Metabolic and Functional Study of γδ T Cells in Critically Ill Patients

Union Hospital, Tongji Medical College, Huazhong University of Science and Technology105 enrolled

Overview

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.

Study Type

OBSERVATIONAL

Enrollment

105

Conditions

Sepsis Critical Illness Immunosuppression MODS

Eligibility

Sex: ALLMin age: 18 YearsMax age: 80 YearsHealthy volunteers:

Medical Language ↔ Plain English

Inclusion Criteria: 1. Healthy Control Group (NHC): * Age ≥ 18 years. * No acute or chronic major diseases. * Provide written informed consent. 2. Non-septic Critical Illness Group (CI-NS): * Age ≥ 18 years. * Admitted to the ICU and meeting the definition of critical illness. * Excluded from sepsis according to the Sepsis-3 criteria (infection + ΔSOFA ≥ 2 points). * Written informed consent provided by the participant or legally authorized representative. 3. Septic Critical Illness Group (CI-Sep): * Age ≥ 18 years. * Admitted to the ICU and meeting the Sepsis-3 criteria (infection + ΔSOFA ≥ 2 points). * Written informed consent provided by the participant or legally authorized representative. Exclusion Criteria: * Age \< 18 years. * Known immunodeficiency, HIV infection, active hematologic malignancy, or history of hematopoietic stem cell or solid organ transplantation within the past 3 months. * Receipt of T-cell-targeted immunosuppressive therapy (e.g., antithymocyte globulin, calcineurin inhibitors, mycophenolate mofetil, methotrexate, or high-dose corticosteroids \>1 mg/kg/day prednisone equivalent) before ICU admission or within 24 hours after ICU admission. * Use of immune checkpoint inhibitors (e.g., anti-PD-1/PD-L1/CTLA-4 antibodies) within the past 6 weeks. * Expected ICU stay \< 24 hours or imminent risk of death (moribund state). * Pregnancy or breastfeeding. * Active major bleeding. * Inability to obtain informed consent.

Outcomes

Primary Outcomes

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.

Time frame: Day 2 post-ICU admission

Secondary Outcomes

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).

Time frame: Day 2 post-ICU admission

Change in Cytotoxic Molecule Expression in Cytotoxic γδT Cells

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⁺).

Time frame: 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⁺).

Time frame: 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⁺).

Time frame: 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⁺).

Time frame: 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⁺).

Time frame: 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⁺).

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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).

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: 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.

Time frame: Up to 90 days post-discharge

Metabolic and Functional Study of γδ T Cells in Critically Ill Patients

Overview

Conditions

Eligibility

Locations (1)

Outcomes

Primary Outcomes

Secondary Outcomes