescents, Peripheral blood mononuclear cells, Seahorse XFp analyzer

Subject terms: Immunology, Health care, Medical research

Introduction

The outbreak of respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus that causes coronavirus disease 2019 (COVID-19), is a major health problem worldwide, including in Thailand. Since January 2022, more than 349.6 million people have been infected, and more than 5.6 million people have died from COVID-19 infection, particularly patients with chronic disease or a compromised immune system1. Vaccinations play an important role in global health and help prevent numerous deadly and infectious diseases. Currently, vaccines are playing an important role in preventing COVID-19 disease in children2, adults, and elderly people3,4. However, there is limited research on the effectiveness and safety of COVID-19 vaccines for adolescent children, including adolescents undergoing liver transplantation (LT)5. Liver transplantation is a critical therapeutic intervention for end-stage liver disease or acute liver failure. However, lifelong immunosuppression is required to prevent organ rejection, which can lead to impaired immune responses and increased susceptibility to infections6. The BNT162b2 mRNA vaccine, developed by Pfizer-BioNTech7, has been successful in protecting against COVID-19 in various age groups. In addition, the BNT162b2 mRNA vaccine has been authorized for use in adolescents older than 12 years since May 20218–10. However, there is a lack of information on the efficacy and safety of the BNT162b2 mRNA vaccine, particularly in immunocompromised hosts, including LT adolescents.

Blood-based assays for assessing cellular metabolism are highly accurate and require minimal invasiveness when used to assess underlying disease conditions. Peripheral blood mononuclear cells (PBMCs) are utilized as a model system for monitoring diseases through bioenergetic analyses. PBMCs play a pivotal role in the immune response because they consist of lymphocytes (T cells, B cells) and monocytes that are essential for pathogen recognition and elimination. Moreover, PBMCs offer an accessible supply of mitochondria and other delicate biomarkers. Notably, PBMCs can reflect overall changes within the body and have been studied in various chronic diseases11–13 and non-diseases14,15. Metabolic impairments in PBMCs have been observed under various conditions and can significantly affect their functionality. Although both mitochondrial respiration and glycolysis are essential for fundamental cellular metabolism, post-vaccination studies on PBMC metabolism following BNT162b2 mRNA vaccination among LT adolescents are lacking.

PBMCs contain immune cells such as T cells that play a crucial role in the adaptive immune response and protection against viral infections. T cell function is associated with cellular metabolism, particularly through oxidative phosphorylation within mitochondria and glycolysis16,17. Mitochondria serve as energy-generating powerhouses of cells and play a critical role in generating ATP through oxidative phosphorylation during T cell metabolism and function. The energy requirements and functional capabilities of T cells mainly rely on mitochondrial metabolism18. Glycolysis is an alternative metabolic pathway and provides a rapid energy source for T cells during activation and proliferation processes19,20.

Assessing the metabolic profile of immune cells can provide valuable insights into their functionality. Stress tests that examine mitochondrial respiration and glycolytic activity, such as the Seahorse Extracellular Flux Analyzer (EFA), have been employed to evaluate the metabolic status of cells21. These assays allow the measurement of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which represent mitochondrial respiration and glycolytic activity, respectively. Changes in these metabolic parameters can reflect alterations in immune cell function and may contribute to the impaired immune response observed in LT adolescents.

Therefore, this study aimed to investigate how the cellular metabolism affects peripheral blood mononuclear cell (PBMC) mitochondrial respiration and glycolysis in LT adolescents compared to healthy adolescents after receiving the BNT162b2 mRNA vaccine. The alterations in mitochondrial respiration and glycolysis metabolism in PBMCs may provide insights into the cellular mechanisms underlying the impaired immune response observed in LT adolescents compared to healthy adolescents after BNT162b2 mRNA vaccination.

Results

Loss of SARS-CoV-2-specific T-cell responses in liver-transplantation adolescents after BNT162b2 vaccination

We examined the quantity of IFN-γ-secreting SARS-CoV-2-specific T-cell responses from PBMCs in 15 liver-transplanted (LT) adolescents aged between 12 and 17 years (median age: 13) and 15 healthy adolescents aged between 12 and 18 years (median age: 15). Among the LT adolescents, 33% were female and 67% were male, whereas among the healthy adolescents, 60% were female and 40% were male (Table 1). Age and sex distributions were compared between groups; no significant differences were detected. All participants received 30 µg of BNT162b2 on day 0 and day 21. Blood was collected at baseline (day 0; D0) and at 21 (D21) and 35 (D35) days after vaccination. Fresh PBMCs were tested for SARS-CoV-2-specific T-cell responses by the human interferon gamma (IFN-γ) ELISpot assay (Fig. 1A). Among 15 LT adolescents, 8 (53%) experienced SARS-CoV-2-specific T-cell responses after receiving the first dose of the vaccine, and 4 (27%) experienced T-cell responses after receiving the second dose. In contrast, all healthy adolescents (100%) had SARS-CoV-2-specific T-cell responses following both the first and second doses. The number of spot-forming cells (SFCs)/106 PBMCs ranged from 0 to 28 SFCs/106 PBMCs at baseline (D0), 0-348 SFCs/106 PBMCs at D21 and 0-712 SFCs/106 PBMCs at D35 (median 0, 32 and 4 SFCs/106 PBMCs at D0, D21 and D35, respectively) from LT adolescents (Fig. 1B), and the number of 0–44 SFCs/106 PBMCs at D0, 44–616 SFCs/106 PBMCs at D21 and 84–848 SFCs/106 PBMCs at D35 (median 4, 244 and 280 SFCs/106 PBMCs at D0, D21 and D35, respectively) from healthy adolescents (Fig. 1C). We found that SARS-CoV-2 spike peptide pool (S1 and S2)-specific T-cell responses were significantly greater in the number of IFN-γ-secreting cells after the first and second doses of BNT162b2 at D21 and D35 than at baseline in both LT adolescents (p = 0.0028 and 0.0450, respectively, Fig. 1B) and healthy adolescents (P = 0.0005 and < 0.0001, respectively, Fig. 1C). In addition, the number of SARS-CoV-2-specific T cells in LT adolescents was significantly lower than that in healthy adolescents on both D21 and D35 after BNT162b2 vaccination (p = 0.0002 and 0.0003, respectively, Fig. 1D). These data suggested an impaired T-cell response in LT adolescents after BNT162b2 vaccination.

