TCR activation mimics CD127lowPD-1high phenotype and functional alterations of T lymphocytes from septic shock patients.

BACKGROUND: Sepsis is the leading cause of mortality for critically ill patients worldwide. Patients develop T lymphocyte dysfunctions leading to T-cell exhaustion associated with increased risk of death. As interleukin-7 (IL-7) is currently tested in clinical trials to reverse these dysfunctions, it is important to evaluate the expression of its specific CD127 receptor on the T-cell surface of patients with septic shock. Moreover, the CD127lowPD-1high phenotype has been proposed as a T-cell exhaustion marker in chronic viral infections but has never been evaluated in sepsis. The objective of this study was first to evaluate CD127 and CD127lowPD-1high phenotype in septic shock in parallel with functional T-cell alterations. Second, we aimed to reproduce septic shock-induced T-cell alterations in an ex vivo model. METHODS: CD127 expression was followed at the protein and mRNA levels in patients with septic shock and healthy volunteers. CD127lowPD-1high phenotype was also evaluated in parallel with T-cell functional alterations after ex vivo activation. To reproduce T-cell alterations observed in patients, purified T cells from healthy volunteers were activated ex vivo and their phenotype and function were evaluated. RESULTS: In patients, neither CD127 expression nor its corresponding mRNA transcript level was modified compared with normal values. However, the percentage of CD127lowPD-1high T cells was increased while T cells also presented functional alterations. CD127lowPD-1high T cells co-expressed HLA-DR, an activation marker, suggesting a role for T-cell activation in the development of this phenotype. Indeed, T-cell receptor (TCR) activation of normal T lymphocytes ex vivo reproduced the increase of CD127lowPD-1high T cells and functional alterations following a second stimulation, as observed in patients. Finally, in this model, as observed in patients, IL-7 could improve T-cell proliferation. CONCLUSIONS: The proportion of CD127lowPD-1high T cells in patients was increased compared with healthy volunteers, although no global CD127 regulation was observed. Our results suggest that TCR activation participates in the occurrence of this T-cell population and in the development of T-cell alterations in septic shock. Furthermore, we provide an ex vivo model for the investigation of the pathophysiology of sepsis-induced T-cell immunosuppression and the testing of innovative immunostimulant treatments.

Low Interleukin-7 Receptor Messenger RNA Expression Is Independently Associated With Day 28 Mortality in Septic Shock Patients.

OBJECTIVES: Septic shock is the primary cause of death in ICUs. A better comprehension of its pathophysiology, in particular, the immune alteration mechanisms, opened new therapeutic perspectives such as the recombinant interleukin-7. The use of biomarkers could improve the identification of eligible patients for this therapy. The soluble form of the interleukin-7 appears as a promising candidate in this regard since an association between its high plasmatic level and mortality in critically ill patients has been demonstrated. Because there are no data available on the transcriptional regulation of the interleukin-7 receptor in such patients, this study aimed to explore the expression level of different interleukin-7 receptor transcripts after septic shock and evaluate their association with mortality. DESIGN: Retrospective discovery cohort (30 patients) and validation cohort (177 patients). SETTING: Two French ICUs (discovery study) and six French ICUs (validation study). PATIENTS: Adult septic shock patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The quantification of several interleukin-7 receptor transcripts using specific reverse transcription quantitative polymerase chain reaction designs allowed for global evaluation of interleukin-7 receptor gene expression in whole blood. In the discovery cohort, all interleukin-7 receptor transcripts studied were expressed at lower levels in septic shock patients than in healthy volunteers. Interleukin-7 receptor gene expression at day 3 after septic shock diagnosis was associated with day 28 mortality. Patients at a lower risk of death showed higher expression levels. These results were confirmed in the independent validation cohort. Interestingly, using a threshold obtained on the discovery cohort, we observed in the validation cohort a high negative predictive value for day 28 mortality for the transcript encoding the membrane form of interleukin-7 receptor (0.86; 95% CI, 0.79-0.93). CONCLUSIONS: Interleukin-7 receptor transcripts appear as biomarkers of impaired adaptive immune response in septic shock patients and as a promising tool for patient stratification in clinical trials evaluating immunoadjuvant therapies.

An optimized protocol for adenosine triphosphate quantification in T lymphocytes of lymphopenic patients.

In several clinical contexts, the measurement of ATP concentration in T lymphocytes has been proposed as a biomarker of immune status, predictive of secondary infections. However, the use of such biomarker in lymphopenic patients requires some adaptations in the ATP dosage protocol. We used blood from healthy volunteers to determine the optimal experimental settings. We investigated technical aspects such as the type of anticoagulant for blood sampling, the effect of freeze and thaw cycles, the reagent and sample mixing sequence, and the optimal dilution buffer. We also shortened the incubation time to 8h, and even showed that a 30min incubation may be sufficient. To evaluate the ATP rise upon lymphocyte activation, the optimal dose of stimulant was defined to be 4µg/mL of phytohaemagglutinin. Lastly, we determined that the number of T cells needed for this measurement was as low as 50,000, which is compatible with the existing lymphopenia in clinical settings. This optimized protocol appears ready to be assessed in lymphopenic patients to further investigate the interconnection between T lymphocyte metabolism and impaired phenotype and functions.