Enhanced susceptibility of T lymphocytes to oxidative stress in the absence of the cellular prion protein

Abstract

The cellular prion glycoprotein (PrP(C)) is ubiquitously expressed but its physiologic functions remain enigmatic, particularly in the immune system. Here, we demonstrate in vitro and in vivo that PrP(C) is involved in T lymphocytes response to oxidative stress. By monitoring the intracellular level of reduced glutathione, we show that PrP(-/-) thymocytes display a higher susceptibility to H(2)O(2) exposure than PrP(+/+) cells. Furthermore, we find that in mice fed with a restricted diet, a regimen known to increase the intracellular level of ROS, PrP(-/-) thymocytes are more sensitive to oxidative stress. PrP(C) function appears to be specific for oxidative stress, since no significant differences are observed between PrP(-/-) and PrP(+/+) mice exposed to other kinds of stress. We also show a marked evolution of the redox status of T cells throughout differentiation in the thymus. Taken together, our results clearly ascribe to PrP(C) a protective function in thymocytes against oxidative stress.

Fig. 1

Measurement of the intracellular reduced glutathione. a Thymocytes from C57BL/6 mice were labeled with anti-CD4, anti-CD8, anti-CD25 and anti-CD44 antibodies to define the different T cell subpopulations, and then incubated with monochlorobimane to evaluate their redox state, as described in “Materials and methods”. The data presented here are representative of four different experiments. b Comparison of the level of GSH between C57BL/6 (black histograms) and PrP^−/− (gray line) thymocytes prepared and analyzed as in (a). The data shown here are representative of six different experiments

Fig. 2

Effect of an oxidative stress on C57BL/6 and PrP-deficient thymocytes. a Control cells or cells that have been incubated with H₂O₂ were stained with mCB and analyzed by flow cytometry as described in “Materials and methods”. The data presented here are representative of six different experiments on PrP^+/+ C57BL/6 or PrP^−/− mice. b GRase activity in C57BL/6 and PrP^−/− thymocytes: GRase activity was measured before (control) and after incubation of cells in the presence of H₂O₂ (*P < 0.05; n = 3)

Fig. 3

Effect of a copper supplementation on the level of GSH. Copper was given for 5 weeks in the drinking water of supplemented mice. Thymocytes were then recovered, submitted or not to H₂O₂ and the level of mCB staining was determined as in Fig. 2a

Fig. 4

PrP^−/− mice are more affected by a redox stress than C57BL/6 mice. a Mice were allowed a free access to food and water for 2 h per day for 7 days. After this period, they recovered a free access to food and water. The cellularity of the thymus was evaluated every day. b T cell number in each thymic subpopulation was determined by FACS analysis at day 8 after the initiation of the diet. Data are representative of n = 4 for control mice and n = 3 for the stressed mice