Fig. 1.

Fig. 1

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Loss of T cell responses in liver transplantation (LT) after BNT162b2 vaccination (A) The spots from human IFN-γ ELISpot assay in LT and healthy adolescents stimulated with spike SARS-CoV-2, R10 as negative control and PHA as positive control, (B) Human IFN-γ ELISpot data of spike SARS-CoV-2 specific T cell responses in LT adolescents, (C) Human IFN-γ ELISpot data of spike SARS-CoV-2 specific T cell responses in healthy adolescents, and (D) Comparison of human IFN-γ ELISpot data of spike SARS-CoV-2 specific T cell responses in LT and healthy. Symbols represent data from individual participants. Statistical analyses were performed using Mann-Whitney test and Friedman test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Mitochondrial function is reduced at baseline and remains impaired after BNT162b2 vaccination in LT adolescents

To determine whether impaired T-cell function in LT adolescents is caused by altered oxygen consumption rates and bioenergetic parameters, we used a Seahorse XFp analyzer in a live cellular environment to measure PBMCs in both LT and healthy adolescents before and after BNT162b2 vaccination. We measured the oxygen consumption rates (OCRs) of healthy adolescents (n = 15) and LT adolescents (n = 15) collected on day 0, day 21 and day 35 before and after the addition of oligomycin, FCCP and rotenone/antimycin A, respectively. We found that mitochondrial respiration in LT adolescents was lower than that in healthy adolescents on days 0, D21 and D35 post-BNT162b2 vaccination (Fig. 2A). In addition, we found that PBMCs isolated from healthy adolescents had significantly higher basal respiration than those from LT adolescents at each time points (D0, 32.16 ± 13.75 vs. 12.17 ± 7.98; D21, 27.28 ± 10.26 vs. 13.91 ± 12.20; and D35, 31.75 ± 21.36 vs. 13.70 ± 6.99 pmol/min/300K cells, respectively), indicating that more exogenous ATP is required in LT cells (Fig. 2B). Moreover, we found that basal respiration was lower in LT adolescents than in healthy adolescents at day 0, day 21, and day 35, indicating that ATP-linked OCR and maximal respiration were also reduced in the LT adolescents.

Fig. 2.

