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. 2006 Aug 22;103(34):12831-6.
doi: 10.1073/pnas.0604571103. Epub 2006 Aug 14.

T cell surface redox levels determine T cell reactivity and arthritis susceptibility

Kyra A Gelderman  1 , Malin Hultqvist, Jens Holmberg, Peter Olofsson, Rikard Holmdahl
Affiliations

T cell surface redox levels determine T cell reactivity and arthritis susceptibility

Kyra A Gelderman et al. Proc Natl Acad Sci U S A. .

Abstract

Rats and mice with a lower capacity to produce reactive oxygen species (ROS) because of allelic polymorphisms in the Ncf1 gene (which encodes neutrophil cytosolic factor 1) are more susceptible to develop severe arthritis. These data suggest that ROS are involved in regulating the immune response. We now show that the lower capacity to produce ROS is associated with an increased number of reduced thiol groups (-SH) on T cell membrane surfaces. Artificially increasing the number of reduced thiols on T cells from animals with arthritis-protective Ncf1 alleles by glutathione treatment lowered the threshold for T cell reactivity and enhanced proliferative responses in vitro and in vivo. Importantly, T cells from immunized congenic rats with an E3-derived Ncf1 allele (DA.Ncf1E3 rats) that cannot transfer arthritis to rats with an arthritis-associated Dark Agouti (DA)-derived mutated Ncf1 allele (DA.Ncf1DA rats) became arthritogenic after increasing cell surface thiol levels. This finding was confirmed by the reverse experiment, in which oxidized T cells from DA.Ncf1DA rats induced less severe arthritis compared with controls. Therefore, we conclude that ROS production as controlled by Ncf1 is important in regulating surface redox levels of T cells and thereby suppresses autoreactivity and arthritis development.

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Conflict of interest statement

Conflict of interest statement: P.O. is employed by Biovitrum, which holds the rights in a patent application related to the use of oxidants for therapy. Both P.O. and R.H. are named as inventors in this application.

Figures

Fig. 1.
Fig. 1.
Ncf1 mutated rats and mice have increased numbers of cell surface thiol groups. (a) T cells do not exert oxidative burst upon PMA stimulation, in contrast to granulocytes (Gr-1+ cells). DHR123 staining in blood TCR+CD4+ cells and granulocytes was determined by flow cytometry after stimulation with PMA or without stimulation. (b) Ncf1 expression was determined by flow cytometry and expressed in geomean. (c) The relative number of cell surface thiol (−SH) groups was determined by Alexa Fluor 633-maleimide staining and expressed in geomean. Rats with a polymorphism in Ncf1 and lower ROS production (DA.Ncf1DA) have higher numbers of cell surface thiol groups compared with congenic rats expressing WT Ncf1 (DA.Ncf1E3). (d) Intracellular thiol levels were determined by staining with MCB, detected by FACS, and expressed in geomean. No differences in intracellular GSH levels were observed. (e) Plasma thiol groups were determined with 5,5′-dithiobis(2-nitrobenzoic acid) and expressed in relative numbers compared with a standard of GSH. Plasma of mutated rats contained more reduced −SH groups. Means ± SEM are shown. The number of animals per group ranged from three to five per experiment; at least three experiments were performed. ∗, P < 0.05.
Fig. 2.
Fig. 2.
The number of cell surface thiols can be increased by GSH. (a and b) Treatment with GSH or NAC increases TCR+CD4+ T cell surface thiol levels in rats with decreased (DA.Ncf1DA) and normal (DA.Ncf1E3) ROS production. Number of cell surface −SH groups is expressed in geomean. (c) Treatment with GSH does not affect intracellular thiol levels, in contrast to NAC, as determined by staining with MCB. Means ± SEM are shown. Four animals per group were used per experiment; at least three experiments were performed. ∗, P < 0.05. (d) GSH treatment does not affect viability of blood cells at the concentration of 4 mM as determined by titration of GSH and double staining with Alexa Fluor 633-maleimide and PI. A representative experiment is shown.
Fig. 3.
Fig. 3.
T cells with increased numbers of surface thiols are more activated. (a) In vitro T cell activation after increasing the number of cell surface thiols was studied in a mouse model with hybridoma T cells recognizing CII. When cell surface thiol numbers on T cells were increased by GSH (GSH-treated T cells), T cells produced more IL-2, which depended on T cell APC interaction. This was not the case if APCs were treated with GSH (GSH-treated APCs). Experimental values minus control values (T cells and APCs without antigen) are shown. (b) These data were confirmed in vivo in the rat: GSH treatment results in increased T cell proliferation in vivo; the geomean of CFSE staining of CD4+ and PI− (living) cells was 758 in the rats receiving PBS-treated cells compared with 454 in the GSH group. Means with SDs are shown for three experiments. b shows a representative experiment of four.
Fig. 4.
Fig. 4.
T cells from DA.Ncf1E3 rats can only transfer arthritis when cell surface thiol numbers are increased. (a) Spleen cells from 14-day immunized DA.Ncf1DA rats can transfer arthritis to naïve recipient DA.Ncf1DA rats. DA.Ncf1E3 spleen cells can only transfer arthritis when the number of cell surface thiols is increased by GSH treatment. (b) This effect is mediated by CD4+ cells; a transfer with purified CD4+ T cells from inguinal LNs provides similar results. (c) Number of cell surface thiols of all TCR+CD4+ cells in recipients did not differ after transfer. (d) In the allotransfer experiment, all cells positive for the donor MHC class I (RT1Aa+; M1) were CD4+; RT1Aa+cells gated by M1 are shown in Right. This finding confirms that CD4 T cells are responsible for transferring disease. Means (with SEM) are shown for representative experiments with five to eight rats per group. All experiments showed similar significant results. ∗, 0.05 > P > 0.005; ∗∗, 0.005 > P > 0.0005; ∗∗∗, P < 0.0005.
Fig. 5.
Fig. 5.
GSH-treated T cells become effector cells. (a) In vivo, cell surface −SH groups of CD4+RT1Aa+ cells in tissues from the transfer experiment retained high levels of −SH groups in blood and inguinal LNs but not in other tissues. To investigate the longevity of GSH treatment on T cells, HCQ10 T cells were cultured in 2-mercaptoethanol-free medium after treatment with PBS or 4 mM GSH and labeling with CFSE. The number of cell surface −SH groups and the relative CFSE staining intensity were followed over time. (b) Cell surface −SH group levels were higher in the GSH-treated groups and only started to decrease after approximately two divisions, reaching normal levels after six divisions. (c) No differences in proliferation kinetics were observed. Representative experiments of two to three are shown. ∗, P < 0.05.
Fig. 6.
Fig. 6.
Decreasing the number of cell surface thiol groups on T cells decreases arthritogenicity. (a) Treatment of blood with GSSG decreases the levels of cell surface −SH groups on CD4+ T cells. (b) In the mouse T cell activation assay, treatment of the T hybridoma cells with GSSG leads to a decrease in IL-2 production. The shown values are corrected for control values in the absence of antigen. GSSG treatment by itself, in the absence of antigen, did not decrease levels of IL-2 production. (c) In a spleen cell transfer from DA.Ncf1DA rats to DA.Ncf1DA recipients, GSSG treatment of cells led to decreased arthritis severity compared with PBS-treated cells. Means with SDs are shown. ∗, P < 0.05.
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