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. 2019 Dec 23;7(1):74.
doi: 10.1186/s40635-019-0287-5.

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

Charles de Roquetaillade  1   2 , Khalil Kandara  1   3 , Morgane Gossez  1   3 , Estelle Peronnet  1   4 , Céline Monard  5 , Martin Cour  6 , Thomas Rimmelé  1   5 , Laurent Argaud  6 , Guillaume Monneret  7   8 , Fabienne Venet  1   3
Affiliations

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

Charles de Roquetaillade et al. Intensive Care Med Exp. .
No abstract available

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Conflict of interest statement

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

Figures

Fig. 1
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
Fig. 2
Fig. 2
PI3/Akt/mTOR pathway activation in CD4+ T cells from septic patients and controls upon TCR ligation. PBMCs were isolated from septic patients and controls, stimulated for 7 min with anti-CD2-CD3-CD28 Ab-coated beads (bead/cell ratio = 3/1) and stained with anti-CD4 and anti-CD3z, anti-pS6, anti-pAkt, anti-pERk, and anti-pAMPK antibodies protein phosphorylation was assessed by flow cytometry. Data represent the percentage of positive cells based on FMO staining and as individual values and means +/− SD in 9 septic patients and 9 healthy volunteers (controls) before (baseline, BL) and after TCR stimulation (TCR stim). *p < 0.05 (Wilcoxon paired test, baseline vs TCR stim), #p < 0.05 (Mann-Whitney, healthy volunteers vs patients after stimulation)
See this image and copyright information in PMC

References

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