Fig. 2

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The mitochondria stress test of PBMC from liver transplantation (LT) and healthy adolescents after BNT162b2 vaccination. (A) Oxygen comsumption rates (OCR) of healthy adolescents (n = 15) and LT adolescents (n = 15) recorded at day0, day21 and day35 before and after adding oligomycin, FCCP and retenone/antimycin A, respectively. Mitochondrial respiration from LT adolescents showed lower OCR at day0, day21 and day35 post BNT162b2 vaccination compared to healthy adolescents. The assay reports significantly higher (B) basal respiration (C) ATP production and (D) maximal respiration in all time points in PBMCs of healthy adolescents compared to LT adolescents. Both groups have similar levels of (E) proton leak (F) non-mitochondrial respiration and (G) coupling efficiency were analyzed using these parameters. (H) The spare respiratory capacity in healthy adolescents showed higher than LT adolescents in all time points but significantly only at day0. (D, F, G) PBMC at maximum respiration, non-mitochondrial oxygen consumption and coupling efficiency at pre-vaccination were significantly higher than at day35 post vaccination whereas (E) proton leak at day0 was significantly lower than at day35 post vaccination. Symbols represent data from individual participants. Statistical analyses were performed using Mann-Whitney test and Friedman test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Then, PBMCs were treated with oligomycin, which decreases electron flow through the electron transport chain (ETC) by inhibiting ATP synthase. The resulting reduction in mitochondrial respiration after oligomycin injection isolates the OCR linked to ATP production (Fig. 2A). The oxygen consumption rate that is used to produce mitochondrial ATP was significantly greater in healthy PBMCs than in those isolated from LT adolescents at each time point (D0, 27.88 ± 13.06 vs. 8.90 ± 5.50; D21, 21.83 ± 9.24 vs. 10.24 ± 4.37; and D35, 23.27 ± 12.6 vs. 9.15 ± 4.88 pmol/min/300K cells, respectively; Fig. 2C), reflecting the reduced ability of LT PBMCs to use membrane potential, which serves as a driving force for ATP synthesis15. In addition, we found that ATP production in healthy individuals tended to decrease from day 0 to day 35, suggesting a reduced ability of PBMCs to use membrane potential after vaccination. However, the number of PBMCs in LT adolescents showing changes in mitochondrial membrane potential did not differ at each time point, indicating that their mitochondrial function remained consistency impaired before and after vaccination. Subsequent treatment with a mitochondrial uncoupler, FCCP, shuttles protons across the inner membrane, causing the ETC to operate at maximal capacity16. The effect was the maximum respiration, maintaining the proton motive force at the highest levels (Fig. 2D). PBMCs from healthy adolescents were significantly higher maximal respiration following FCCP treatment than those from LT adolescents at each time point (D0, 54.77 ± 34.55 vs. 16.45 ± 12.15; D21, 46.06 ± 28.17 vs. 24.32 ± 24.30; and D35, 47.31 ± 45.19 vs. 16.97 ± 9.97 pmol/min/300K cells) (Fig. 2D). The maximum respiration in healthy adolescents at baseline was significantly greater than that at day 35, suggesting that the levels of oxidative respiration decreased after BNT162b2 mRNA vaccination but not in LT adolescents (Fig. 2D). We also measured energy dissipation as proton leakage (Fig. 2E) and found that both groups demonstrated similar proton leak-linked respiration rates at each time point (D0, 4.28 ± 4.48 vs. 3.27 ± 6.08; D21, 5.46 ± 4.62 vs. 3.67 ± 11.29; and D35, 8.48 ± 11.18 vs. 4.55 ± 5.77 pmol/min/300K cells in healthy PBMCs and liver-transplanted participants, respectively).

As a final step of the mitochondrial stress test, rotenone and antimycin A were added to block complexes I and III, respectively. Together, these agents fully inhibit mitochondrial electron transfer, and the residual OCR measured can be attributed to non-mitochondrial oxidases. This residual respiration was greater in PBMCs isolated from healthy adolescents than in those from LT adolescents at all time points, but the difference was not significant (D0, 12.83 ± 5.76 vs. 10.22 ± 6.43; D21, 10.17 ± 4.57 vs. 13.03 ± 8.84; D35, 9.67 ± 6.45 vs. 8.69 ± 5.29 pmol/min/300K cells; and healthy PBMCs and LT adolescents, respectively; Fig. 2F). In addition, we found that the non-mitochondrial oxidases in healthy controls at day 0 were greater than those at day 21 and day 35, indicating that non-mitochondrial respiration contributes to overall respiration to a greater extent in healthy PBMCs than in LT PBMCs prior to vaccination.

We found that the coupling efficiency tended to decrease but not significantly in the PBMCs from the LT adolescents compared with those from the healthy adolescents at each time point (D0, 85.76 ± 12.22 vs. 61.59 ± 51.06; D21, 80.90 ± 14.07 vs. 54.10 ± 55.40; and D35, 76.45 ± 13.86 vs. 79.05 ± 41.01 pmol/min/300K cells) (Fig. 2G). Moreover, we found that the coupling efficiency in healthy adolescent PBMCs at day 21 and day 35 was significantly lower than that at day 0, suggesting that the mitochondrial efficiency of substrate oxidation is lower in those cells after the first and second doses of the BNT162b mRNA vaccine.

The spare respiratory capacity was calculated as the maximal respiration minus the basal respiration. We found that the spare respiratory capacity was significantly greater in PBMCs isolated from healthy adolescents than in those from LT adolescents at day 0 but not at day 21 or day 35, even though it was greater than that in those from LT adolescents (D0, 22.61 ± 23.39 vs. 4.28 ± 11.16; D21, 18.78 ± 21.18 vs. 10.40 ± 19.92; and D35, 15.56 ± 24.51 vs. 3.27 ± 8.17 pmol/min/300K cells; in healthy PBMCs and LT adolescents, respectively; Fig. 2H).

Impairment of glycolytic function in LT adolescents after BNT162b2 vaccination

To determine whether glycolysis affects T-cell function in LT adolescents by measuring the extracellular acidification rate (ECAR) in PBMCs, which reflects the level of lactic acid generated by glycolysis (Fig. 3A), we used the Seahorse XFp Analyzer in a live cellular environment to measure lactic acid in both LT and healthy adolescents before and after BNT162b2 vaccination. First, glucose was injected as the primary energy source, leading to an increase in the ECAR in PBMCs. Subsequently, we measured the amount of glycolysis that was upregulated in response to the elimination of non-glycolytic acidification. PBMCs from healthy adolescents had a more significant response to glycolysis only at day 0 and day 35 and tended to increase at day 21 (D0; 6.89 ± 3.62 vs. 4.39 ± 1.67, D21; 5.7 ± 2.15 vs. 4.21 ± 1.64, and D35; 5.65 ± 2.94 vs. 3.50 ± 1.35 pmol/min/300K cells, in healthy PBMCs and LT adolescents, respectively, P < 0.05, Fig. 3B) than did PBMCs from LT adolescents. When expressed as fold change relative to baseline (D0), glycolytic activity in healthy adolescents increased at day 21 (1.19 ± 0.15) and day 35 (1.16 ± 0.12), whereas LT adolescents showed minimal change at day 21 (1.03 ± 0.11) and day 35 (0.98 ± 0.09) (P < 0.05, between groups). These results indicate the loss of glycolysis energy in LT adolescents after BNT162b vaccination.

