Intracellular calcium signaling and phospho-antigen measurements reveal functional proximal TCR activation in lymphocytes from septic shock patients

* Correspondence: guillaume. monneret@chu-lyon.fr Charles de Roquetaillade and Khalil Kandara contributed equally to the work. EA 7426, Pathophysiology of Injury-Induced Immunosuppression (Université Claude Bernard Lyon 1 Hospices Civils de Lyon bioMérieux), Edouard Herriot Hospital, 69437 Lyon, France Immunology Laboratory, Hôpital E. Herriot Hospices Civils de Lyon, 69437 Lyon, France Full list of author information is available at the end of the article

To the editor, Sepsis deeply perturbs immune homeostasis by inducing a complex immune response that varies over time and associates a tremendous systemic inflammatory response to anti-inflammatory mechanisms. As a delayed consequence, some septic patients enter a state of profound immunosuppression [1]. As the latter may persist for weeks, leaving the patient at increased risk of secondary infections, immunostimulation recently appeared as a reasonable therapeutic option in patients with signs of persistent and severe immunosuppression [2].
Septic patients develop marked T lymphocyte dysfunctions such as profound lymphopenia, increased expression of inhibitory co-receptor molecules, decreased repertoire diversity, and reduced functionality (proliferation and cytokine production). These alterations have been repeatedly associated with deleterious outcomes [1,2]. However, mechanisms leading to these alterations are only partially understood. For instance, while the role of deactivated mTORC1 is established [3], the intrinsic capacity of T cell receptor (TCR) to be activated and to transduce intracellular signalling remains unexplored. Among determinants of T cell response, immediate calcium signaling following TCR ligation is of paramount importance and serves crucial effector functions. Thus, we developed a flow cytometry protocol to follow calcium flux after TCR stimulation in patients' CD4+ T lymphocytes. In addition, phosphorylation of molecules from the proximal and downstream TCR signaling cascade was analyzed (Additional file 1). We included patients with septic shock (according to SEPSIS-3 definition) presenting with features of immunosuppression, i.e., decreased monocyte HLA-DR and lymphocyte count (clinical and immunological characteristics in Additional file 1: Table S1).
We show that immediate signaling downstream TCR stimulation was not altered in circulating CD4 lymphocytes from septic shock patients. Indeed, cells exhibited no deregulation of intracytoplasmic calcium influx after TCR ligation compared with healthy controls (OKT3 response, Fig. 1). In agreement, we observed a significant CD3ζ phosphorylation (one of the first molecules to be phosphorylated after TCR engagement) after T cell stimulation in both cells from septic patients and controls (Fig. 2). This showed that immediate response after TCR activation was unaffected after sepsis.
In contrast, activation of more distal molecules in the TCR signaling cascade was impacted. For example, stimulation-induced rise of pAkt and pERK was affected leading to a limited mTORC1 activation capacity as measured by S6 phosphorylation after stimulation. In contrast, activation of AMPK, an inhibitor of mTORC1 was mostly unaltered in patients compared to controls (Fig. 2).
In conclusion, the present results obtained in septic shock patients show that proximal TCR signaling remains functional in circulating CD4+ T cells from septic shock patients while downstream activation of mTORC1 pathway is markedly diminished. PI3K-Akt pathway integrates signals from both co-activating/inhibitory receptors and an increased expression of such co-inhibitory receptors has been described on circulating T cells from septic patients [4]. In that respect, this suggests Fig. 1 Intracellular calcium signaling in CD4+ T cells from septic patients and controls upon TCR ligation. Peripheral blood mononuclear cells (PBMC) were isolated from patients and controls and loaded with Fluo-4AM for 30 min at 37°C. Cells were analyzed by flow cytometry for 5 min at baseline then stimulated by biotinylated anti-CD3 (OKT3) antibody during 90 s before addition of streptavidin to induced TCR stimulation and analyzed for another 5 min. As a positive control, ionomycin was added at the end of experiment. Left, representative example of overtime intracellular calcium staining in one healthy volunteer. Right, intracellular calcium staining following TCR ligation in septic patients (n = 7) and healthy volunteers (controls, age-matched, n = 7) before (baseline), after TCR stimulation (biotinylated anti-CD3 antibodies + streptavidin) and after ionomycin addition. MFI of Fluo-4AM was measured in CD4+ T lymphocytes on 3 periods of 100 s at baseline and after complete TCR stimulation. For each stimulation condition, the maximum MFI was considered. Data are represented as means +/− SD. *p < 0.05 compared to baseline, Wilcoxon paired test that inhibitory receptors known to block downstream signaling are likely of utmost importance in sepsis-induced T lymphocyte dysfunctions. As TCR from septic lymphocytes remains actionable [5], the present results reinforce the rational for blocking co-inhibitors (e.g., with anti-PD-1) or stimulating mTORC1 (for example with rhIL-7) as reasonable immunoadjuvant approaches to tackle sepsis-induced immunosuppression [6][7][8].
Additional file 1: Table S1. Clinical and biological data from septic shock patients. Funding This work was supported by Université Lyon 1, Hospices Civils de Lyon, and bioMerieux. bioMérieux had no role in the study design; in the collection, analysis, and interpretation of data; and in writing the manuscript.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Septic shock patients: this project was approved by our Institutional Review Board for ethics ("Comité de Protection des Personnes Sud-Est II", number 11236).This study is registered at the French Ministry of Research and Teaching (#DC-2008-509), at the Commission Nationale de l'Informatique et des Libertés, and on ClinicalTrials.gov (NCT02803346). Oral information and non-opposition to inclusion in the study were mandatory and recorded in patients' clinical files. Healthy volunteers: peripheral blood from healthy volunteers was provided by the "Etablissement Français du Sang" from Lyon. According to EFS standardized procedures for blood donation and to provisions of the articles R.1243-49 and following ones of the French public health codes, a written non-opposition to the use of donated blood for research purposes was obtained from HV. The blood donors' personal data were anonymized before being transferred to our research laboratory.

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Competing interests
EP is an employee of bioMérieux. All other authors declare that they have no competing interests.
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