Blood samples and ethical concerns
Blood samples were provided by the French blood bank institution (“Etablissement Français du Sang”). All donors gave their explicit written consent for the use of their blood for biomedical research purposes, according to local legislation. Blood samples were collected in EDTA-anticoagulated tubes, anonymized upon donation, and were sent to the laboratory at room temperature, to be processed on the same day. Technical optimizations were performed on blood from eight healthy volunteers. Blood from 10 other healthy donors served as controls for comparison with septic shock patients’ blood. Median [Q1-Q3] age of these last 10 donors was 33 [30–40] years, and 50% were male.
Septic shock was defined according to the diagnostic criteria of the ACCPS/SCCM , as this study was conducted before the newest definitions were published . Septic shock adult patients (n = 7) without pre-existing immunosuppressive disease or treatment were included in this study, as a part of a global study on injury-induced immune dysfunctions. It has been approved by our institutional ethical board (“Comité de Protection des Personnes Sud-Est II”, #IRB 11236). This study is also registered at the French Ministry of Research and Teaching (#DC-2008-509) and recorded at the national informatics committee (“Commission Nationale de l’Informatique et des Libertés”). Although the ethical board waived the need for informed consent—because the study was observational and measurements were made on residual blood samples after completion of routine follow-up—a non-opposition to inclusion in the study was recorded for each patient.
T cell purification from whole blood
Purification of T lymphocytes from whole blood was performed through an antibody-based negative selection according to manufacturer’s instructions. A human T cell enrichment antibody cocktail (RosetteSep™, StemCell Technologies, Grenoble, France)—containing a mix of anti-CD16 (present on NK cells, monocytes, macrophages, and neutrophils), anti-CD19 (B lymphocytes), anti-CD36 (platelets, red blood cells, monocytes), anti-CD56 (NK cells), and anti-CD66b (granulocytes) antibodies—was added to the blood sample.
After incubation and centrifugation over density gradient medium (Biocoll®, Biochrom, Berlin, Germany), T lymphocytes were isolated. Residual red blood cells were lysed with Versalyse™ (Beckman Coulter, Brea, CA, USA). After purification, T cells were either processed directly to measure intracellular lactate concentration or cultured in complete culture medium, with or without stimulation (see below).
T cell count and subset determination
At the end of the purification process, cells were counted with a flow cytometer (Navios, Beckman Coulter) using LDS (LDS 751, Molecular Probes, Life Technologies, Carlsbad, CA, USA) and calibration beads (Flow-Count™ Fluorospheres, Beckman Coulter).
Lymphocyte subset proportions were determined using a commercially available antibody mix containing anti-CD45, anti-CD3, anti-CD4, and anti-CD8 antibodies stained with fluorochromes (TetraChrome™ CD3-4-8-45, Beckman Coulter). Lymphocytes were identified on a size/CD45 plot then T cell purity was defined as the proportion of CD3+ cells among lymphocytes. T cell purity was routinely above 90%. Percentages of CD4+, CD8+, and CD4−/8− T cells were measured among CD3+ T cells.
Median [Q1–Q3] percentages were 65.8 [59.3–71.8] and 72.0 [59.9–80.9] % of CD4+, 28.2 [23.6–32.8] and 14.8 [11.0–26.5] % of CD8+, and 5.5 [4.4–7.1] and 3.1 [1.8–8.2] % of CD4−/8− T cells for healthy volunteers and septic shock patients, respectively. None of these differences were statistically significant; however, this could only be due to the low number of patients included in this study.
Ex vivo culture and stimulation
In some experiments, purified T cells were diluted in RPMI (Roswell Park Memorial Institute) complete medium to achieve a concentration of 1 × 106 cells/mL. RPMI complete medium is a RPMI 1640 medium (Eurobio, Les Ulis, France), supplemented with 10% human AB serum (Life Technologies), 200 μg/mL amphotericin B (Gibco), 1000 IU/mL penicillin (Eurobio), 1000 μg/mL streptomycin (Eurobio), and 200 mM L-glutamine (Eurobio).