Fig. 3.

Fig. 3

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The glycolysis test of PBMC from liver transplantation (LT) and healthy adolescents after BNT162b2 vaccination. (A) Extracellular acidification rates (ECAR) of healthy adolescents (n = 15) and LT adolescents (n = 15) recorded at day 0, day 2 and day 35 before and after adding glucose, oligomycin and 2-DG, respectively. Glycolysis from LT participants showed lower ECAR at day 0, day 21 and day 35 post BNT162b2 vaccination compared to healthy adolescents. The assay reports significantly higher (B) glycolysis at day 0 and day 35 (C) glycolytic capacity in all time points (D) non-glycolytic acidification at day 0 and (E) glycolytic reverse at day 0 and day 35 in PBMCs of healthy adolescents compared to LT adolescents. (E) PBMC at glycolytic reverse at pre-vaccination was significantly higher than at day 35 post vaccination (P < 0.05). Symbols represent data from individual participants. Statistical analyses were performed using Mann-Whitney test and Friedman test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Next, we assessed alterations in the ECAR (ΔECAR), which represents the overall glycolytic capacity, in response to oligomycin using a comprehensive plot of the ECAR. Compared with those from LT adolescents, PBMCs from healthy adolescents responded significantly more robustly to oligomycin at all time points (D0: 6.28 ± 2.40 vs. 6.28 ± 2.40; D21: 20.20 ± 11.05 vs. 6.61 ± 3.37; and D35: 16.85 ± 5.39 vs. 5.2 ± 2.4 pmol/min/300K cells, respectively; Fig. 3C). These findings indicate that the switch to glycolysis is more efficient in PBMCs from healthy adolescents when ATP synthase is inhibited.

As the final step of the glycolysis stress test, 2DG was introduced to inhibit glucose production, effectively blocking the glycolysis pathway. The remaining ECAR measured under these conditions was referred to as non-glycolytic acidification. Notably, at baseline, the residual respiration was significantly greater in PBMCs isolated from healthy adolescents than in those from LT adolescents (D0; 5.01 ± 1.84 vs. 3.87 ± 1.79 pmol/min/300K cells in healthy and LT adolescents, respectively; Fig. 3D). However, this difference was not significant between day 21 and day 35 (D21: 3.86 ± 1.25 vs. 4.06 ± 2.23 and D35: 4.44 ± 1.67 vs. 3.58 ± 1.87 pmol/min/300K cells in healthy and LT adolescents, respectively; Fig. 3D). These results suggested that non-glycolytic acidification contributes more significantly to overall respiration in healthy adolescents than in LT adolescents before and after vaccination.

In addition, the glycolytic reverse was calculated from the ratio of glycolytic capacity to glycolysis. We found that the glycolytic reversal rate was significantly higher in PBMCs isolated from healthy adolescents than in those from LT adolescents at day 0 and day 35 but not at day 21, even though it was greater than that in those from LT adolescents (D0, 4.24 ± 1.77 vs. 1.89 ± 1.24; D21, 3.68 ± 1.83 vs. 2.40 ± 2.23; D35, 3.34 ± 1.1 vs. 1.7 ± 1.36 pmol/min/300K cells; and healthy PBMCs and LT adolescents, respectively; Fig. 3E). We also found that the extent of glycolytic reversal in healthy adolescents at baseline was significantly greater than that at day 35, suggesting that glycolytic reversal decreased after BNT162b vaccination.

Decreased overall metabolism with a greater proportion of ATP derived from mitochondria in LT adolescents

The evidence of lower oxidative stress in LT adolescent points to possible mitochondrial and glycolytic damage. To examine the overall metabolism that affects mitochondrial and glycolytic functions, we assessed the respiration profiles of the PBMCs using a Seahorse XFp Analyzer. The metabolomics data showed that a smaller proportion of ATP was generated by glycolysis, whereas a greater proportion of ATP was generated by mitochondria at all time points (glycolysis/mitochondria in healthy adolescents; D0: 6.89/32.16%, D21: 5.70/27.29% and D35: 5.65/31.75% and in LT adolescents; D0: 4.39/12.44%, D21: 4.21/14.89% and D35: 3.50/13.70%, respectively) in both LT and healthy adolescents (Fig. 4A). Overall metabolic flux in both mitochondria and glycolysis was decreased in LT adolescents compared to healthy adolescents (Fig. 4A). There were significant differences in the overall metabolic flux between LT adolescents and healthy adolescents at all time points (P < 0.05).

Fig. 4.

