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This prospective observational cohort study investigates the subset-specific metabolic adaptation and functional remodeling of cytotoxic γδT cells in critically ill patients with and without sepsis. Emerging evidence indicates that γδT cells, as a bridge between innate and adaptive immunity, play a critical role in early anti-infection defense during sepsis. However, the functional status and underlying regulatory mechanisms of cytotoxic γδT cells in septic patients remain incompletely understood. Our preliminary single-cell transcriptomic analysis revealed that cytotoxic γδT cells from septic patients exhibit significant alterations in cytotoxicity-associated molecules (GZMB, PRF1, GNLY) and mitochondrial oxidative phosphorylation (OXPHOS) pathway genes, particularly COX6C, which correlates with cytotoxic effector molecule expression. This study aims to systematically characterize the proportion, cytotoxicity, and mitochondrial metabolic function of circulating cytotoxic γδT cells across three cohorts: healthy controls, critically ill non-septic patients, and critically ill septic patients. By integrating flow cytometry, mitochondrial function assays, and functional validation experiments, we seek to elucidate the role of COX6C-mediated mitochondrial metabolic abnormalities in cytotoxic γδT cell dysfunction, providing theoretical basis for understanding immune dysregulation in sepsis and identifying novel therapeutic targets.
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| Label | Type | Description | Intervention Names |
|---|---|---|---|
| Healthy Controls (NHCs) | Healthy volunteers without acute or chronic major diseases, aged ≥18 years, who have provided written informed consent. Participants undergo a single blood draw and rectal swab collection. | ||
| Critically Ill Non-Septic Patients (CI-NS) | Patients admitted to the ICU meeting the criteria for critical illness but without sepsis, as determined by the Sepsis-3 criteria (infection with ΔSOFA ≥ 2 points excluded). Participants are aged ≥18 years, with informed consent obtained from the patient or legally authorized representative. Blood and rectal swab samples are collected at three time points: within 24 hours of ICU admission (D0), Day 2 (D2), and Day 7 (D7). Clinical follow-up extends to 90 days. | ||
| Critically Ill Septic Patients (CI-Sep) | Patients admitted to the ICU meeting the Sepsis-3 criteria (infection with ΔSOFA ≥ 2 points). Participants are aged ≥18 years, with informed consent obtained from the patient or legally authorized representative. Blood and rectal swab samples are collected at three time points: within 24 hours of ICU admission (D0), Day 2 (D2), and Day 7 (D7). Clinical follow-up extends to 90 days. |
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| Measure | Description | Time Frame |
|---|---|---|
| Change in the Proportion of Cytotoxic γδT Cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) Among Total γδT Cells | Flow cytometric quantification of the frequency of Cytotoxic γδT cells, defined as TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺ cells, as a percentage of total γδT cells (TCRγδ⁺) in peripheral blood. This subset represents the cytotoxic effector population critical for early anti-infection defense. | Day 2 post-ICU admission |
| Measure | Description | Time Frame |
|---|---|---|
| Change in COX6C Expression in Cytotoxic γδT Cells | Measurement of COX6C (mitochondrial respiratory chain complex IV subunit) expression levels in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) at the mRNA level (by single-cell RNA sequencing) and protein level (by flow cytometry). | Day 2 post-ICU admission |
| Change in Cytotoxic Molecule Expression in Cytotoxic γδT Cells |
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Inclusion Criteria:
Healthy Control Group (NHC):
Non-septic Critical Illness Group (CI-NS):
Septic Critical Illness Group (CI-Sep):
Exclusion Criteria:
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The study population comprises three distinct groups: healthy volunteers, non-septic critically ill patients, and septic critically ill patients admitted to the intensive care unit. Sepsis is defined according to the Sepsis-3 criteria (infection with an acute increase in SOFA score ≥2 points), while critical illness is based on standard ICU admission criteria. All participants must be aged ≥18 years and provide written informed consent (from the participant or a legally authorized representative for those unable to consent). Key exclusion criteria include known immunodeficiency, recent use of T-cell-targeted immunosuppressants or immune checkpoint inhibitors, expected ICU stay <24 hours, pregnancy, active major bleeding, or inability to obtain consent. Consecutive enrollment will take place at the Department of Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, with follow-up through 90 days after enrollment.