Cells were then stimulated with 4 μg/mL PHA (phytohaemagglutinin, Remel-Oxoid, Dardilly, France) or left untreated (non-stimulated condition). Indeed, PHA stimulation is a standard mitogenic stimulus for T cell activation and induction of proliferation, and PHA-induced T cell functions are altered in septic shock patients [14, 15]. Duplicates were made whenever possible. Cell culture plates were then incubated in standard conditions (37 °C, 5% CO2). After a 40-h culture, supernatants were collected. Remaining cells were recovered after centrifugation and both samples were then deproteinised and stored at − 80 °C until metabolite measurement.
Cell lysis and sample deproteinisation
Immediately after purification or cell culture, 250,000 T cells were aliquoted and lysed with the buffer supplied in the L-Lactate assay kit (ref ab65330, Abcam, Cambridge, UK). Efficacy of this cell lysis protocol was verified on two consecutive experiments using Trypan blue exclusion test (data not shown).
To avoid further modifications of lactate concentration in the samples due to the potential presence of enzymes, we decided to deproteinate the obtained cell lysates or cell culture supernatants. We used a chemical process, according to manufacturer’s instructions. The whole process took place on ice and in a cold 4 °C centrifuge. Perchloric acid was added to a final concentration of 1 M, resulting in protein denaturation. After centrifugation, supernatant was added with potassium hydroxide to normalize sample pH. This reaction produced potassium perchlorate, which was pelleted by centrifugation. The remaining deproteinised supernatant was collected and stored at − 80 °C. Efficacy of this deproteinisation protocol was checked with the Bradford technique (data not shown).
Lactate and pyruvate quantitative determination
An enzymatic method followed by a colorimetric measure was used to determine lactate concentration in samples, with a commercially available kit (L-Lactate Assay Kit, ref. ab65330, Abcam).
T cell samples did not need any dilution, whereas supernatants were diluted 10 times before reading. A reaction mix prepared according to manufacturer’s instructions was mixed with the samples and incubated for 30 min. The emitted light was measured with a spectrophotometer (Victor™ X4 Multilabel Plate Reader, Perkin Elmer, Waltham, MA, USA) at a wavelength of 570 nm for 1 s. Samples and standard curves were always processed in duplicates. Means of duplicates were used to calculate lactate concentration in samples and standards. The mean measured luminescence of the blank, corresponding to the background luminescence, was subtracted to all measurements. Lactate concentrations were calculated from the standard curve (ranging from 0 to 10 nmol/well) generated from the lactate standard according to manufacturer’s instructions.
As lactate concentration was not detectable in samples containing 250,000 cells either from septic shock patients or healthy volunteers when measured by colorimetry, we used a fluorometric measurement (Ex/Em 540/590 nm) for this experiment. The Lactate Assay Kit (ref ab65330, Abcam) indeed allows for both measurement techniques, the fluorometric one being more sensitive for low lactate concentrations. This fluorometric measurement relies on the same basic principle than the colorimetric described above.
Pyruvate concentration was measured using a commercially available kit from the same manufacturer (Pyruvate Assay Kit, ref. ab65342, Abcam) followed by a fluorometric measurement (Ex/Em 540/590 nm).
mHLA-DR, CD4+, and T regulatory lymphocytes count determination
These parameters were measured on whole blood by flow cytometry. HLA-DR expression on monocytes (mHLA-DR) was determined using the Quantibrite™ assay (ref 340827, BD Biosciences, Franklin Lakes, NJ, USA). HLA-DR-positive cells were selected among monocytes, identified as CD14+ cells. Results were expressed as numbers of antibodies bound per cells (ab/c) using standardized beads (BD Quantibrite™ beads) as described previously .
Absolute number of CD4+ lymphocytes and percentage of T regulatory cells were determined by a combined staining protocol with anti-CD4, anti-CD25, and anti-CD127 antibodies stained with fluorochromes. Calibration beads (Flow-Count™ Fluorospheres, Beckman Coulter) were added to be able to determine the absolute concentration of CD4+ lymphocytes, expressed as numbers of cells per μL of blood. Tregs were defined as CD4 + CD25highCD127−/low cells as described previously . Percentage of Tregs was calculated among CD4+ cells.
Means of the duplicates were used for all calculations. Statistical analyses were performed with R Studio® software (RStudio Inc., Boston, MA, USA). Comparisons between groups used non parametric Wilcoxon tests. Paired tests were performed for matched data. Differences with p values lower than 0.05 were considered statistically significant.