Fig. 4

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Overall metabolism in liver transplantation (LT) and healthy adolescents after BNT162b2 vaccination. (A) The ATP production rate shows a decrease in mitochondrail of LT adolescents compared to healthy adolescents, (B) The metabolic index in LT and healthy adolescents, (C) The overall energy map in LT and healthy adolescents at day 0, day 21 and day 35 post BNT162b2 vaccination. Statistical analyses were performed using Mann-Whitney test and Friedman test. *P < 0.05, **P < 0.01.

The metabolic index representing the ratio of the mitochondrial ATP production rate to the glycolytic ATP production rate in healthy adolescents was greater than that in LT adolescents at all time points (D0, 5.53 ± 3.45 vs. 3.13 ± 2.57; D21, 5.17 ± 2.41 vs. 4.20 ± 3.93 and D35, 6.81 ± 5.04 vs. 5.11 ± 4.42; in healthy and LT adolescents, respectively; Fig. 4B). Notably, the metabolic index in healthy adolescents was significantly greater than that in LT adolescents only at baseline (P < 0.05), suggesting that a greater metabolic activity and efficiency in ATP production are associated with mitochondrial function at baseline in healthy adolescents.

Energetic maps of LT patients and healthy adolescents were generated by comparing mitochondrial ATP (mitoATP) to glycolysis-generated ATP (glycoATP). When comparing the energy phenotypes inherent to LTs and healthy adolescents at all time points, it was observed that LT adolescents were more quiescent, as they had less oxygen consumption and glycolysis activity, whereas healthy adolescents were more energetic, as they had more oxygen consumption and glycolysis activity at all time points (Fig. 4C). In addition, the PBMCs of both LT adolescents and healthy adolescents did not exhibit any alterations in oxygen consumption after BNT162b2 vaccination (D0 vs. D21 vs. D35). These data indicated that LT adolescent PBMCs already have decreased mitochondrial and glycolytic functions compared to those of healthy adolescent PBMCs at baseline.

Inhibition of the glycolytic pathway impairs SARS-CoV-2-specific T-cell responses in healthy adolescents

To determine whether the immune response of SARS-CoV-2-specific T cells involves the activation of glycolysis pathways, we incubated PBMCs from healthy adolescents with 2-deoxy-D-glucose to block the glycolysis pathway (2-DG, a glycolytic inhibitor). We observed that in all 10 healthy adolescents, IFN-γ secretion from SARS-CoV-2-specific T cells was significantly reduced by more than 80% after stimulation with spike peptides and 2-DG compared to stimulation with peptides alone. The median number of spot-forming cells (SFCs)/106 PBMCs decreased from 28 to 4 SFCs/106 (86%, D0), 48 to 4 SFCs/106 (92%, D21), and 90 to 16 SFCs/106 (82%, D35) (Fig. 5A). In addition, IFN-γ secretion after stimulation with spike peptides and 2-DG was significantly lower than that after stimulation with spike peptides alone at all time points (P < 0.01), indicating that glycolysis was effectively inhibited (Fig. 5B). These data indicate that 2-DG effectively inhibits glycolysis pathways and impairs the cellular metabolic pathways crucial for the proliferation and function of SARS-CoV-2-specific T cells.

Fig. 5.

Fig. 5

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Decreased IFN-γ secretion after stimulation with spike peptides with 2-DG in healthy adolescents after BNT162b2 vaccination. (A) The spots from frozen PBMCs stimulated with spike SARS-CoV-2 with and without 2-DG by human IFN-γ ELISpot assay (N = 2), (B) Human IFN-γ ELISpot data of spike SARS-CoV-2 specific T cell responses with and without 2-DG from healthy adolescents at day 0, day 21 and day 35 post BNT162b2 vaccination. Symbols represent data from individual participants. Statistical analyses were performed using Kruskal-Wallis test and Wilcoxon matched-pairs test. *P < 0.05, **P < 0.01.

Discussion

This prospective study provides comprehensive information on PBMCs, including T cells, responses and metabolic pathways, in liver transplantation (LT) adolescents after BNT162b2 mRNA vaccination. First, our study determined the T-cell responses using an ex vivo human IFN-γ ELISpot assay in PBMCs from LT adolescents and healthy adolescents after two doses of the BNT162b2 mRNA vaccine. The results showed an increase in SARS-CoV-2 spike peptide pool (S1 and S2)-specific T-cell responses after the first (D21) and second (D35) doses of the BNT162b2 vaccine compared to baseline in both groups. These findings support the findings that vaccinated individuals exhibit hallmark features of induced T-cell responses22. However, the SARS-CoV-2-specific T-cell responses in LT adolescents at D21 and D35 were lower than those in healthy adolescents, suggesting an impaired T-cell response in LT adolescents after vaccination. Previous studies indicate that memory T cells play an important role in protection after primary SARS-CoV-2 vaccination23, although our study did not directly characterize memory T-cell subsets. The observed boosting effect after the second dose is in line with the generation of SARS-CoV-2-specific T-cell responses, which persisted after vaccination24. Similar to our results, other transplantation recipients, such as those of the lung, heart, and kidney, exhibited a decrease in cellular immune responses14,15,25. Moreover, clinical factors such as impaired renal function and increased use of immunosuppressive medications may further contribute to reducing humoral and cellular immune responses in LT adolescents following treatment with the Pfizer-BioNTech SARS-CoV-2 mRNA-based vaccine26,27.