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| ID | Term |
|---|---|
| D018805 | Sepsis |
| D016638 | Critical Illness |
| D009102 | Multiple Organ Failure |
| D028361 | Mitochondrial Diseases |
| D064806 | Dysbiosis |
| ID | Term |
|---|---|
| D007239 | Infections |
| D018746 | Systemic Inflammatory Response Syndrome |
| D007249 | Inflammation |
| D010335 | Pathologic Processes |
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Flow cytometric quantification of the mean fluorescence intensity (MFI) of cytotoxic effector molecules including Granzyme B (GZMB), Perforin (PRF1), and Granulysin (GNLY) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺). |
| Day 2 post-ICU admission |
| Change in Mitochondrial Mass in Cytotoxic γδT Cell | Flow cytometric quantification of mitochondrial mass using MitoTracker Green FM staining in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺). | Day 2 post-ICU admission |
| Change in Mitochondrial Membrane Potential (TMRE) in Cytotoxic γδT Cells | Flow cytometric quantification of mitochondrial membrane potential using tetramethylrhodamine ethyl ester (TMRE) staining in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺). | Day 2 post-ICU admission |
| Change in Mitochondrial Membrane Potential (JC-1) in Cytotoxic γδT Cells | Flow cytometric quantification of mitochondrial membrane potential using JC-1 dye (red/green fluorescence ratio) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺). | Day 2 post-ICU admission |
| Change in Oxygen Consumption Rate (OCR) in Cytotoxic γδT Cells | Measurement of basal oxygen consumption rate reflecting oxidative phosphorylation (OXPHOS) capacity in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺). | Day 2 post-ICU admission |
| Change in Extracellular Acidification Rate (ECAR) in Cytotoxic γδT Cells | Measurement of basal extracellular acidification rate reflecting glycolytic metabolic activity in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺). | Day 2 post-ICU admission |
| Change in Glycolytic Capacity of Cytotoxic γδT Cells at Serial Time Points | Longitudinal flow cytometric puromycin-incorporation assay (SCENITH method) to measure glycolytic capacity in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) at Day 0, Day 2, and Day 7 post-ICU admission, to track the temporal evolution of metabolic flexibility and stress-adaptive glycolytic reprogramming during the course of critical illness. | Day 0, Day 2, and Day 7 post-ICU admission |
| Change in Mitochondrial ROS Levels in Cytotoxic γδT Cells at Serial Time Points | Longitudinal flow cytometric quantification of mitochondrial reactive oxygen species (mitochondrial ROS) levels using MitoSOX Green staining in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) at Day 0, Day 2, and Day 7 post-ICU admission, reflecting oxidative stress dynamics. | Day 0, Day 2, and Day 7 post-ICU admission |
| Change in Cytotoxic γδT Cell Migratory Function | Assessment of Cytotoxic γδT cell (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) chemotactic and migratory capacity using Transwell migration assay. | Day 2 post-ICU admission |
| Change in Total γδT Cell Proportion at Serial Time Points | Longitudinal flow cytometric quantification of the frequency of total γδT cells (TCRγδ⁺) as a percentage of total live CD3⁺ T cells in peripheral blood at Day 0, Day 2, and Day 7 post-ICU admission. | Day 0, Day 2, and Day 7 post-ICU admission |
| Change in Cytotoxic γδT Cell Proportion at Serial Time Points | Longitudinal flow cytometric quantification of the frequency of Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) as a percentage of total γδT cells at Day 0, Day 2, and Day 7 post-ICU admission, to track the temporal evolution of this cell subset. | Day 0, Day 2, and Day 7 post-ICU admission |
| Change in COX6C Expression in Cytotoxic γδT Cells at Serial Time Points | Longitudinal measurement of COX6C expression levels in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) at Day 0, Day 2, and Day 7 post-ICU admission, to assess the temporal dynamics of mitochondrial metabolic gene expression. | Day 0, Day 2, and Day 7 post-ICU admission |
| Correlation Between Cytotoxic γδT Cell Glycolytic Capacity and APACHE II Score | Spearman rank correlation analysis between glycolytic capacity (measured by puromycin incorporation assay at Day 2) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) with APACHE II (Acute Physiology and Chronic Health Evaluation II) score measured within the first 48 hours of ICU admission. | Day 2 post-ICU admission |
| Correlation Between Cytotoxic γδT Cell Glycolytic Capacity and SOFA Score | Spearman rank correlation analysis between glycolytic capacity (measured by puromycin incorporation assay at Day 2) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) with SOFA (Sequential Organ Failure Assessment) score measured within the first 48 hours of ICU admission. | Day 2 post-ICU admission |
| Correlation Between Cytotoxic γδT Cell Metabolic Parameters and vasopressor dose | Correlation analysis between COX6C expression, glycolytic capacity, and mitochondrial ROS levels in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) with vasopressor dose (reported as norepinephrine equivalent dose, NEE). | Day 2 post-ICU admission |
| Correlation Between Cytotoxic γδT Cell Metabolic Parameters and plasma lactate levels | Correlation analysis between COX6C expression, glycolytic capacity, and mitochondrial ROS levels in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) with plasma lactate levels. | Day 2 post-ICU admission |
| Association Between Cytotoxic γδT Cell Glycolytic Capacity and 28-day all-cause mortality | Analysis of association between glycolytic capacity (measured at Day 2) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) and 28-day all-cause mortality. Patients will be stratified by vital status (survivors vs. non-survivors) and by hospital length of stay (above vs. below median) for comparative analysis. | Up to 90 days post-discharge |
| Association Between Cytotoxic γδT Cell Glycolytic Capacity and 90-day all-cause mortality | Analysis of association between glycolytic capacity (measured at Day 2) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) and 90-day all-cause mortality. Patients will be stratified by vital status (survivors vs. non-survivors) and by hospital length of stay (above vs. below median) for comparative analysis. | Up to 90 days post-discharge |
| Association Between Cytotoxic γδT Cell Glycolytic Capacity and in-hospital mortality | Analysis of association between glycolytic capacity (measured at Day 2) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) and in-hospital mortality. Patients will be stratified by vital status (survivors vs. non-survivors) and by hospital length of stay (above vs. below median) for comparative analysis. | Up to 90 days post-discharge |
| Association Between Cytotoxic γδT Cell Glycolytic Capacity and ICU mortality | Analysis of association between glycolytic capacity (measured at Day 2) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) and ICU mortality. Patients will be stratified by vital status (survivors vs. non-survivors) and by hospital length of stay (above vs. below median) for comparative analysis. | Up to 90 days post-discharge |
| Association Between Cytotoxic γδT Cell Glycolytic Capacity and hospital length of stay | Analysis of association between glycolytic capacity (measured at Day 2) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) and hospital length of stay (days). Patients will be stratified by vital status (survivors vs. non-survivors) and by hospital length of stay (above vs. below median) for comparative analysis. | Up to 90 days post-discharge |
| D013568 |
| Pathological Conditions, Signs and Symptoms |
| D020969 | Disease Attributes |
| D012769 | Shock |
| D008659 | Metabolic Diseases |
| D009750 | Nutritional and Metabolic Diseases |