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

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.

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).

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⁺).

Flow cytometric quantification of mitochondrial mass using MitoTracker Green FM staining in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺).

Flow cytometric quantification of mitochondrial membrane potential using tetramethylrhodamine ethyl ester (TMRE) staining in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺).

Flow cytometric quantification of mitochondrial membrane potential using JC-1 dye (red/green fluorescence ratio) in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺).

Measurement of basal oxygen consumption rate reflecting oxidative phosphorylation (OXPHOS) capacity in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺).

Measurement of basal extracellular acidification rate reflecting glycolytic metabolic activity in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺).

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.

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.

Assessment of Cytotoxic γδT cell (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) chemotactic and migratory capacity using Transwell migration assay.

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.

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.

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.

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.

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.

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).

Correlation analysis between COX6C expression, glycolytic capacity, and mitochondrial ROS levels in Cytotoxic γδT cells (TCRγδ⁺GZMB⁺PRF1⁺GNLY⁺) with plasma lactate levels.

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.

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.

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.

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.

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.