Currently, cellular metabolism in various diseases, such as Parkinson’s disease28, HIV29, and renal disease30, has been investigated with a Seahorse analyzer using PBMCs. We examined cellular metabolism via mitochondrial and glycolytic stress tests in the PBMCs of LT adolescents compared to those of healthy adolescents after two doses of the BNT162b2 mRNA vaccine. In this study, we determined the oxygen consumption rate (OCR), which is associated with mitochondrial respiration, in peripheral blood mononuclear cells (PBMCs) isolated from liver transplantation (LT) adolescents. We found that the basal, ATP-linked and maximal oxygen consumption rates in LT adolescents were lower than those in healthy adolescents at all time points, suggesting that impaired mitochondrial function led to a reduction in the components of the electron transport chain (ETC) machinery and a decrease in oxidative phosphorylation in LT adolescents. While oxidative phosphorylation is typically closely linked to electron transport under normal conditions, the energy generated from substrate oxidation does not completely contribute to ATP synthesis. This is because some protons, which are transported from the mitochondrial matrix to the intermembrane space during the ETC, can leak back across the mitochondrial inner membrane through uncoupling proteins31. The level of proton leakage was similar in both healthy and LT adolescents at all time points, suggesting that it does not significantly affect mitochondrial performance. PBMCs obtained from LT adolescents exhibited a diminished ability to reach the same levels of oxidative phosphorylation observed in healthy adolescent PBMCs, which might be due to reduced mitochondrial mass or compromised integrity32. The coupling efficiency did not differ between healthy and LT adolescents. However, the coupling efficiency in healthy adolescents after the first and second doses of BNT162b mRNA vaccination was lower than that at baseline because their respiration is linked to alternative proton conductance pathways, leading to heat generation instead of ATP synthesis33. In addition, spare respiratory capacity represents a crucial parameter that affects the ability of cells to enhance their capacity for mitochondrial ATP synthesis34. This was measured by observing the change in oxidative phosphorylation following uncoupler stimulation. PBMCs from healthy adolescents demonstrated a greater capacity to generate energy reserves and adjust to unanticipated increases in energy demand at baseline that were greater than those of the first and second doses of the BNT162b mRNA vaccine.

Additionally, we assessed extracellular acidification rates, which are attributed to glycolysis. Glycolysis is considerably less efficient in terms of ATP production; glycolysis generates 2 ATP molecules, whereas mitochondrial respiration produces 36 ATP molecules35. In terms of the baseline glycolytic rate (measured by the ECAR), we found that the overall baseline glycolytic rate in LT adolescents was significantly lower than that in healthy adolescents (D0 and D35), except for the D29 trend toward a decrease, but the difference was not significant. This results in fewer PBMCs from LT adolescents using glycolysis for energy than do PBMCs from healthy individuals. Unlike type 1 diabetes those of T1D participants, the PBMCs of T1D participants exhibited greater baseline glycolysis than did those of healthy participants36. In addition, Amy M. Smith reported an increase in glycolysis in both Parkinson’s disease and rapid eye movement-sleep behavior disorder participants28. This is likely because our cohort consisted of LT adolescents who were not disease-challenged individuals, suggesting that the glycolytic pathway was less preferred across LT adolescents after BNT162b vaccination.

Due to the limited availability of LT adolescent samples, we collected only healthy adolescent PBMCs with 2-DG. To explain the role of glucose utilization for IFN-γ-secreting T cells specific to the BNT162b vaccine, a study was designed to inhibit glycolysis using the glucose competitive inhibitor 2-deoxy-D-glucose (2DG) in vitro. Our study showed that the inhibition of glucose metabolism with 2-DG treatment decreased the frequency of IFN-γ-producing SARS-CoV-2-specific T cells in healthy adolescents, indicating a glycolytic requirement for their effector function. Similar findings were also obtained from other studies37,38. Our study is among the first to link impaired PBMC metabolism with loss of SARS-CoV-2-specific T-cell responses in LT adolescents after BNT162b2 vaccination. Strengths include the use of paired metabolic and functional assays and a well-defined clinical cohort. However, several limitations should be noted: the relatively small sample size (n = 15 per group) reduces power for subgroup analysis, potential confounders such as immunosuppressive regimens and time since transplantation. The human IFN-γ ELISpot assay provides a functional readout of antigen-specific cytokine production but does not allow quantification of SARS-CoV-2-specific T-cell frequencies. Moreover, the extracellular flux assays were performed on bulk PBMCs, which represent a heterogeneous mix of lymphoid and myeloid subsets. Because different immune populations (e.g., T cells, B cells, NK cells) have distinct metabolic requirements, our results provide only a global readout of immunometabolism function. Although baseline age/sex comparisons showed no differences, larger studies with multivariate models are needed to confirm these observations.

In summary, this research highlights the mitochondrial and glycolytic dysfunction observed in liver-transplanted (LT) adolescents compared to their healthy counterparts following BNT162b vaccination. This study shows the potential of peripheral blood mononuclear cells (PBMCs) as a readily accessible cell type for quantitatively assessing early physiological alterations, thereby providing valuable insights into human cellular function. Our finding indicate that LT adolescents may have a reduced capacity to generate energy through both mitochondrial and glycolysis pathways, which may contribute to their impaired immune responses to SARS-CoV-2 vaccination. These results have clinical implications for vaccination strategies in immunocompromised adolescents, suggesting that booster doses or adjuvant approaches may be required to achieve adequate protection. Future studies with larger cohort, flow cytometry-based approaches such as activation-induced marker (AIM) assays or intracellular cytokine staining (ICS), metabolic probes or single cell metabolic profiling, and integration of immunosuppressive drug regimens will be essential to provide orthogonal validation and extend these observations.

Methodology

Study design and study population

The cryopreserved peripheral blood mononuclear cells (PBMCs) from 30 individuals who had been vaccinated with two doses of BNT162b2 (day 1 and day 21) and followed up on day 35, as part of Clinical Trial TCTR20210830002, were used in this study. All methods were performed in accordance with the relevant guidelines and regulations. Fifteen individuals were liver-transplanted adolescents who had regular follow-ups at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand, and 15 healthy adolescents aged between 12 and 18 years old. All individuals who provided demographic and clinical data are included in Table 1. The indications for liver transplantation included biliary atresia (66.6%), Budd–Chiari syndrome (13.3%), acute liver failure (6.7%), cryptogenic cirrhosis (6.7%), and autoimmune hepatitis (6.7%). The median time from liver transplantation to BNT162b2 vaccination was 9.25 years (range 0.5–13.33 years). At the time of vaccination, 60% were maintained on tacrolimus and 40% on cyclosporin. In addition, 53.4% received a single immunosuppressant, 13.3% received dual therapy, and 33.3% received triple immunosuppressive therapy. No immunosuppressive therapy was paused during the vaccination. The healthy adolescent group had no history of liver disease, transplantation, or prior SARS-CoV-2 infection. Baseline BMI was similar between groups LT: 17.72 kg/m² vs. healthy: 19.38 kg/m² (p = 0.2854). This cohort is under the Chulalongkorn University (Bangkok, Thailand) IRB-approved clinical study (COA No. 0044/2022, IRB. No. 874/64). Informed consent was obtained from all participants and/or their legal guardians. No organs or tissues were procured from prisoners.

Exclusion criteria for LT adolescents included liver transplantation within the last 6 months, a history of previous or active SARS-CoV-2 infection, and clinical instability at the time of vaccination (defined as fever > 38 °C). For healthy adolescents, the exclusion criterion was a history of previous or active SARS-CoV-2 infection.

Sample size estimation was performed using a two-proportion test39,40. Assuming a two-sided significance level of α = 0.05 and 80% power to detect a 50-percentage-point difference in ELISpot responder rates (70% in healthy controls vs. 20% in LT adolescents), a minimum of 15 participants per group was required.

Peripheral blood mononuclear cell (PBMC) preparation

Peripheral blood mononuclear cells (PBMCs) were isolated from 10 ml of acid citrate dextrose (ACD) blood using Ficoll density gradient centrifugation (Isoprep, Robbins Scientific Corporation, Sunnyvate, CA). After centrifugation at 2200 rpm for 20 min at room temperature (RT) with the break-off, PBMCs were collected and transferred into a new 15 ml conical tube. Then, PBMCs were washed twice with RPMI 1640 medium (Gibco). After counte cells, PBMCs were resuspended in freezing medium (90% FBS, 10% DMSO), transferred to cryovials and stored at -80 °C and then liquid nitrogen tank until use.

Preparation of PBMCs for assays

On the day of experiment, PBMCs were rapidly thawed at 37 °C, diluted in cold R10 medium (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA), 100 U/ml penicillin and 100 µg/ml streptomycin). After washed twice by centrifugation at 1500 rpm for 5 min, PBMCs were resuspended in fresh culture medium and counted before assays.

Human IFN-γ ELISpot assay

The ability of SARS-CoV-2-specific cells to secrete human IFN-γ was determined ex vivo using human IFN-γ ELISpot assays (Mabtech, Stockholm, Sweden). In brief, precoated ELISpot plates were washed and blocked with R10 medium for 30 min at RT. A total of 2.5 × 105 PBMCs/well were stimulated with the SARS-CoV-2 spike peptide pool (2 µg/ml/peptide); R10 medium as a negative control, and phytohemagglutinin (PHA, Sigma, USA) as a positive control. After 40 h incubation, the plates were washed with PBS, and a secondary anti-IFN-γ antibody directly conjugated to alkaline phosphatase (7-B6-ALP) (1:200) in filtered PBS containing 0.5% FBS was added for 2 h at RT. After being washed, the plates were incubated with 100 µl/well of substrate solution (BCIP/NBT-plus) until distinct spots emerged. The reaction was stopped by washing extensively in tap water and rinsing the underside of the membrane. The plate was left to dry. Spots were counted using ImmunoSpot® Analyzer (Germany). Results were expressed as spot-forming cells (SFCs)/106 PBMCs following the subtraction background. Positive responses were defined as > 30 SFCs/106 PBMCs and at least 2SD above background.

Extracellular flux analysis by mito stress test kit

PBMC metabolism was analyzed using the Agilent Seahorse XFp analyzer and Mito Stress Test kit (Agilent). In brief, the Seahorse 8-well culture plate was coated with poly-L-ornithine solution for 20 min. After washed twice with distilled water (DW), plate was left in a biosafety cabinet overnight. At the same time, the Seahorse XFp sensor cartridge (FluxPak, Agilent) was hydrated by filling each well with 200 µl of DW and placed in a non-CO2 incubator at 37 °C overnight. On the day of the experiment, the FluxPak was removed from the non-CO2 incubator, and the DW was replaced with calibrants before being placed in a non-CO2 incubator set to 37 °C for 1 h. Thawing PBMCs were counted, washed and resuspended in the assay medium (Seahorse XFp RPMI medium, pH 7.4 with 1 mM sodium pyruvate, 2 mM glutamine, and 10 mM glucose). PBMCs were loaded at 300,000 cells per well in 180 µl. The plate was centrifuge at 200 × g for 1 min with no break and incubated in a non-CO2 incubator at 37 °C for 45–60 min. The FluxPak was filled with the following compounds: oligomycin (an inhibitor of ATP synthase), fluorocarbonyl cyanide phenylhydrazone (FCCP, a potent uncoupler of oxidative phosphorylation in mitochondria), and rotenone/antimycin A (inhibitors of mitochondrial electron transport). These compounds were prepared to final well concentrations of 1.5 µM oligomycin, 0.75 µM FCCP and 0.5 µM of each rotenone and antimycin A. The loading volumes of each 10X compound into the injection ports were 20 µl of oligomycin, 22 µl of FCCP, and 25 µl of rotenone/antimycin A. After calibration of the Seahorse analyzer, the plate containing the cells was loaded and the assay was run. Oxygen consumption rate (OCR) parameters included basal respiration, ATP production, maximal respiration, proton leak, coupling efficiency and spare capacity.

Extracellular flux analysis by glycolysis stress test kit

The day before the experiment, a procedure similar to that used for the Mito Stress Test Kit was used. On the experimental day, the FluxPak was removed from the non-CO2 incubator, and the DW was replaced with calibrants before being placed in a non-CO2 incubator set to 37 °C. After 1 h of incubation, the injection ports were loaded with glucose, oligomycin (an inhibitor of oxidative phosphorylation), and 2-deoxy-D-glucose (2-DG; an inhibitor of glycolysis). These compounds were prepared to final well concentrations of 10 mM glucose, 1.0 µM oligomycin and 50 mM 2-DG for each specimen. The loading volumes of each 10X compound into the injection ports were 20 µl of glucose, 22 µl of oligomycin and 25 µl of 2-DG. After calibration of the Seahorse analyzer, the plate containing the cells was loaded and the assay was run. Extracellular acidification rate (ECAR) parameters included non-glycolytic acidification, basal glycolysis, glycolytic capacity, and glycolytic reserve.

Inhibition of the glycolytic pathway in healthy adolescents

To block the glycolysis pathway, frozen PBMCs from 10 healthy adolescents were thawed and stimulated with spike antigen in the presence or absence of 50 µM 2-deoxy-D-glucose (2-DG; a glycolytic inhibitor). IFN-γ production was assessed by IFN-γ ELISpot assay as described above.

Statistical analysis

All the statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA). Code and raw data have been shown in supplementary Table 1. The results of the mitochondrial and glycolysis functional analyses were performed in duplicate, and the results were averaged for each vaccinated group. The data are presented as the means ± standard errors of the means (SEMs). Comparisons between LT and healthy adolescents were performed using the Mann‒Whitney test for independent groups and the Friedman test for repeated measures. For paired assay, Wilcoxon match pairs test was used. All P values < 0.05 were considered significant; *P < 0.05, **P < 0.01, and ***P < 0.001.

Table 1.

Clinical characterization of liver-transplanted (LT) adolescents and healthy adolescents (n = 30)

graphic file with name 41598_2026_53892_Fig1_HTML.jpg

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Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (30.4KB, xlsx)

Acknowledgements

The authors are very grateful to all the LT adolescents and healthy adolescents who participated in this study and to our Center of Excellence in Thai Pediatric Gastroenterology, Hepatology and Immunology (TPGHAI) team for their assistance with sample collection and valuable suggestions. Furthermore, we acknowledge the support of the Chulalongkorn Medical Research Center (Chula MRC), Research Affairs, Faculty of Medicine, Chulalongkorn University, for providing the ELISpot reader and the Seahorse XFp analyzer. This research was funded by the Thailand Science Research and Innovation Fund Chulalongkorn University (HEA663000048).

Author contributions

Conceptualization, SB and NK; methodology, SB and PT; formal analysis, SB and NK; data curation, SB and PS; investigation, SB, NK, AC and PS; draft the manuscript, SB; reviewed and approved the final manuscript, SB, AC and NK; funding acquisition, SB and PS. All the authors have read and agreed with the published version of the manuscript.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (30.4KB, xlsx)

Data Availability Statement

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

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